Photoaffinity labelling of Escherichia coli ribosomes

Pharmac. Ther. Vol. 34, pp. 271-302, 1987 Printed in GreatBritain. All rights reserved

0163-7258/87 $0.00+0.50 Copyright© 1987 PergamonJournalsLtd

Specialist Subject Editor: J. S. FEDAN

PHOTOAFFINITY

LABELING

OF ESCHERICHIA

COLI

RIBOSOMES BARRY S. COOPERMAN Department of Chemistry, University of Pennsylvania, Philadelphia, PA 19104, U.S.A.

ABBREVIATIONS BrsU EF FMN HPLC IF PAGE poly(U) RF RP r-proteins RRF rRNA sau

5-Bromouridylic acid elongation factor Flavinmononucleotide High performance liquid chromatography Initiation factor Polyacrylamide gel electrophoresis Polyuridylic acid Release factor Reverse phase Ribosomal proteins Ribosome release factor Ribosomal RNA 4-Thiouridylic acid

1. INTRODUCTION Ribosomes are complex ribonucleoprotein particles that carry out the crucial function of protein synthesis within the cell. Over the last two decades, an extensive and largely successful effort has resulted in the characterization of both the functional and structural properties of ribosomes (for recent reviews see Nierhaus, 1982; Liljas, 1982; Wittmann, 1983; Noller and Lake, 1984; Giri et al., 1984). More recently, attention has focused on constructing a structure-function map in which different proteins and RNA regions would be located within the ribosome structure and assigned specific roles in the overall process of protein synthesis. Photoaffinity labeling, with its intrinsic capability of defining the components of ligand binding sites, has played a large role in efforts to attain this goal. While ribosomes isolated from several sources are currently under active study, the Escherichia cull ribosome is by far the best understood and this article will concern itself exclusively with this particle. However, it should be pointed out many of the functional and structural properties of ribosomes appear to be evolutionarily conserved, and that studies highly analogous to those herein reviewed have been carried out on ribosomes isolated from other organisms, both procaryotic and eucaryotic. In what follows, the current status of functional and structural studies of the E. coli ribosome is briefly described before beginning the review of photoaffinity labeling studies.

2. C U R R E N T S T A T U S O F F U N C T I O N A N D S T R U C T U R E STUDIES O F T H E E. COLI R I B O S O M E 2.1. REACTION SEQUENCE IN PROTEIN BIOSYNTHESIS As with any polymerization process, it is convenient to divide protein synthesis into three discrete phases: initiation, elongation and termination. Initiation (Maitra et al., 1982) may in turn be divided into three reactions ( 1 - 3 ) . In the first, 271

272

B. S. COOPERMAN TABLE 1. Occupancy of tRNA Sites on the Ribosome*

Complex

Site on the Ribosome E

P

A

R

----

fMet-tRNAMet fMet-tRNAMet fMet-tRNAMet tRNAMet fMet-aa2-tRNA~ a

--aa2-tRNA~a fMet-aaz-tRNA~a _

-aa2-tRNA~a- EF-Tu' GTP _ _ _

--

aa3-tRNA~a. EF-Tu. GTP

I II III IV V

tRNAMet

lla

--

fMet-aa2-tRNA~a

*A and R sites believed to overlap substantially. See text. 70S IF-l, IF-~ 30S + 50S

(1)

30S + fMet-tRNA~ et + m R N A + GTP ~.F-2,IF-3, 3 0 S f M e t - t R N A ~ et. mRNA" GTP

(2)

3 0 S ' f M e t - t R N A f M e t - m R N A - G T P + 50S, 7 0 S ' f M e t - t R N A ~ e t ' m R N A + GDP + Pi P-site

(3)

protein initiation factors IF-11 and IF-3 act together both to shift the equilibrium of reaction (1) toward dissociation of the 70S ribosome into 30S and 50S subunits, and to increase the rate of such dissociation. In the second, factors IF-2 and IF-3 aid in the binding of f M e t - t R N A ~ et and m R N A (containing an A U G codon for f M e t - t R N A fMet respectively. In the third, a 50S subunit binds to form a 70S ribosome in which the f M e t - t R N A ~ a~t is in the so-called ' P ' (for peptidyl-tRNA) site. Elongation takes place completely on the 70S ribosome and may be conveniently described as involving the interconversion of a series of five complexes in which various forms of tRNA are bound to one or more of a total of four distinct (though in part overlapping) sites on the ribosome (Table 1) (Nierhaus, 1984; Spirin, 1985). In the first cycle of elongation, complex I corresponds to the final complex formed in initiation. Complex II is formed when the ternary complex, aa2-tRNA ~ . EF-Tu. GTP (here aa2 represents the second amino acid in the sequence), is bound in the ' R ' (recognition) site in response to the appropriate codon sequence in the mRNA. GTP hydrolysis and release of the binary EF-Tu. GDP complex leads to the formation of complex III, in which aa2-tRNA~a "is bound in the ' A ' (aminoacyl-tRNA) site. The A and R sites are believed to have substantial steric overlap. Conversion of complex ffl to complex IV corresponds to the peptidyl transferase step, as a result of which the growing peptide chain is attached to a tRNA bound in the A site. The translocation step follows, in which, in response to the binding o f the binary elongation factor G (EF-G) complex with GTP, and the subsequent hydrolysis of GTP to GDP, the discharged t R N A ~ et migrates to the ' E ' (exit) site and the peptidyl-tRNA migrates to the P site (complex V). Complex V is converted to complex IIa by the binding of aa3-tRNA~ a. EF-Tu. GTP to the R site which is thought to occur essentially simultaneously with the release of t R N A ~ et from the low affinity E site. The ribosome is now poised to undergo another cycle of elongation. It is worth noting that there is strong evidence for direct c o d o n - a n t i c o d o n interactions in the R,A and P sites, but not in the E site. The final phase of protein synthesis, termination, can be divided into two steps. In the first, proteins called release factors (RF) and either G D P or GTP are bound to the A site in response to the nonsense codons U A A (RF1 or R F 2 ) , U A G (RF1) or U G A (RF2). The presence of either of these factors leads to hydrolysis of peptidyl-tRNA to the polypeptide, which dissociates into solution, and the tRNA, which remains bound to the ribosome. In the second reaction, which is only incompletely understood, both the discharged tRNA and m R N A are dissociated from the ribosome in a reaction requiring a protein called ribosome release factor (RRF), GTP hydrolysis, and EF-G (Ryoji et a l . , 1981). A fourth protein factor, RF3, appears to stimulate various release reactions.

Escherichia coli ribosomes

273

From this brief description, it is not inappropriate to think of a precisely choreographed play as a metaphor for protein synthesis, in which the ribosome is the stage, and the various ligands (tRNAs, initiation, elongation and termination factors) appear at specific times and in specific places, following the script that is contained in the mRNA.

2.2. STRUCTUREOF THE RIBOSOME 2.2.1. Chemical Composition and Overall Structural Organization The E. coli 70S ribosome has a total molecular weight of 2.5 × 106 daltons and consists of two dissociable subunits, denoted 30S (MW - 0 . 8 × 106) and 50S (MW - 1.7 × 106). The 30S subunit is composed of 21 proteins, designated S 1 - $21, and one strand of RNA, denoted 16S RNA, that is 1542 bases long. The 50S subunit consists of 33 proteins designated L1 - L 3 4 (there is no L8) and two strands of RNA, 23S RNA which is 2904 bases long and 5S RNA, 120 bases long. The complete sequences of each of the RNAs (Noller, 1984) and each of the proteins have been determined (Giri et al., 1984). A number of methods have been used in determining the overall shape of the ribosome. Although different methods give somewhat different dimensions, the following dimensions (in ,~) may be considered at least approximately correct: 30S subunits, 5 0 - 8 0 × 100 × 200; 50S subunits, 160 × 200 x 230; 70S ribosomes, 170 × 230 × 250. Three-dimensional models of structure, based on electron micrographs, have been proposed for 30S, 50S and 70S particles (Liljas, 1982; Nierhaus, 1982; Wittmann, 1983; Noller and Lake, 1984; St6ffler and St6fflerMeilicke, 1984; Vasiliev and Shatsky, 1984). Until recently such models were based on inspection of large numbers of micrographs showing two-dimensional projections of ribosomes in different orientations. In the past several years, however, more rigorous three-dimensional reconstruction techniques have been applied, and some of the past disagreements regarding particle shapes have been resolved (Verschoor et al., 1984,1985,1986). In addition, procedures for crystallization of 30S and 50S subunits and 70S ribosomes have recently been published. Analysis of such crystals should also yield improved three-dimensional structures (Clark et al., 1982; Yonath et al., 1986).

2.2.2. Protein Topography A variety of techniques are being used to investigate protein topography within the ribosome. Most of the recent work is treated in reviews already cited and will be discussed only briefly in this section. The technique that has yielded the most easily interpretable results is that of immunoelectron microscopy, using antibodies to individual ribosomal proteins (St6ffier and St6ffler-Meilicke, 1984; Noller and Lake, 1984; Giri et al., 1984). Distances between ribosomal proteins within the 30S subunit are also being mapped using neutron scattering, with reconstituted subunits containing pairs of deuterated proteins. These studies have allowed, by triangulation, generation of a model placing proteins in a three-dimensional matrix (Ramakrishnan etal., 1984). Distances between ribosomal proteins have also been estimated by fluorescence energy transfer (Huang et al., 1975; Deng et al., 1986). Other information on the relative placement of proteins within the ribosome comes from cross-linking studies (Traut et al., 1980), using either bifunctional reagents, direct u.v. irradiation or u.v. irradiation in the presence of a photosensitizer. Isolation of cross-linked protein pairs or triplexes can be accomplished by gel electrophoresis. The proteins in such complexes can be identified by means of specific antibodies, or, if cleavable cross-linking reagents are used, by diagonal two-dimensional gel electrophoresis, in which the first dimension is run before and the second dimension after cleavage of the cross-link. Yet another approach useful for inferring protein-protein interaction on the ribosome is the detection of specific complexes of ribosomal proteins in solution. For example, L7, L12 and L10 spontaneously form a protein complex (Pettersson et al., 1976). Combination of the results obtained by these techniques has allowed formulation of models for the placement of 30S proteins within the 30S structure and more complete models will JPT

34:2-I

274

B.S. COOPERMAN

undoubtedly be forthcoming. Similar efforts are also underway for the 50S subunit, but here the available information is much less complete (St6ffler and St6ffler-Meilicke, 1984). Although immunoelectron microscopy has provided the most important single contribution, it is important to note that many of the results of this technique are controversial, with different laboratories putting forward what are at times different and incompatible protein localizations, as discussed in Prince et al. (1983). Much of the controversy appears to arise from the use of impure ribosomal proteins as immunogens, resulting in antibody preparations with specificities for more than one ribosomal protein.

2.2.3. R N A Structure The availability of the primary sequences of 5S, 16S and 23S RNA has led to the formulation of models for their 2 ° structures as recently reviewed by Noller and others (Noller, 1984; Gutell et al., 1985). These models are based on: (a) the Tinoco et al. (1973) rules for base-pairing; Co) chemical modification of specific bases by reagents specific for single-stranded RNA; (c) invariance of certain residues or structural elements in RNAs from different organisms; and (d) specific nuclease cleavage of both single-stranded and double-stranded regions. A limited amount of information is also available regarding 3 ° structure, based largely on the analysis of intra-RNA cross-links (Stiege et al., 1983; Wollenzein et al., 1985; Atmadja et al., 1986). In addition to the 2 ° and 3 ° structures, an increased body of information has accumulated with respect to the three-dimensional locations of specific sites in ribosomal RNA. Thus, specific sites have been localized by immunoelectron microscopy using antibodies both to modified bases such as N6,N6-dimethyladenosine (Politz and Glitz, 1977,1980) and 7-methylguanosine (Trempe et al., 1982) that occur at unique locations in the RNA sequence as well as to haptens which can be chemically attached to the 3' or 5' ends of the RNA molecules (St6ffler and St6fflerMeilicke, 1984). Other electron microscopic probes have been applied to localize short ( - 10 bases) deoxyoligonucleotides that are complementary to specific sequences in rRNA (Oakes et al., 1986). In addition, distances between the 3' ends of 5S, 16S and 23S RNA and between distinct regions of tRNAs bound in the A and P sites (Robbins and Hardesty, 1983; Wintermeyer et al., 1984; Paulsen and Wintermeyer, 1986) have also been measured by fluorescence energy transfer. Here, energy donor and acceptor groups have generally been introduced by chemical modification, although in the case of tRNAs the intrinsic fluorescence of the naturally occurring wybutine base has also been exploited (Paulsen et al., 1983).

2.2.4. Functional Sites A variety of approaches have been used to localize functional sites involved in protein synthesis to specific areas of the ribosome. In addition to affinity labeling, these include immunoelectron microscopy, reconstitution, genetic alteration and antibody binding, or some combination of these approaches. Several recent reviews have appeared summarizing work in these areas (Nierhaus, 1982; Wittmann, 1983; St6ffler and St6ffler-Meilicke, 1984; Noller and Lake, 1984; Vasiliev and Shatsky, 1984). Reconstitution experiments are based on the results that 30S and 50S subunits may be fully reconstituted, either from the individual proteins and RNA strands, or by combination of core particles and 'split' (i.e. dissociated) proteins, both produced by high salt washing of the subunits. In reconstitution experiments, the functional properties of a ribosomal subunit reconstituted either with one or a small number of proteins omitted, or with a single protein chemically modified, are tested, and changes in or loss of function are attributed to the proteins in question. Using genetic alteration, the source of a phenotypic change in ribosomal function in a mutant strain may be traced to a particular protein, or group of proteins, both by identifying altered proteins by changes in electrophoretic migration and/or column (e.g. HPLC) retention, and by reconstituting hybrid ribosomes containing both wild type and mutant proteins. An alternative approach, exploited particularly by Dabbs et al. (1983), is to demonstrate the non-essentiality

Escherichia coli ribosomes

275

of certain ribosomal proteins by isolating ribosomes from mutant strains of E. coli that lack such proteins. Finally, with antibody binding, the effects on ribosomal function of a monovalent antibody fragment specific for a particular ribosomal protein is measured. That an antibody fragment inhibits a given function is taken as evidence that the protein to which it binds is involved in that function.

3. PHOTOAFFINITY LABELING RESULTS Although approximately 100 papers have appeared in the last 12 years describing the results of photoaffinity labeling studies on E. coli ribosomes, in only a portion of these published studies has the analysis of results been pushed far enough to permit some measure of confidence that the goal of identifying ribosomal components at the ligand binding site has been achieved. For a photoaffinity labeling experiment to be successful it is necessary first that the labeled ribosomal components are unambiguously identified and second, that evidence is obtained demonstrating that labeling has occurred at a functionally significant site. However, in a large number of published studies either one or both of these conditions has (or have) not been met, resulting in a large body of data of uncertain significance. Below the experimental approaches used in photoaffinity labeling experiments on the E. coli ribosome are considered first, followed by a presentation of the results obtained, concentrating both on those experiments that best meet the conditions posed above and on more recent experiments. Earlier work on both electrophilic and photolabile affinity labels has been reviewed elsewhere by myself (Cooperman, 1978,1980) and others (Keuchler and Ofengand, 1979; Ofengand, 1980).

3.1. EXPERIMENTALAPPROACHESFOR THE IDENTIFICATIONOF COVALENTLY-LABELEDSITES 3.1.1. Identification of Labeled Proteins

In virtually all cases, photoaffinity labeling experiments on ribosomes have been performed using radioactive photoaffinity labels and photoaffinity-labeled proteins have been identified by the radioactivity incorporated in them. Three predominant methods have been used in performing the requisite protein separations: electrophoresis, using both one- and two-dimensional (Kaltschmidt and Wittman, 1970; Howard and Traut, 1974; Kenny et al., 1979) polyacrylamide gels; immunological analysis, using purified rabbit antisera to specific ribosomal proteins and either specific immunoprecipitation via 'sandwiches' employing goat antibody to rabbit -r-globulin (Grant et al., 1979a), or immunodiffusion through high porosity agarose gels (Pongs et al., 1975a), or cosedimentation in a sucrose gradient (Fiser et al., 1975b); and most recently, reversed phase HPLC chromatography (Kerlavage et al., 1983a,b; Weitzmann and Cooperman, 1985). One-dimensional PAGE analysis, though insufficient to fully resolve all ribosomal proteins, has nevertheless been extensively used because it does allow relatively rapid and quantitative measurement of a labeling pattern and the response of that pattern to changes in experimental protocol (Jaynes et al., 1978). Two-dimensional PAGE analysis allows full resolution of all ribosomal proteins but is both more time-consuming to perform and shows more variability in the yields of recovered material, thus complicating analysis of affinity labeling experiments. The advent of HPLC separations of ribosomal proteins has provided a technique for ribosomal protein analysis that supersedes PAGE analysis in that it is at the same time easier to perform, more rapid, more precise and affords higher yields and better reproducibility. Moreover, although typical analyses of ribosomal protein by HPLC give somewhat lower resolution than is possible via two-dimensional PAGE analysis, it is possible to manipulate elution conditions so as to afford very high resolution in a region of particular interest. This point is illustrated by comparing the chromatogram in Fig. 1, which displays a typical analysis of total protein extracted from a 50S particle (TP50) using what is for the most part a linear gradient of acetonitrile as the eluant, and Fig. 2, in which the gradient is made much shallower in open specific region, with the result that proteins L22, L23 and L29, which are hardy separated in Fig. 1, are fully resolved in Fig. 2.

276

B . S . COOPERMAN

A

0.24

14 Ig 30

',g

0.16

32 33 2r

28

40

151

4

16 2Z.Z~

< ZS 5

20

12

I

0.08 ZS

} _L,v0

20

q

I

l

I

I

J

I

40

60

80

IO0

120

140

E L U T I O N TIME (rain)

FIG. 1. R P - H P L C of TP50 (Kerlavage et al., 1983B). Numbers next to peaks correspond to L proteins.

An inherent problem in the use of either two-dimensional PAGE or HPLC analysis to identify labeled proteins is that the photoaffinity-labeled protein may migrate (or elute) sufficiently differently from the native protein that its location in a gel (or its elution volume) may not be adequate to allow for its unambiguous identification by PAGE or HPLC analysis. This can be particularly problematic in crowded regions of the gel or chromatogram. Three different approaches have been successfully employed in confronting this problem. The first is to obtain an unambiguous identification through an immunological approach, as described above. The major practical problem with this approach is the tediousness of preparing all 52 ribosomal protein antisera in pure (i.e. non-cross-reacting) form, although once such materials are available analysis is straightforward. The second is to identify the photoaffinity-

I 32


2,3 13,17

4,10

14 21

6 9 15 16 I

3327

ij

28 24

18

I0

3b

5?

7'0

ELUTION TrME (mini

FIG. 2. R P - H P L C of TP50 with high resolution in the area of proteins L18, L22, L23 and L29 (Kerlavage et al., 1984). Reprinted from Kerlavage et al. (1984). Copyright Elsevier Science Publishers, B.V.

Escherichia coli ribosomes

277

labeled protein by classic protein characterization methods, such as tryptic fingerprints or partial N-terminal sequence determination. The advantage of such an approach is the ease with which it can be applied directly, provided that it is possible to prepare a photoaffinity-labeled protein that is not contaminated by other ribosomal proteins. Recent results in our own laboratory would indicate that such preparations should generally be obtainable via HPLC (Kerlavage et al., 1984,1986; Cooperman et al., 1986; Kerlavage and Cooperman, 1986). The third method is indirect, but can nevertheless be quite powerful. It consists of comparing the results of applying two different high resolution methods to the analysis of a given sample of photoaffinity-labeled ribosomal proteins. The underlying idea behind this approach is that the cohort of proteins close to which a labeled protein elutes (or migrates) will sometimes be different for the two different methods, thus eliminating the ambiguity in the identification of the labeled protein. Suitable pairs of methods would be R P - H P L C and two dimensional PAGE, or R P - H P L C and ion exchange HPLC. An additional problem for the identification of labeled proteins is that some of them, in particular S1, $2, $21 and L7/12, are easily lost from 30S or 50S subunits either during subunit separation or as a result of the wash procedures typically employed to remove excess reagent. As a result, labeling of these proteins is frequently not monitored.

3.1.2. Identification of Labeled Regions of RNA The ultimate goal of work directed toward the localization of sites of photoaffinity labeling of rRNA is the identification of the base or bases into which incorporation has occurred. In recent years, two approaches have been successfully applied in achieving this end, although, as applied to date, both have significant limitations. The first consists in determining the extent of labeling in oligonucleotides produced on full ribonuclease (e. g. T 1) digestion of photoaffinitylabeled rRNA and the purification and sequencing of the labeled oligonucleotide or oligonucleotides. The drawbacks to this approach are that it is not only quite tedious to perform, but it also has the requirement that the isolated, labeled oligonucleotide must be of sufficient length to be uniquely placed in the primary structure of either 16S or 23S rRNA. Thus, while it has worked in some cases (e.g. Prince et al., 1982) it has failed in others (e.g. Eckermann and Symons, 1978; Leitner et al., 1982). The second approach is based on two steps. In the first, rDNA restriction fragments are used as hybridization probes of photoaffinity labeled rRNA to fairly rapidly localize labeling to RNA sequences of 100-200 bases (Barta et al., 1984; Hall et al., 1985). A schematic of this step is presented in Fig. 3. In the second, the photoaffinity labeled rRNA is hybridized with singlestranded oligodeoxynucleotide complementary to a region of rRNA that is past (to the 3' side) the region of rRNA labeling found in the first step. This heteroduplex then serves as a substrate for reverse transcriptase. Using a classic sequencing gel technique as the method of measurement, a position where the reverse transcriptase is found to halt or pause when the photoaffinity-labeled sample is being analyzed, but where there is no corresponding halt or pause on analysis of unlabeled rRNA, is deemed to be a site of photoaffinity labeling (Barta et al., 1984). The first step in this approach is both easy to perform and totally general in its applicability and may be considered the method of choice for partial localization of sites of rRNA photoaffinity labeling. Here it should be noted that localization to the level of 100-200 bases will usually be sufficient for analysis of the labeling process as a function of several variables such as light flux, photoaffinity label concentration, and wavelength of irradiation. The second step is both conceptually more elegant and easier to perform than earlier techniques, but has three significant problems limiting the generality with which it can be applied: first, it falls to distinguish between indirect and direct effects of photoaffinity labeling on the reverse transcriptase assay -- for example, a photoaffinity label, in forming a non-covalent complex with the ribosome, might act to photosensitize a change in rRNA structure without actually photoincorporating; second, this approach is essentially blind at those sites of unmodified rRNA at which reverse transcriptase halts or pauses; and third, it is not clear that all photoincorporation reactions inhibit reverse transcriptase activity. From this discussion it seems clear that combining these two approaches

278

B. S. COOPERMAN

Q

I.

Restriction andonuclem digmtion

pKK 3535 11. Hybridization with exceu (3H)-Iabeled RNA X b

x b

4

"=''°

2

I11. S1 nucleateand RN/~ TI digestion

=''" 3 • ------o I

i

x

...°..°.~..o...o. ....°..=~ ........ ....o...~o

.......

Hybri~

Single-stranded DNA (non-complimentary to rRNA)

J IV. PAGEanalysis

-I

r ,=====lunal=~ 1 V.

==.=8=..~ 2 ....... x

Gel dicing and counting

1

2 3

I

"1-

I

I

4 I

hybrid positions

3

"'~'=4 Gel Slice Number (migrations)

DNA ............

RNA

X - group contains radioactive label

FIG. 3. Schematic of method for localizing sites of labeling within ribosomal RNA (Hall et al., 1985). Note that pKK 3535 contains the entire rrnB gene. Reprinted from Hall et al. (1985). Copyright ACS.

AcceptorStem 1~

End

~

T~,CStem .4~

J

54

,~,~,.__.~ ~ -

DStem5 ~

T~C

DLoop V~r;:~le

~;~"i'~-'-- Anticodon :

Anticodon _

_

8

~

~

Stem

2

34

FIG. 4. Three-dimensional structure of a typical tRNA (yeast tRNA-Phe) showing positions (as shaded circles) from which photocross-linldng to ribosomes has been achieved. These are: aminoacyl group and bases 8, 20, 32. 34, 37 and 47.

TABLE 2. Photoaffinity Labeling of 30S and 70S Particles with tRNAs Derivatized at Specific Positions Derivatization position tRNA binding site occupied Ribosomal component RNA

N~
23S RNA; U-2584, U-2585" 23S RNA; 2445-2668t

S proteins 3 7

11 18

70S, A or P site¶

Position 8 70S, A site**

Position 32 or 60 70S, P sitet t

16S RNA; C-1400

+

10 14 19 L proteins

30S§

Position 34 (anticodon)

~r

+~ +~

*Barta et al. (1984). tHall et al. (1985). ~Hsiung and Cantor (1974); Hsiung et al. (1974). Girshovich et al. (1974a,b). Prince et al. (1982); Ciesiolka et al. (1985). **Linet al. (1984). ttRiehl et al. (1982). The assignment of position 32 or 60 is speculative.

~

to

to

TABLE 3. Photoaffinity Labeling of 30S and 70S Particles Using Either Underivatized tRNA or tRNA Derivatized Randomly on G Residues with An Aryl Azide* Underivatized tRNAJ" Target Site Occupied Cross-linked tRNA

70S A Phe-tRNAPhe(l); N-AcPhePhe-

70S P N-AcPhetRNA Phe

Aryl Azide t R N A $

70S R

70S S(E)

P h e - t R N A Phe EF-Tu. GMPPCP

tRNA Phe

tRNA Phc (2)

30S N-AcPhe-tRNAPhe(1); f M e t - t R N A Met (2);

70S A

70S P

P h e - t R N A Phc

tRNA Phe A site empty (1); a site full

P h e - t R N A Phe (3);

70S P Phen_tRNAPhe A site empty

~o

(2) tRNA Phe (4) Labeled proteins S 4 5 6 7 8 9 or 9/11 10 11 12 13 14 or 13/14 15 16

+ + +

+ (1,2)

z

+ (1,2,3,4) + (1,2,3,4) + (3,4)

+ (1,2)

+

+

+

(2)

+ (3,4) + (1) + (1,2)

+

+ (1,2,3,4)

+

+ (2)

+ (2) + (4)

¢3 © ©

+

+ (1)

+

+ + + +

+

(1,2) (1,2) (1,2) (2)

+

+ (2)

15/16/17 18 19 20 21 L 2 4 5 6 7/12 8/9 11 13 14 15 16 17 24 27 31 32 33

+

+ (1,2)

+ + +

+ (1,2) + (1) + (1)

+ + +

+ (1,2)

+

+ + (2) + (1) + (1,2)

2.

+ +

*The number in parentheses indicates which form of tRNA is in the site in question. An underlined protein is a minor product. tAbdurashidova et al. (1979, 1981, 1985a,b,c); Broude et al. (1985). :~Babkina et al. (1984a,b,c); Bausk et al. (1985).

+ + + + +

(1,2) (1,2) (1,2) (2) (1)

+

~r o

282

B.S. COOPERMAN

by carrying out total ribonuclease digestions on labeled RNA regions prepared by hybridization would yield a more general method for labeled base localization.

3.2. SURVEY OF PHOTOAFFINITYLABELINGRESULTS 3.2.1. tRNA

A major challenge for photoaffinity labeling studies is the determination of the complete loci for tRNA binding sites on the ribosome. Two general approaches have been employed. The first consists in derivatizing tRNAs with photoabile reagents at different specific locations in the tRNA molecules, one position at a time, in order to be able to determine the parts of the ribosome in contact with these locations when the tRNA is bound in the A, P, R or E site (see Section 2.1). Specific derivatives have been prepared by exploiting the unique chemical reactivities at various positions in tRNA molecules. For essentially all tRNA molecules, appropriate derivatives can be made both through reaction at the ct-amino group of 3'-aminoacyl tRNA and by replacement of the dihydrouridine ring at position 20 with a photolabile group. For some tRNA molecules additional derivatives can be made at positions containing appropriate naturallyoccurring modified bases, including (i) the 4-thiouridine at position 8, (ii) the 2-thiocytidine at position 32, (iii) the 5-carboxymethoxyuridine at position 34, and (iv) the 3-(3-amino, 3-carboxypropyl)uridine at position 47. In addition, the intrinsic photoreactivities of the 5-carboxymethoxyuridine or 5-methoxyuridine at position 34 and of the wyebutine base at position 37 which are found in some tRNAs have been exploited in direct photoincorporation studies. The position of these bases within the typical three-dimensional structure of a tRNA molecule are shown in Fig. 4. The second approach consists of attempting to simultaneously determine many points of contact of tRNA with the ribosome, either via direct photolysis of the native tRNA ribosome complex using relatively high light fluxes to compensate for the modest photoreactivity of most nucleoside bases, or via photolysis of a complex of the ribosome and tRNA in which the tRNA has first been randomly derivatized on reactive guanosine or cytidine bases with an aryl azide-containing reagent. The results obtained in these studies are summarized in Tables 2 and 3 and discussed below.

3.2.1.1. N~-Derivatives ofaminoacyl tRNA. Photoaffinity labels prepared by acylating the ~amino group of aminoacyl-tRNA have been shown to photoincorporate into both r-proteins and rRNA on photolysis of their complexes with ribosomes. Cantor and his co-workers, using two different arylazide derivatives of PhetRNA Phc, have obtained quite strong evidence that two proteins, L11 and L18, are labeled from the peptidyl donor, or P' site (defined as the portion of the P site in which the 3'-terminus of the peptidyl-tRNA is bound), located in the peptidyl transferase center of the ribosome (Hsiung and Cantor, 1974; Hsiung et al., 1974). As twodimensional PAGE analysis alone was used in identifying the labeled proteins, there remains some question as to whether the labeled proteins were correctly identified. Here it should be noted that both L11 and L18 migrate in crowded regions of the two-dimensional gel. On the other hand the evidence for labeling at a functional site is compelling. Not only does labeling depend on the presence of mRNA (in this case poly(U)), but also ribosomes that have been photoaffinity labeled with these derivatives have been shown to react with a peptidyl acceptor. Thus, if ribosomes are first photoaffinity labeled with a nonradioactive N~-arylazide derivative of Phe-tRNA Phe and then treated with radioactive [3H]Phe-tRNA Pne, radioactivity is found incorporated into proteins L l l and L18, as identified by two-dimensional PAGE. The other major study resulting in r-protein labeling is that of Girshovich et al. (1974a,b) who, again using two-dimensional PAGE, identified proteins $3, $7 and S 14 as being the major proteins labeled by a third arylazide derivative of Phe-tRNA Phe. Unfortunately in this case very little evidence was presented for the functional site specificity of such labeling. Although several early studies clearly showed labeling of 23S rRNA by a variety of photolabile A~-derivatives of aminoacyl tRNA (Bispink and Matthaei, 1973; Girshovich et al., 1974a,b;

Escherichia coli ribosomes

283

Barta et al., 1975; Sonnenberg et al., 1975), it is only quite recently that the sites of labeling by such derivatives within 23S RNA have been determined. Thus, both Barta et al. (1984), using a benzophenone derivative, and Hall et al. (1985), using an arylazide derivative, have shown the major labeling site for each derivative to fall within the base sequence 2445-2668. In addition, Barta et al. (1984) have provided further evidence, using the reverse transcriptase procedure described above, that the major sites of labeling in their experiment are uridines 2584 and 2585. Controls for both experiments, similar to those described above, provide strong evidence for the functional site specificity of the labeling obtained. In a related study the derivative prepared by coupling tRNA Phe, periodate-oxidized at its 3'-end, with p-azidobenzoylglycylhydrazide, was employed as a photoaffinity label for the ribosome (Leitner et al., 1982). In this work most of the labeling was shown to occur within 23S rRNA, and a labeled oligonucleotide, G*AAGC was isolated, but this sequence was too short to place unambiguously within 23S rRNA.

3.2.1.2. Anticodon loop. Important results have been obtained involving photoincorporation from positions 32, 34 and 37 (the anticodon bases are positions 34-36) within tRNAs bound in both the A and P sites on the ribosome. Although significant r-protein labeling has been found in several cases, the precise localization of labeling has been confined to sites within rRNA and it is these results that will be emphasized. The most extensive set of results have been obtained using E. coli tRNA1 val. This tRNA, when charged* with N-acetylvaline and bound in the P site, has been shown, on photolysis, to form a cross-link to 16S RNA (Ofengand et al., 1979). An elegant series of experiments in the laboratories of both Ofengand and Zimmermann has clearly shown this cross-link to consist of cyclobutane formation between the 5-carboxymethoxyuridine-34 of the tRNA and the C-1400 of 16S RNA (Prince et al., 1982). The chemical characterization of the cross-link, though indirect, is quite convincing. It is based, first, on the absolute dependence of cross-linking on the presence of a 5-carboxymethoxyuridine-34 (or 5-methoxyuridine-34) in a series of tRNAs that were investigated; second, on the observation that the cross-link can be formed by irradiation at 320 nm and is cleaved on irradiation of 254 nm, two reactions that it has in common with cyclobutane formation between pyrimidines; and third, on the observation that RNase T1 digestion of the tRNA-16S RNA cross-linked species permits isolation of a cross-linked oligonucleotide that, on photoreversal at 254 nm and subsequent analysis of the small oligonucleotides released with RNase A and U2, is shown to have contained a cross-link between positions 34 (tRNA) and 1400 (16S RNA). The functional site specificity of this cross-link is shown by the dependence of cross-link formation on the correct positioning of the tRNA in the P site and by the reactivity toward puromycin of covalently bound N-acetylVal-tRNAv~. An interesting aspect of the crosslinking reaction is that it only occurs when there is incorrect base pairing between 5-carboxymethoxyuridine-34 and the corresponding codon base (Ofengand and Liou, 1981). Correct base pairing would presumably make base 34 unavailable for the cross-linking reaction. In a related study, Ofengand and his co-workers have prepared several different photoabile derivatives of tRNAI val by attaching a nitrophenylazide to the carboxyl group of 5-carboxymethoxyuridine-34 via side chains varying in length from 18 to 24 A (Gornicki et al., 1985). Although all of the derivatives tested were found to photoaffinity label E. coli ribosomes when bound either in the A (as Val-tRNA v~) or P (as N-acetylVal-tRNAval) sites, identification of the sites of labeling have only been carried out for two derivatives bound in the A site. Each of these derivatives has a side chain of 2 3 - 2 4 A. and each cross-links to C-1400 of 16S RNA as the major site of photoincorporation (Ciesiolka et al., 1985). By contrast, when such derivatives are bound in the P site and photolyzed, the only photoincorporation found apparently takes place into 30S proteins. Chen et al. (1985a) have prepared several photoaffinity labels of E. coli tRNA~arg by selectively alkylating it on the 2-thiocytidine at position 32 with different arylazide-containing

*Charging is the term used to describethe tRNA synthetasc-catalyzed¢sterificationof a tRNA with the appropriate aminoacidon the 3'-terminalbase. Acetylationofthe aminogroupis performedchemicallyfollowingthe chargingreaction.

284

B.S. COOPERMAN

reagents. These derivatives, when bound in the P site in the form of modified N-acetylArgtRNA1Arg and irradiated, photoincorporate into both the protein and RNA fractions of the 30S subunit. The individual proteins labeled have not as yet been identified, although the major site of incorporation of at least one of the derivatives into 16S RNA has been shown to fall within bases 918-1497. Given the proximity of bases 32 and 34 in the tRNA molecule, this result is at least consistent with the tRNAt val photoincorporation results presented above. Finally, Kuechler and his co-workers (Matzke et al., 1980; Steiner et al., 1984) have shown that on irradiation at 320 nm, yeast Phe-tRNA Ph~ which is bound to the A site of E. coli ribosomes in the form of a ternary complex with EF-Tu and GTP incorporates covalently via its photolabile wybutine base at position 37 into the 5' position of the mRNA codon triplet at the A site.

3.2.1.3. Other positions of specific modification. In addition to tRNAs modified at the 3' terminus and anticodon loop, photoaffinity labeling studies have been carried out with tRNAs modified at several other positions. Of these, the most complete results have been obtained by Ofengand and his co-workers in experiments conducted using E. coli Phe-tRNA Phe derivatized at the 4-thiouridine in position 8 with an arylazide-containing reagent (Hsu et al., 1984). They have shown that when such a derivative is bound in the A site and photolyzed, protein S19 is labeled virtually exclusively. Here identification was by immunological analysis (Lin et al., 1984). Little covalent incorporation is obtained when the derivative is positioned in the P site. In more preliminary work, Chen et al. (1985b) have exploited the lability of the dihydrouridine residue at position 20 of E. coli tRNAI ~ly to specifically remove this residue via reduction (Yang and $6ll, 1974) and to replace it with an arylazide-containing hydrazide. When this modified tRNA, charged with an N-acetylglycl residue, is bound in the P site and photolyzed, 16S RNA is the major species labeled. Partial RNase digestion suggests that incorporation proceeds into more than one site, but a full characterization of incorporation sites has not yet been reported. One further specifically-modified tRNA that has been studied as a photoaffinity label for the ribosome is the derivative formed on acylation of the 3-(3-amino-3-carboxypropyl) uridine at position 47 of E. coli tRNA Phe with N-(4-azido-2-nitrophenyl) glycine-N-hydroxysuccinimide ester (Schwartz and Ofengand, 1982). When it is charged with an N-acetylPhe and bound in the P site, this derivative is found to photoincorporate into both 30S and 50S subunits in a poly(U)dependent manner. Here again no detailed localization results have yet been reported.

3.2.1.4. Unmodified tRNA. Budowsky and his co-workers (Abdurashidova et al., 1979,1981, 1985a,b,c; Broude et al., 1985) have exploited the result that [32p]-tRNAPhe bound to ribosomes in the presence of poly(U) will covalently incorporate into ribosomal proteins on irradiation at 254 nm, to determine which ribosomal proteins are in contact with bound tRNAs. With 70S ribosomes as the target, their work has focused on determining the contacts made in the A and P sites as well as in the E (which they call the 'S') and R sites. With 30S subunits as the target, they have asked the question whether the nature of the tRNA bound alters the identities of the proteins cross-linking to tRNA. They have employed two methods to identify cross-linked proteins. In the first, used in the earlier work, the covalently labeled ribosomes are extensively digested with RNases A and T1, and the radioactivity-labeled proteins are separated by twodimensional PAGE and detected by autoradiography. Such analysis is, however, sometimes insufficient to identify the labeled proteins because of the influence of attached oligonucleotides on protein mobility. Accordingly, when required, labeled proteins are further characterized by iodination with 1251 and comparison of their tryptic peptide fingerprint analyses with those obtained for known proteins. It should be noted that while this procedure is appropriate in principle, its successful application is critically dependent on the protein purity of the 32p-labeled proteins, since contamination of a labeled protein with a different unlabeled protein could lead to incorrect identification of the contaminant as the labeled protein. Unfortunately, this point is not addressed in the published work. In the second method for identification of cross-linked proteins, the tRNA is derivatized with

Escherichia coli ribosomes

285

Hg (such derivatization has no effect on tRNA binding to the ribosome) and the protein-tRNA covalent complexes are specifically-bound to a thiopropyl Sepharose column. The bound proteins are then iodinated with 1251,cleaved from the tRNA by ribonuclease treatment, resolved by twodimensional PAGE, and located using a multi-channel proportional detector. Of course, here too the change in the electrophoretic mobility of proteins on modification poses a potential problem for identification. Another potential problem with this work, which the authors consider, is that cross-linking might occur on light-denatured rather than native tRNA. ribosome complexes. Three lines of evidence indicate that this is not so for the light fluences used in this work ( < 50 quanta per nucleotide). First, the loss of ribosomal tRNA-binding activity following irradiation is < 30%; second, the structural integrity of the ribosome is maintained, as shown by the lack of any change in sedimentation coefficient of the 70S particle and the lack of any photo-induced cleavage of the ribosomal RNA; third, and most importantly, the rate of overall cross-linking follows apparently 'single-hit' kinetics. Important as these criteria are, it is important to point out that because 'single-hit' kinetics were not demonstrated for each labeled protein separately (for example, by two-dimensional PAGE analysis of labeled proteins as a function of light fluence) the possibility remains that some proteins are labeled by 'multi-hit' kinetics, i.e. within a lightdenatured ribosome.tRNA complex. Despite these caveats, the results of this work are rather striking in showing quite different sets of proteins labeled depending on whether tRNA is bound in the A, P, R or S(E) site (for studies on the 70S ribosome), or whether charged tRNA or uncharged tRNA is bound (for studies on the 30S subunit). These results are summarized in Table 3. In related work, Kruse et aL (1982) have effected ravin mononucleotide-sensitized crosslinking between [3H]Phe-tRNAahe bound at different sites on the ribosome to both ribosomal RNA and r-protein. Although no detailed determination of labeled components was performed, it was nevertheless clear from the large difference in distribution of radioactive label betwen the protein and RNA fractions of the 30S and 50S subunits that labeling was occurring at distinct sites.

3.2.1.5. Randomly-modified tRNA. Experiments directed toward the identification of ribosomal proteins coming into contact with tRNA have also been carried out by the Novosibirsk group (Babkina et al., 1984a,b,c; Bausk et al., 1985). Although the overall logic of their experiments is very similar to that of the Budowsky group, discussed above, the approach is somewhat different in that cross-linking is carried out via irradiation of tRNAPhe-ribosome complexes in which the tRNA Phe has been derivatized at the N7-position of guanosine residues with an arylazidecontaining reagent. The derivatized tRNA phe is prepared by alkylation with the nitrogen mustard [14C]-N-2-chloroethyl-N-methylamino-benzyl amine followed by arylation of the benzyl amine moiety with 2,4-dinitro-5-fluorophenyl azide (Vladimirov et al., 1981). In this way, derivatized tRNAs are prepared containing 2 to 3 arylazides distributed among the available N 7 atoms of the guanine residues in tRNA phe. The advantage of this approach is that irradiation leading to photoincorporation can be performed at wavelengths >_350 nm that have no denaturing effect on ribosomes. Two preparations are employed, azido-tRNAPh%I, in which guanines involved in 2 ° structure are protected from derivatization, and azido tRNAPhe-II, in which guanines involved in either 2 ° or 3 ° structure are protected from derivatization. In this work, the great majority of the incorporation takes place into proteins. Following nuclease digestion of covalently-labeled ribosomes, the labeled proteins are identified by twodimensional PAGE analysis. An improvement over the procedure used by Abdurashidova et al., with unmodified tRNA (as outlined above), is that E. coli phosphomonoesterase is used in the digestion step in addition to RNases A and T 1. As a result, no more than one nucleotide should be covalently incorporated into any labeled protein and the mobility of labeled protein should be closer to that of native protein. Although even this improvement falls short of providing totally unambiguous identification of all labeled proteins it is nevertheless clear that in this work, as with that of the Budowsky group, different sets of proteins are labeled depending on whether

286

B.S. COOPERMAN

derivatized tRNA is bound in the A or P sites, or for derivatized tRNA bound in the P site, on whether or not it is charged with a peptide group or whether the A site is occupied or empty. The results of a number of such labeling experiments are summarized in Table 3. A third approach to the identification of ribosomal components interacting along the tRNA binding surface is due to Barritault et al. (1981). They convert cytidine residues within tRNA into 4-thiouridines by treatment with H2S and exploit the photoreactivity of 4-thiouridine to form covalent bonds with portions of the ribosome on irradiation of the complex between the ribosome and the modified tRNA at 335 nm. Although the potential of this approach for yielding covalent incorporation was clearly shown in this first paper, it is the subsequent work of Riehl et al. (1982) that led to the identification of a labeled ribosomal component. Using yeast tRNA Phe these latter authors first removed the 3'-CCA end enzymatically, then thiolated the tRNA, and reincorporated the CCA end using [lnC]-labeled CTP and ATP. This rather sophisticated bit of affinity label design not only prevents loss in activity of the functional 3'-end due to thiolation, but also provides a convenient method for introducing radioactivity into the tRNA. The modified tRNA, when charged with an N-acetylPhe group and bound to the P site, was shown to photoincorporate exclusively into a single ribosomal protein, which was identified as protein S10 by two-dimensional PAGE analysis (and therefore subject to the uncertainty already discussed above) of an RNase A and RNase T 1-treated sample of 30S protein. The specificity of the photoincorporation reaction for a single protein leads the authors to speculate that the thiolation reaction was confined to a limited number of cytidine residues. One possibility is that modification occurs only on C-residues not involved in base pairing, which according to the known X-ray structure of yeast tRNA Phe (Fig. 4) would restrict modification to C-32 and C-60.

3.2.2. m R N A Photoaffinity labeling studies directly toward the identification of ribosomal proteins involved in mRNA binding have been carded out using both synthetic polynucleotides [poly(U) (Schenkman et al., 1974; Fiser et al., 1975a), poly(BrSU) (Pongs et al., 1975b) and poly(sau) (Fiser et al., 1974,1975b, 1977)], natural polynucleotides [Q/3RNA (Broude et al., 1980) and MS2 phage RNA (Broude et al., 1983)] and oligonucleotides containing arylazide groups at both the 3'-end (Towbin and Elson, 1978) and the 5'-end (Gimautdinova et al., 1984). In our previous review of the work with poly(U) and poly(S4U) (Cooperman, 1980), we concluded that good evidence had been presented for the functional site labeling of protein S 1 by each of the polynucleotides. For example, such labeling was inhibited in the presence of R17 mRNA, presumably via a competition mechanism, and stimulated in the presence of the cognate tRNA Phe. More recently, Broude et al. (1983) have carried out similar studies by irradiating complexes of 30S subunits with MS2132p]mRNA at 254 nm at light fluences _<20 quanta/nucleotide. Proteins cross-linked to RNA were iodinated with 1251and, following digestion of the RNA with nucleases, identified by two-dimensional PAGE. Although the authors were unable to determine the extent of crosslinking to protein S1, which is especially unfortunate given the previous results quoted above, they were able to show virtually exclusive cross-linking to protein $9 on irradiation of the simple mRNA. 30S complex at 0°C. Addition of IF-3, and/or carrying out photolysis at 37°C led to mRNA cross-linking to proteins identified as $3, $4, $5, $7 and S18, although $9 remained the major protein labeled. IF-3 was also found to cross-link to mRNA. It is important to note that other than the effects of IF-3, little evidence was presented for the functional site specificity of the observed cross-links. In early work, Towbin and Elson (1978) demonstrated that photolabile oligonucleotides, in which a nitrophenyl azide is attached to the 3'-end of (Ap) 6 or (Ap) 7, form complexes with 70S ribosomes and photoincorporate into proteins $3 and $5. Furthermore, such photoincorporation is stimulated by the addition of the cognate tRNA Lys. More recently, Gimautdinova et al. (1984) have studied the photoincorporation of (Up)a, (Up)7 and (Up)8 oligonucleotides that have been derivatized with a dinitrophenyl azido group attached at the 5'-end through a phosphoramide linkage. After separation of cross-linked from unmodified proteins by ion exchange chromatography and removal of the attached oligonucleotides by acid hydrolysis

Escherichia coli ribosomes

287

TABLE4. Proteins Most Strongly Implicated at the mRNA Binding Center by Photoaffinity Labeling Studies Ligand

poly(U)

poly(S4U)

Reagent type

native*

S4Ut

G,FTP

G,I,FCP,FLO

MS2 RNA native:~

(AP)6,7-3'Azide

5'-Azido-(Up),l,7,s

aryl azide§

aryl aziddi

S protein

1 3 4 5 9 18

n.d.** G,FTO G,FTO G, FTO G,FTO G,FTO

n.d°

G,FTP,FCO,FGO

G,FTO,FCP G,FTO,FCP

G,FTP,FCO,FGO G, FTO,FCP

*Fiser et al. (1975a,b); Margaritella and Kuechler (1978). tFiser et al. (1977). *Broude et al. (1983). §Towbin and Elson (1978). ~]Gimatudinova et al. (1984). **n.d., not determined. Abbreviations Used -- Protein identification: G, gel electrophoresis; I, immunoprecipitation; H, high performance liquid chromatography. Site specificity (protein, overall): SSP,SSO; functional site (protein, overall) -- binding constant: FKP,FKO: competition: FCP,FCO; ternary complex: FTP,FTO; gain of function: FGP,FGO; loss of function: FLP,FLO.

of the phosphoramide linkage, the originally cross-linked proteins were made radioactive through reductive methylation with tritiated borohydride (NaB3H4) and their identities determined by two-dimensional PAGE analysis. Unfortunately, the ion exchange separation employed has as a consequence that it does not permit the detection of cross-links with the acidic proteins S1, $2, $6, L7/L12, L8/L9, L4, L10 and L21. Furthermore, high background radioactivity is observed during PAGE analysis, making the identification of cross-linked proteins somewhat uncertain. In this work, photoincorporation was carried out on complexes of the derivatized oligo U and 70S ribosomes. For the tetranucleotide, the cognate tRNA Phe was bound in the P site to increase functional site binding. For the hepta- and octanucleotides, which span both tRNA binding sites, tRNAPhe was found in both the A and P sites. In addition, evidence for functional site incorporation was provided by the finding that incorporation was substantially inhibited in the presence of the oligonucleotide (Up)14. Despite the difficulties noted above it is nevertheless clear that different proteins are cross-linked depending on the size of the oligonucleotide photoaffinity label. Thus, the major labeled proteins are, for (Up)4, $3, S11, S14, L2 and L32; for (Up)7, $3, S12, S17, L2, L32 and L33; and for (Up)a, $4, $9, S19, $20, L13, L16 and L27. A summary of the most significant mRNA photoaffinity labeling results is presented in Table 4.

3.2.3. Protein Factors and GTP Photoaffinity labeling experiments have been carried out with two protein factors, IF-3 and EF-G. Cross-linking has been achieved via heterobifunctional cross-linking reagents or via direct" or photosensitized photolysis. In the first approach the proteins are first derivatized via a lightindependent reaction with reagents containing an electrophilic center as well as a photolabile group. The resulting photolabile protein derivatives, usually in radioactive form, are then complexed to the ribosome and photolyzed in the usual manner. A typical example of such an approach is provided by the work of Maasen and M611er (1981), as described in reactions (4) and (5). The radioactive, photolabile derivative of EF-G, which they called [3H]-alcohol-G, is found to cross-link to ribosomal proteins. Cleavage of the cross-link with the base permits identification of the cross-linked ribosomal proteins by two-dimensional PAGE analysis. In this way proteins L1, L7, L11, $3 and $4 were identified as major cross-linked proteins (technical problems prevented the assessment of cross-linking into proteins S1 and $2). The identities of L7 and L11 were independently confirmed by immunoprecipitation. Controls demonstrating the

288

B. S. COOPERMAN TABLE5. Proteins Most Strongly Implicated in Translocation by Photoaffinity Labeling Studies

Ligand

EF-G

GTP

Macrolide

Reagent type

aryl azide*

aryl azidet

native +

L

S

protein 1 11 14/15 18

G,FTP,FCP G , I , F T P , F C P G,SSP,FCO G,SSP,FCO G,SSP,FCO

G,SSO,FTP

protein 3 4

G,FTP,FCP G,FTP,FCP

G,SSO,FTP G,SSO,FTP

G,SSO,FTP G,SSO,FTP

Streptomycin

Tobramycin

dinitrophenylgroup §

aryl azideq

G,I,SSP G,I,SSP**

G,SSO,FTO

*Maasen and M611er (1981). ?Maasen and M611er (1978). ~:Siegrist et al. (1985). §Luddy (1982). ~]Tangy et al. (1983). **L18 or L22 -- antibody preparation was a mixture. Letter code used: see Table 4. site-specificity of labeling were provided by the inhibition of cross-linking by either native EFG or by thiostrepton, an antibiotic known to interfere with EF-G binding to the ribosome. Protein $4 has also been implicated in EF-G binding by Tejedor and Ballesta (1985c), who showed that ribosome-bound EF-G protects $4 from reaction with dissolved iodine. NH

N3

EF-G--NH 2 + CH30--C--(CH2)3--O--

NH ~

N3

EF-G--NH-- UC--(CH2)3--O- ( ~ CHO

(4)

EF-G--NH--C-- (CH2)30--

CHO NH

N3

CHO NH

+ NaB3H4

N3

(5)

EF-G--NH--C--(CH2)3--O[3H]-alcohol-G

C H2OH

In related work, Maasen and M611er (1978) have studied the EF-G dependent photoincorporation of an arylazide derivative of GTP formed by esterification of the 3,-phosphate. The process of translocation normally proceeds via the formation of a ternary EF-G. GTP. ribosome complex in which GTP is hydrolyzed to GDP and Pi. The derivative used in this study is not only a good competitor of EF-G-dependent GTP hydrolysis but also inhibits EF-G-dependent binding of non-hydrolyzable GTP analogues to the ribosome. Photolysis of complexes of the 3H-labeled arylazide GTP derivative, EF-G, and 70S ribosomes affords incorporation of radioactivity into a large number of ribosomal proteins, as well as into ribosomal RNA. However, specific labeling, which Maasen and M611er define as that labeling which is decreased on addition of the nonhydrolyzable analogue guanyl-5'-yl imidodiphosphate, occurs into only a limited number of ribosomal proteins, and not at all into RNA. As identified by two-dimensional PAGE analysis, L11 is by far the major protein labeled, and there is also significant labeling of proteins L5, L18 and either L14 or L15. Photoaffinity labeling results obtained with EF-G and GTP are summarized in Table 5. Using an approach similar to that described above for EF-G, Cooperman e t al. (1981) have employed N-p-nitrobenzylmaleimide to cross-link 3H-labeled IF-3 to 30S subunits, and have compared the cross-linked proteins so obtained, as identified by immunoprecipitation, to those found when FMN is used to photosensitize cross-link formation between IF-3 and 30S proteins. In closely related work, Schwartz and his co-workers have shown that IF-3 can be cross-linked to ribosomal proteins through direct photolysis of both IF-3.30S (Mackeen e t a l . , 1980) and IF-3.50S (Schwartz et a l . , 1983) complexes. In each of these studies, evidence for the functional

289

Escherichia coli r i b o s o m e s TABt.E 6. 30S Proteins Photocross-linked to IF-3*

S protein

Direct irradiationt

1

+

2 3 7 11 12 13 18 21

+ ++ ++ ++

Photocross-linkingprocedure FMN Prior derivatization photosensitization:~ of IF3 with PNBM:~

In situ addition

of PNBM :~ +

++

++

+

+++

++

+

++

++

++ +

++ ++

++ +

*Number of plusses indicates relativeamount of cross-linking where such comparisonscan be made. tMackeen et al. (1980). ~:Coopermanet al. (1981). PNBM: N-p-nitrobenzylmaleimide.

site specificity of cross-linking is that aurintricarboxylic acid and 0.5 M NH4C1, each of which interferes with IF-3 binding, abolish cross-linking. A summary of the results obtained with the 30S subunits is presented in Table 6, from which it is clear that there is a fair amount of consistency in the identities of the 30S proteins cross-linked via different approaches, particularly proteins S12, S l l and $2. Evidence has also been obtained in these and related studies for photocross-linking of EF-G to 23S RNA and of IF-3 to 16S RNA, but no data is available regarding the placement of the cross-links within these RNAs.

3.2.4. Antibiotics Antibiotics differ functionally from the ligands that have thus far been considered in that they inhibit ribosome-catalyzed protein synthesis rather than participating in it. They also differ structurally in that they are, for the most part, relatively small molecules ( < 500 daltons) that are thought to bind to the ribosome at rather discrete sites. The importance of the use of antibiotics as photoaffinity labels is that identifying the site of interaction with the ribosome of an antibiotic of known inhibitory function provides strong evidence for the identification of the functional site. A potential problem for this approach is that many antibiotics bind to more than one site on the ribosome, some of which may not be directly involved in protein synthesis. Thus, identifying an antibiotic binding site may be insufficient for identifying a functional site, and additional evidence may be needed before such an identification can be made.

3.2.4.1. Puromycin. Puromycin is a close structural analogue of the 3'-end of Tyr-tRNATyr and is a functional analogue as well, participating as a peptidyl acceptor from P-site bound peptidyltRNA. In fact, it is the substrate activity of puromycin in ribosome-catalyzed peptidyl transferase, leading to the formation of aborted peptide chains, that is the basis for its antibiotic action (Alien and Zamecnik, 1962; Nathans, 1964). Both [3H]-puromycin itself (Cooperman et al., 1975; Jaynes et al., 1978; Grant et al., 1979a,b; Weitzmann and Cooperman, 1985) and [3H]-p-azidopuromycin, a functionally competent derivative in which a p-azidophenyl group replaces the p-methoxyphenyl group in puromycin, have been used as photoaffinity labels (Nicholson et al., 1982a,b; Symons et al., 1978; Krassnig et al., 1978). Puromycin photoincorporates into both protein and RNA, but only protein labeling has been examined in detail. As shown by one- and two dimensional PAGE (Jaynes et al. ,1978), RP-HPLC (Weitzmann and Cooperman, 1985), and specific immunoprecipitation (Grant et al,, 1979a) analyses, the major protein labeled is L23, and there is lesser though still significant JP~ ]4: 2-J

290

B.S. COOPERMAN

labeling of protein S 14 and $7. Studies examining the dependence of such labeling on puromyicn concentration, on the presence of several structural analogues of puromycin, and on whether the 70S ribosome or either the 30S or 50S subunit is the target of photoaffinity labeling demonstrate that labeling of each protein takes place from a different site. Furthermore, only the site leading to L23 labeling has properties consistent with those expected for the A' site (defined as the site of binding of the 3'-end of aminoacyl-tRNA within the peptidyl transferase center). Most notably, the inhibitory effects of puromycin analogues on puromycin photoincorporation into L23 parallel very closely the inhibitory effects of these same analogues on ribosome-catalyzed peptidyl transfer to puromycin (Nicholson et al., 1982b; Weitzmann and Cooperman, 1985). It is interesting to note that puromycin aminonucleoside, which lacks the O-methyl tyrosine moiety of puromycin, photoincorporates into all three proteins with about the same distribution pattern as does puromycin, thus demonstrating that puromycin photoincorporation proceeds through its adenosyl moiety (Weitzmann and Cooperman, 1985). The S 14 site has considerably higher affinity for puromycin than does the $7 site and a recent experiment has provided evidence for the direct involvement of S14 in binding to the 3'-end of Phe-tRNA Phe in the 30S Phe-tRNA Phe complex (Cooperman et al., 1986; Kerlavage and Cooperman, 1986). In this work, puromycin-labeled S 14 was resolved from native S 14 by RPHPLC and used in place of native S 14 in a 30S reconstitution experiment, which otherwise used only native ribosomal components. Relative to a 30S subunit reconstituted with native S14, the subunit reconstituted with puromycin-labeled S14 was found to have much reduced PhetRNA Phe binding, at a level similar to that obtained when S14 was omitted entirely. Additional support for the role of S14 in binding to the 3'-end of aminoacylated tRNA comes from the photoaffinity labeling results of Girshovich et al. (1974a,b), showing that S14 is labeled by an N~-aryl azide derivative of Phe-tRNA phe (see Table 2). Using experimental approaches similar to those described above, protein L23 was also shown to be the major 50S protein labeled by p-azidopuromycin (Nicholson et al., 1982a). Such labeling, which proceeds via an azide-dependent photochemistry not available to puromycin, provides strong evidence for the site specificity of L23 labeling. When the experiment is carried out in the presence of i3-mercaptoethanol, added as a scavenger to decrease labeling coming from pazidopuromycin in solution or via a slowly reacting photogenerated intermediate, the other major labeled proteins are L18/22 and L15. These three proteins appear to be labeled from the same site. Preliminary results have also been obtained for the identification of sites of RNA labeling by p-azidopuromycin (Hall, C.C., Johnson, D. and Cooperman,B. S., manuscript in preparation). In particular, the major site of labeling was shown to fall within bases 2445 - 2 6 6 8 of 23S RNA, in very good agreement with the results obtained with N~-derivatives of aminoacyl tRNA (see Table 2). Such derivatives are also directed toward the peptidyl transferase center.

3.2.4.2. Chloramphenicol. Chloramphenicol is an inhibitor of peptidyl transferase. It is currently believed that such inhibition results from its interference with proper binding of the 3'-terminus of aminoacyl tRNA at the A' site, although the exact nature of such inhibition is unclear. For example, chloramphenicol does not appear to compete directly for the functional puromycin site. Chloramphenicol has a single high affinity site (Ko - 2 x 10 -6 M), located on the 50S subunit, presumably at the peptidyl transferase center, and at least one site of lower affinity (Ko - 2 × 10 -4 M) located on the 30S subunit and of unknown function (Lessard and Pestka, 1972; Grant et al., 1979b). Although several biologically active photolabile derivatives of chloramphenicol have been reported (Nielsen et al., 1978; Bouthier de la Tour et al., 1983), detailed localization results have only been presented for the photoincorporation of the unmodified drug itself (Le Goffic et al., 1980). In this work, ribosomes were irradiated in the presence of [3H]-chloramphenicol, with a light source having maximal output at 360 nm. As identified by two-dimensional PAGE, the proteins most labeled by the D-threo diastereomer of chloramphenicol, which is the active antibiotic form, are $4 > $3 > L11, L1. Although labeling is reduced in the presence of unlabeled chloramphenicol, thus demonstrating site specificity, three factors combine to render uncertain the functional significance of the labeling. First, the major proteins labeled are on the 30S subunit rather than the 50S subunit, which contains the

Escherichia coli ribosomes

291

peptidyl transferase center. Second, labeling of ribosomal protein by the inactive D-erythro diastereomer proceeds to a much larger stoiehiometric extent, albeit with reduced specificity, than is found with the threo form. This contrasts to the results obtained with tetracycline and 4-epitetracycline (see below). Third, chloramphenicol photodecomposes to p-nitrobenzaldehyde and other photoproducts during the course of the photoincorporation experiment, but the contribution of such photoproducts to the observed labeling results has not been determined.

3.2.4.3. Lincomycin. Lincomycin is also an inhibitor of peptidyl transferase. Although its precise mode of action is unclear, the weight of the evidence suggests that it acts at one or both of the A and P sites. Minnella and Cooperman have synthesized a diazo derivative of 7-thiolincomycin by alkylation of the parent thiol with ethyl-2-diazo-4-iodoacetoacetate (Cooperman et al., 1979). This derivative is actually somewhat more potent than lincomycin in inhibiting peptidyl transferase (concentration for 50% inhibition: lincomycin - 15/~M; diazo derivative -3/~M) and also shows tighter binding to the ribosome. Photolysis of the 3H-labeled derivative in the presence of ribosomes leads to apparent incorporation into both ribosomal protein and ribosomal RNA. Specific immunoprecipitation and two-dimensional PAGE analysis indicate the major labeled proteins to be L11, L14, L15, L17 and $3 and $7, but the site-specificity of such labeling has not been established (Minnella,A., Strycharz,W.A. and Cooperman,B.S., unpublished observations).

3.2.4.4. Macrolides. Macrolides are potent inhibitors of ribosome-catalyzed protein synthesis. Erythromycin, the best characterized macrolide, has a high affinity binding site on the 50S subunit (Kd --10 -7 M), and although the evidence is equivocal, is thought to act by blocking translocation, rather than inhibiting peptidyl transferase itself. Several macrolides contain both an o~,/3-unsaturated ketone and an isolated aldehyde, and this latter functionality is not necessary for antibiotic action. In recent work, three such macrolides, rosaramicin (Siegrist et al., 1985) and carbomycin A and niddamycin (Tejedor and Ballesta, 1985a,b), have been converted to their respective 3H-labeled dihydro derivatives through reduction of their aldehyde functions with NaB3H4, and directly photoincorporated into ribosomes, presumably via their ct,/3unsaturated ketones. Although all three dihydromacrolides share the common property of photoincorporating predominantly into ribosomal protein rather than ribosomal RNA, it is disappointing that the major proteins labeled by the two different groups, as determined by two-dimensional PAGE analysis, differ markedly. Thus, L27 is the major protein labeled when either the 50S subunit or the 70S ribosome is the target for photoincorporation by both dihydrocarbomycin A and dihydroniddamycin, and both of these macrolides give substantial photoaffinity labeling of S12 as well. By contrast, dihydrorosaramicin, even when employed at rather low concentration (2 #M), photoincorporates into 70S ribosomes into a large number of proteins, most prominently L1, LS, L6, L15, L18, L19, $1, $3, $4, $5 and $9. Neither L27 nor $12 is labeled to a significant extent. Interestingly, addition of puromycin (0.1-0.2 mM) not only induces a 100% stimulation of dihydrosaramicin binding to E. coli ribosomes, but also leads to an increase of photoincorporation into L18 and L19, while at the same time decreasing photoincorporation into most other ribosomal proteins. These results are interpreted as showing that L18 and L19 are labeled from a high affinity site, whereas the labeling of the other proteins takes place from low affinity sites or from solution. One possible interpretation of the disparity between these results is that the two different macrolides, despite their similar structures, bind to different sites on the ribosome, and it is clear that competitive binding experiments would be useful in examining this possibility. However, it also must be pointed out that in neither case has compelling evidence been presented for the site-specificity of photoincorporation into individual proteins (although overall incorporation does appear to be site-specific). Moreover, in both experiments there is extensive formation of photoproducts during the course of the photoincorporation experiment, and in neither case has labeling by native antibiotic been clearly distinguished from labeling by photoproduct.

292

B.S. COOPERMAN

3.2.4.5. Tetracycline. Tetracycline inhibits aminoacyl-tRNA binding to the A-site of the ribosome. The weight of the evidence is that such inhibition is due to tetracycline binding to a unique high affinity site located on the 30S subunit. In addition to this site tetracycline also binds to a large number of low affinity sites located on both the 30S and 50S subunits. Tetracycline is a photolabile molecule. Goldman et al. (1983) have shown that unmodified [3HI-tetracycline can be used as photoaffinity label for the ribosome, but that the labeling obtained on irradiation of tetracycline in the presence of ribosomes, which takes place virtually exclusively into ribosomal proteins, is due not only to the photoincorporation of native tetracycline, but also to both the light-independent and light-dependent incorporation of tetracycline photoproducts. By determining both the rate of photoproduct formation and the labeling pattern due to photoproduct alone, it was possible to factor the overall labeling pattern obtained on irradiation of tetracycline and ribosomes into contributions from the photoincorporation of native tetracycline and contributions from incorporation of tetracycline photoproduct. In this way protein $7 was shown to be the major protein photolabeled by tetracycline. Evidence that $7 is labeled from a high affinity and functionally significant site was provided by (a) the decrease in $7 labeling observed on addition of nonradioactive tetracycline, (b) the insensitivity of $7 labeling to addition of the quenching reagent/3-mercaptoethanol, and (c) the much lower labeling observed when photoincorporation experiments are carried out with 4-epitetracycline. This latter result is expected if labeling of $7 is functionally significant, since 4-epitetracycline, though having the same photochemistry as tetracycline, is both a much weaker inhibitor of aminoacyl-tRNA binding and also binds more weakly to 30S subunits. It is also worth noting that $7 is labeled by tRNA on irradiation of both tRNA-30S and tRNA-70S complexes (Table 3). More recent work with tetracycline derivatives (Hasan et al., 1985) has demonstrated the potential utility of photolabile derivatives such as both 7-azido and 9-azidosancycline. Interestingly, the photoincorporation of such derivatives into ribosomal proteins, which leaves the tetracycline chromophore at 365 nm intact, can be optically detected at this wavelength when analysis is performed by RP-HPLC (Cooperman et al., 1986).

3.2.4.6. Tobramycin. Tobramycin, like other aminoglycosides derived from kanosamine, has several high affinity binding sites on the E. coli ribosome (Ko -0.2/zM) a s well as a larger number of lower affinity site (Ko ~ 10 #M). This class of antibiotics display several effects on ribosomes, including inducing misreading, inhibiting translocation, and, at relatively high concentrations, inhibiting the dissociation of ribosomes into subunits. Tangy et al. (1983) have studied the photoincorporation of a 3H-labeled 6-N-p-azidobenzyl derivative of tobramycin into E. coli ribosomes. This derivative retains antibiotic activity, though it binds considerably less tightly to ribosomes than does the native compound. At a low concentration of derivative (2 #M) more labeling takes place into 30S than 50S subunits, RNA is not labeled and the major proteins labeled, as identified by both one- and two-dimensional PAGE analysis, are $4, $5, S 18, L6, L2 and L13. At a higher concentration of derivative (20 #M) many more proteins are labeled. These results are certainly consistent with the existence of several tobramycin binding sites, although here again little evidence is presented demonstrating which proteins are labeled in a site-specific manner.

3.2.4.7. Streptomycin. Streptomycin, like tobramycin, has several affects on ribosomes. At low concentrations (1 /~M) it induces miscoding, at higher levels (40 #M), it inhibits translocation, and at still higher levels (100 #M) if 'freezes' initiation complexes. Streptomycin has a single high affinity site on the 70S ribosome (Ko 0.1 - 1.0/zM), clearly localized on the 30S subunit. The binding of streptomycin to this site, which is lacking in ribosomes isolated from some streptomycin-resistant cells, is thought to induce misreading. There are also a large number of low affinity streptomycin binding sites, which may be linked to the other effects of streptomycin on ribosomal function. Luddy (1982) and Cooperman have studied the photoincorporation of the 3H-labeled 2,4-dinitrophenylhydrazone of streptomycin into 70S ribosomes. This derivative mimics streptomycin in inducing miscoding, although at considerably higher concentrations.

Escherichia coli ribosomes

293

Most of the labeling takes place into ribosomal protein, with 50S subunits labeled to a higher extent (60-70%) than 30S subunits (30-40%). The major proteins labeled, as identified by both PAGE and specific immunoprecipitation analyses, are, in the 30S subunit, proteins S 1 8 > $ 6 > S 1 , S19, S14, $5 and $7, and in the 50S subunit, L18/22 > LI4 > L15. Unfortunately, none of this labeling appears to take place from the unique high affinity site, as shown by the observations first, that adding streptomycin (at 5 ;tM) does not decrease photoincorporation of the derivative and second, that the labeling pattern obtained with ribosomes isolated from a resistant strain is essentially the same as that obtained using wild-type ribosomes. There is, however, evidence that at least some labeling is occurring site-specifically from lower affinity sites.

3.2.4.8. Pactamycin. Pactamycin inhibits the initial steps of protein synthesis in both procaryotes and eucaryotes. It binds preferentially to the small subunit, having much lower affinity for the whole ribosome and essentially none for the large subunit. Tejedor et al. (1985) have shown that 125I-labeled pactamycin will photoincorporate into both 30S subunits and 70S ribosomes, presumably via its acetophenone moiety. When 30S subunits are the target, about 2/3 of the labeling is into rRNA. The major proteins labeled, as characterized by two-dimensional PAGE, are S18 > $21 > $4 > $2. When 70S ribosomes are the target, 60% of the label is in proteins, the major ones being $4 > S 18, L13. Although tests for site specific labeling of individual proteins were not reported, overall labeling of 70S ribosomes by [12sI]-pactamycin does saturate as a function of [12sI]-pactamycin concentration. The apparent K d (-~ 10 -6 M) for such labeling is comparable to the Ko for both pactamycin and iodopactamycin binding. Furthermore, although no direct studies of possible iodopactamycin photoproduct formation were reported, the labeling pattern of 70S proteins is similar when photolysis times of either 1 h or 3 h are employed, a result that is at least consistent with the absence of secondary incorporation reactions arising from photoproduct formation.

4. PHOTOAFFINITY LABELING STUDIES AND THE IDENTIFICATION OF FUNCTIONAL CENTERS Photoaffinity labeling results have been presented above strictly by ligand type. In this section those photoaffinity labels targeted, at least potentially, toward the same or closely related functional centers are considered together, in an effort to define a minimum set of ribosomal components (based on the available information, these will be, for the most part, ribosomal proteins) at these centers. In making this effort we consider explicitly the method(s) of protein identification utilized, the site specificity of labeling and the evidence for functional site labeling. This information is collected in Tables 4, 5 and 7. It must be stressed that results summarized in these tables include only those proteins for which there is at least some reason to suppose that labeling is significant, either because of direct experiments showing site and/or functional site specificity, or because of an overlap with proteins labeled by a related photoaffinity label. Thus, not all of the proteins labeled by a particular photoaffinity label are necessarily included. As previously (Cooperman, 1980), letter codes are employed to describe the data obtained. A one letter code identifies the method(s) of protein identification utilized, with G referring to gel electrophoresis, H to HPLC and I to immunoprecipitation. SSP refers to a demonstration of site specificity, either by saturation of labeling as a function of photoaffinity label concentration or by competition by native ligand for photoaffinity labeling by a derivative, with incorporation measured at the individual protein level. SSO refers to such a demonstration measured by overall labeling. Triplets beginning with F refer to evidence for functional site labeling as described by the middle letter. K refers to binding constant measurement, C to competition, T to ternary complex formation, and G or L to gain or loss of ribosomal function on affinity labeling. The third letter, O or P, again describes whether evaluation is at the overall or individual protein level. Clearly, evidence obtained at the individual protein level is the more desirable for directly implicating a particular protein as being part of a functional center.

294

B.S. COOPERMAN

TABLE 7. Proteins Most Strongly Implicated at the Peptidyl Transferase Center by Photoaffinity Labeling Studies Ligand

3'-end tRNA

Reagent

aryl azide*

Puromycin native;~aryl azide :~

Chloramphenicol

Lincomycin

native§

diazocarbonyl ¶

Macrolides native**' t t

L protein

11 15 18 23 27

G,SSP,FTP,FGP

G,SSP G,H,I,SSP,FCP,FLO G,H,I,SSP,FCP,FLO§§ G,H,I,SSP,FKP,FCP,FLO Ill¶

G,SSP,FTP,FGP G,SSP,FTP,FGP + ~:

G,I G,I G,SSO,FTP G,SSO

*Hsiung and Cantor (1974); Hsiung et al. (1974). tJaynes et al. (1978); Grant et al. (1979a); Weitzmann and Cooperman (1985). :~Nicholson et al. (1982a,b). §LeGoffic et al. (1980). ¶Minnella,A., Strycharz,W.A. and Cooperman,B.S., unpublished observations. **Tejedor and Ballesta (1985a). "~tSiegrist et al. (1985). :~:~Minor product. §§Labeled by azide photochemistry only. ¶ ¶ Labeled by azide and native photochemistries. Letter code used: see Table 4.

4.1. THE PEPTIDYL TRANSFERASE CENTER

Peptidyl transferase activity is located exclusively on the 50S subunit. The photoaffinity labels directed toward this center include those prepared by acylating the a-amino group of aminoacyltRNA, as well as those derived from antibiotic inhibitors of peptidyl transferase. Proteins labeled by both groups of photoaffinity label are listed in Table 7. Results with the macrolide photoaffinity labels are also included in this table, because of data suggesting that these antibiotics may act in the general vicinity of the peptidyl transferase center. From the collected results it is clear that of the five proteins most strongly implicated, the strength of the evidence falls roughly in the order L 18 > L 11 > L15, L23 > L27. Interestingly, if electrophilic affinity labels directed toward peptidyl transferase are also included for consideration only two additional proteins need be added to the list (Cooperman, 1980), i.e. L2 and L16. In this connection it is worth pointing out that L2 is labeled to a minor extent both by the arylazide derivative of Phe-tRNA Ph~ (Hsiung et al., 1974) and by the macrolide dihydrocarbomycin (Siegrist et al., 1985). Support for the notion that this group of proteins are clustered comes from the work of Traut et al. (1980) showing there to be numerous cross-linking relationships among them, as summarized in Fig. 5. Here it should be noted that L5 is known to neighbor L18 because both of these proteins bind to 5S RNA within the 50S subunit, and because of the proximity of their footprints on

L32

_--=

FIG. 5. Cross-links among proteins linked to the peptidyl transferase center and other related proteins (Traut et al., 1980). The proteins most strongly implicated are circled. Although no L5-L18 cross-link has been reported, these proteins must be neighbors since both bind to 5S RNA.

Escherichia coli ribosomes

295

Fio. 6. Immunoelectron microscopy determinations of the location of the 50S subunit of ribosomal proteins implicated in peptidyl transferase by photoaffinity labeling (StOffler and StOffler-Meilicke, 1984) of covalently bound puromycin and chloramphenicol (Luhrmann et al., 1981; Olson et al., 1982, 1985), and of 5S RNA (Vasiliev and Shatsky, 1984).

5S RNA (Douthwaite et al., 1982). That 5S RNA neighbors the peptidyl transferase center has also been independently demonstrated by immunoelectron microscopy. In this approach, complexes are made between antibody molecules and ribosomal subunits containing corresponding haptens (either naturally occurring, e.g. modified nucleosides, or incorporated into the subunit by chemical or affinity labeling modification), the complexes are visualized by electron microscopy, and the site of attachment of the antibody to the subunit indicates the placement of the hapten. Studies of this kind have been carried out with 50S subunits that (1) have been photoaffinity labeled with both puromycin (Olson et al., 1982) and p-azidopuromycin (Olson et al., 1985), (2) have been labeled with electrophilic derivatives of both puromycin and chloramphenicol (Luhrmann et al., 1981; StSffler and St6ffler-Meilicke, 1984) and (3) contain 5S RNA that has been chemically modified at either the 5' or 3' terminus (Vasiliev and Shatsky, 1984). The major results of these studies are depicted in Fig.6. Recalling the overlap in RNA labeling results between N~-derivatives of aminoacyl-tRNA and p-azidopuromycin, it is fair to conclude that the application of photoaffinity labeling has narrowed the peptidyl transferase center to a limited region of RNA (bases 2445-2668 with preference for bases 2584 and 2585), to a limited number of neighboring ribosomal proteins (Fig. 5) and to specific region of the 50S subunit (Fig. 6). One important direction for further work will be to determine the functional consequences of affinity labeling individual proteins, as was described for puromycin-labeled S14 (Section III-4a).

$10

$2

FIG. 7. Cross-links among proteins linked to the mRNA binding site and other related proteins (Traut et al., 1980). The most strongly implicated are circled.

296

B. S. COOPERMAN

®

O

FIG. 8. Protein map of 30S subunit as determined by neutron diffraction studies (Moore et al., 1986). Reproduced from Moore et al. (1986). Copyright permission of the Biophysical Society.

4.2. THE mRNA BINDING CENTER Just as peptidyl transferase is located on the 50S subunit, mRNA binding is the exclusive property of the 30S subunit. Fragments of mRNA up to 40 bases long are protected from ribonuclease digestion on complexation of mRNA with ribosomes, thus indicating a rather extensive zone of interaction. The proteins most clearly implicated by photoaffinity labeling experiments as forming parts of this zone are collected in Table 4. Related studies with electrophilic affinity labels implicate protein S 12 as an additional member of this group (Pongs e t a l . , 1975a; Babkina e t a l . , 1985). These results may be compared with those obtained using three other mapping techniques, protein-protein cross-linking (Fig. 7), neutron diffraction (Fig. 8) and immunoelectron microscopy (Fig. 9). This comparison clearly shows that while the seven proteins in question generally fall within the neck portion of the 30S particle connecting the head and body (Fig. 9), they appear to be widely spread out within this zone. It would have been interesting to compare labeling results obtained with mRNA photoaffinity labels with those obtained with photoreactive groups placed in the anticodon loop of tRNA. This is not yet directly possible because as of now mRNA labels have led only to labeled protein identification, whereas anticodon-derived labels have afforded, exclusively, the identification of a single labeled nucleotide (C1400). However, it is possible to compare the locations of the labeled proteins with the location of C1400, as revealed by electron microscopy in two recent studies. In the first (Gornicki e t a l . , 1984) a dinitrophenyl group was attached close to the site of the tRNA-C1400 cross-link, and located with anti-DNP antibody. In the second (Oakes et

FIG. 9. Immunoelectronmicroscopydeterminationof protein locations on the 30S subunit (Winkelmann et al., 1982; St6fflerand St6ffler-Meilicke, 1984; Noller and Lake, 1984). Proteins photoaffinitylabeled

by either mRNA or IF-3 are circled.

Escherichia coli ribosomes

297

al., 1986), a single-stranded deoxyoligonucleotide probe complementary to bases 1392-1407

in 16S rRNA and containing a biotin group at its 3'-terminus was allowed to form a specific complex with 30S subunits and its position was localized using avidin. Both of these studies give similar results, localizing C-1400 in the vicinity of S18 shown in Fig. 9. Thus, this region is considered a prime candidate for the site of codon-anticodon interaction within the total mRNA binding zone defined by all seven affinity labeled proteins. It is worth noting that the IF-3 binding site has also been mapped in the neighborhood of this putative codon-interaction site, both by immunoelectron microscopy studies (St6ffler an St6ffler-Meilicke, 1984) and from the locations of several proteins which are photoaffinity labeled by IF-3, especially S11, S12 and $21 (see Table 6).

4.3. TRANSLOCATION

As already mentioned, during normal protein synthesis translocation requires factor EF-G and is accompanied by GTP hydrolysis. In an effort to localize an area of the ribosome involved in translocation, the major proteins labeled by photoaffinity labels for EF-G and GTP are compared with those labeled by photoaffinity labels for antibiotics having inhibitory activity toward translocation (Table 5). As may be seen, the overlap is fairly extensive, lending some credence to the notion that the six proteins listed define a translocation region. Some of the L proteins of Table 5 are also peptidyl transferase proteins (L11, L18 and perhaps L14/15) while both $3 and $4 are at the mRNA binding center. These overlaps lead to the plausible and functionally sensible suggestion that the translocation region is in contact with the two most important binding areas of a tRNA molecule to the ribosome, i.e. the 3'-end and the anticodon.

5. CONCLUSIONS The results presented above leave this author, and perhaps the reader as well, with some contradictory impressions concerning the true efficacity of photoaffinity labeling for defining the location of functionally important sites on the ribosome. On the one hand, there are some clear successes, such as in the cases of the peptidyl transferase center and of the mRNA binding site, in which the identities of proteins labeled by photoaffinity probes are in very good accord with predictions based on the use of other approaches. On the other hand, there are bewildering results in which similar photoaffinity labels used by different groups lead to quite different results. As one example, there is only modest overlap in the identities of the proteins labeled by Budowsky and his co-workers using direct cross-linking by irradiation of native tRNAs bound to different positions on the ribosome, and those labeled by the Soviet group working in Novosibirsk (Babkina et al., 1984a,b,c, 1985; Bausk et al., 1985) using tRNAs derivatized at G positions spread almost randomly over the entire tRNA molecule (see Table 3). A second example is provided by comparing the completely nonoverlapping results obtained by Siegrist et al. (1985) on the one hand and Tejedor and Ballesta (1985a,b) on the other, in which both groups identify different proteins as being photoaffinity labeled by dihydro derivatives of closely related macrolides (Section 3.4.4.). One straightforward way of rationalizing these results is by invoking the possibility that ribosomal ligands are, in general, capable of a variety of binding interactions with the ribosome, at what may be quite different sites. Going a step further, it is possible that the distribution of ligand among these sites could be dependent on experimental variables (e.g. ribosome preparation, ionic strength, pH, temperature) that vary from laboratory to laboratory. Although such sites need not all have functional importance, they may each be capable of being photoaffinitylabeled. Thus, variation in details of the labeling procedure could lead to variation in the identities of the ribosome components that are labeled. The task then of the experimenter using photoaffinity labeling to locate functional sites is not only to correctly identify the ribosomal components that are labeled, but also to obtain evidence showing that labeling is taking place from a site having functional significance. In the rather

298

B.S. COOPERMAN

limited number of cases in which both of these requirements have been met, photoaffinity labeling studies have been clearly successful in locating functional sites within the ribosome.

Acknowledgements -- This review was prepared with support from NIH grant AI 16806 and NSF grant DMB-86~2610. The author wishes to acknowledge the excellent typing skills of Delores Magobet and the cooperation of many researchers who sent me preprints and reprints of their work.

REFERENCES ABDURASHIDOVA,G. G., TURCHINSKY,M. F., ASLANOV,Kh. A. and BUDOWSKY,E. I. (1979) Polynucleotide-protein interactions in the translation system. Identification of proteins interacting with tRNA in the A- and P-sites of E. coli ribosomes. Nucleic Acids Res. 6 : 3 8 9 1 - 3 9 0 9 . ABDURASHIDOVA,G. G. TURCHINSKY,M. F. and BUDOWSKY,E. I. (1981) Ribosomal proteins contacting with deacylated tRNA in the S-site of the translating ribosome. FEES Lett. 129: 5 9 - 6 1 . ABDURASHIDOVA,G. G., NARGIZYAN,M. G., RUDENKO, N. V., TURCHINSKY, M. F. and BUDOWSKY,E. I. (1985a) Contacts of ribosomal proteins with tRNA Phe and 16S RNA in analogs of the 30S initiator complex. Molec. Biol. (Moscow) 19: 4 5 9 - 4 6 3 . ABDURASHIDOVA,G.G., OVSEPYAN,V. A., CHERNII,A. A., KAMINIR,L. B. and BUDOWSKY,E. I. (1985b) Ribosomal proteins interacting with Phe-tRNA during enzymic binding with the translating ribosome before and after release of elongation factor EF-Tu. Molec. Biol. (Moscow) 19:667 - 6 7 1 . ABDURASmOOVA,G. G., OVSEPYAN,V. A. and BUDOWSKY,E. I. (1985c) Proteins contacting peptidyl-tRNA at the Asite of Escherichia coli ribosomes after enzymatic and nonenzymatic binding of aminoacyl-tRNA. Molec. Biol. (Moscow) 19: 9 4 7 - 9 5 0 . ALLEN, D. W. and ZAMECNL~,P. C. (1962) The effect of puromycin on rabbit reticulocyte ribosomes. Biochim. Biophys. Acta 55: 8 6 5 - 8 7 4 . ATMADJA, J., STIEGE, W., ZOBAWA,M., GREUER, B., OSSWALD,M. and BRIMACOMBE,R. (1986) The tertiary folding of Escherichia coli 16S RNA, as studied by in situ intra-RNA sequence cross-linking of 30S ribosomal subunits with bis-(2-chloroethyl)-methylamine. Nucleic Acids Res. 14: 6 5 9 - 6 7 3 . BABKINA, G. T., KARPOVA,G. G. and MATASOVA,N. B. (1984a) Affinity modification of Escherichia coli ribosomes close to the acceptor tRNA-binding site. Molec. Biol. (Moscow) 18: 1045-1053. BAEKINA, G. T., BAUSK, E. V., KARPOVA, G. G., MATASOVA, N. I . and GRAIFER, D. M. (1984b) Photoaffinity modification of Escherichia coli ribosomes close to the tRNA-binding centers by tRNA Phe derivatives bearing arylazido groups on the guanosine residues. Molec. Biol. (Moscow) 18: 1062-1065. BABKINA, G. T., BAUSK, E. V., GRAIFER, D. M., KARPOVA, G. G. and MATASOVA, N. n. (1984c) The effect of aminoacyl- or peptidyl-tRNA at the A-site on the arrangement of deacylated tRNA at the ribosomal P-site. FEBS Lett. 170: 2 9 0 - 2 9 4 . BABKINA. G. T., KARPOVA, G. G., MATASOVA,N. B., BEREZIN, V. M., GREN, E. Ya. and TSIELENS, I. E. (1985) Investigations of the mRNA-binding region of the ribosome at different stages of translation. II. Affinity modification of Escherichia coli ribosomes by benzylidene derivatives of AUGU 6 in the 70S initiation complex. Molec. Biol. (Moscow) 19: 8 9 0 - 8 9 5 . BARRITAULT,D., BUCKINGHAM,R. n . , FAURE, A. and THOMAS, G. (1981) Photocross-linking of thiolated aminoacyltRNA to ribosomal RNA and proteins. Biochimie 63: 5 8 7 - 5 9 3 . BARTA, A., KUECHLER, E., BRANLANT,C., SRIWADADA,J., KROL, A. and EBEL, J. P. (1975) Photoaffinity labeling of 23S RNA at the donor-site of the Escherichia coil ribosome. FEBS Lett. 56: 170-174. BARTA, A., STEINER. G., BROSIUS.J., NOLLER, H. F. and KUECHLER,E. (1984) Identification of a site on 23S ribosomal RNA located at the peptidyl transferase center. Proc. Natn. Acad. Sci. U.S.A. 81: 3607-3611. BAUSK, E. V., GRAIFER, D. M. and KARPOVA,G. G. (1985) Photoaffinity modification of Escherichia coli ribosomes close to the donor tRNA-binding site. Molec. Biol. (Moscow) 19: 4 5 2 - 4 5 8 . BISPINK. L. and MATTHAEI,H. (1973) Photoaffinity labeling of 23S rRNA in Escherichia coli ribosomes with poly(U)coded ethyl-2-diazo-malonyI-Phe-tRNA. FEES Lett. 3 7 : 2 9 1 - 2 9 4 . BOUTHIERDE LATOUR, C., MOREAU,B., BAILLARGI~,M., BLAZEJEWSKI,J.-C., CAPMAU,M.-L. and LEGOFFIC, F. (1983) Synthesis of affinity label structural analogues of chloramphenicol. Eur. J. Med. Chem. -Chim. Ther. 18:315 - 318. BROUDE, N. E., KUSSOVA,K. S. MEDVEDEVA, N. I. and BUDOWSKY,E. I. (1980) Proteins of 30S subunit of E. coli ribosomes involved in the interaction with messenger RNA. Bioorg. Khim. 6: 1303-1306. BROUDE. N. E., KUSSOVA, K. S., MEDVEDEVA, N. I. and BUDOWSKY,E. I. (1983) Proteins of the 30-S subunit of Escherichia coli ribosomes which interact directly with natural mRNA. Eur. J. Biochem. 132: 139-145. BROUDE, N. E., MEDVEDEVA,N. I., KUSOVA.K. S. and BUDOWSKY,E. I. (1985) Ribosomal proteins directly interacting with fMet-tRNA Met within the 30S initiation complex. Molec. Biol. (Moscow) 19: 1040-1043. CHEN, J.-K., FRANKE,L. A., HLXSON,S. S. and ZIMMERMANN,R. A. (1985a) Photochemical cross-linking of tRNA Arg to the 30S ribosomal subunit using aryl azide reagents attached to the anticodon loop. Biochemistry 24:4777 -4784. CHEN, J.-K. KRAUSS, J.H., HlXSON, S.S. and ZIMMERMANN,R.A. (1985b) Covalent cross-linking of tRNA Gly to the ribosomal P site via the dihydrouridine loop. Biochim. Biophys. Acta 825: 161-168. CIESIOLKA, J., GORNICKI, P. and OFENGAND, J. (1985) Identification of the site of cross-linking in 16S rRNA of an aromatic azide photoaffinity probe attached to the 5'-anticodon base of A site bound tRNA. Biochemistry 24: 4931-4938. CLARK, M. W., LEONARD,K. and LAKE, J. A. (1982) Ribosomal crystalline arrays of large subunits from Escherichia coli. Science 216: 9 9 9 - 1 0 0 1 . COOPERMAN,B.S. (1978) Affinity labeling studies on Escherichia coli ribosomes. In: Bioorganic Chemistry. A Treatise

Escherichia coli ribosomes

299

to Supplement Bioorganic Chemistry, An International Journal, Vol. 4, pp. 81-115, VAN TAMELEN, E. (ed.) Academic Press, new York. COOPERMAN, B. S. (1980) Functional sites on the E. coli ribosome as defined by affinity labeling. In: Ribosomes, Structure, Function and Genetics, pp. 531-554, CHAMBLISS,G., CRAVEN,G. R., DAVIES,J., DAVIS,K., KAHAN, L. and NOMURA. M. (eds) University Park Press, Baltimore. COOPERMAN,B. S., JAYNES,E. N.,JR., BRUNSWICK,D. J. and LUDDY, M. A. (1975) Photoincorporation of puromycin and N-(ethyl-2-diazomalonyl) puromycin into Escherichia coli ribosomes. Proc. Natn. Acad. Sci. U.S.A. 72: 2974-2978. COOPERMAN,B. S., GRANT,P. G., GOLDMAN,R. A., LUDDY,M. A., MINNELLA,A., NICHOLSON,A. W. and STRYCHARZ, W. A. (1979) Photoaffinity labeling of ribosomes. Methods Enzymol. 59: 796-815. COOPERMAN,B. S., EXPERT-BENZANCON,A., KAHAN,L., DONDON,J. and GRUNBERG-MANAGO,M. (1981) IF-3 crosslinking to Escherichia coli ribosomal 308 subunits by three different light-dependent procedures: identification of 30S proteins cross-linked to IF-3 -- Utilization of a new two-state cross-linking reagent, p-nitrobenzylmaleimide. Arch. Biochem. Biophys. 208: 554-562. COOPERMAN, B. S., HALL, C. C, KERLAVAGE.A. R., WEITZMANN,C. J., SMITH.J., HASAN, T. and FRIEDLANDER,J. D. (1986) Photoaffinity labeling of Escherichia coil ribosomes: new approaches and results. In: Structure, Function and Genetics of Ribosomes, pp. 362-378, HARDESTY, B. and KRAMER, G. (eds), Springer, New York. DABBS, E. R., HASENBANK,R., KASTNER, B., RAK, K.-H., WARTUSCH,B. and ST6FFLER, G. (1983) Immunological studies of Escherichia coil mutants lacking one or two ribosomal proteins. Molec. Gen. Genet. 192:301-308. DENO, H.-Y., ODOM, O.W. and HARDESTY, B. (1986) Localization of L l l on the Escherichia coli ribosome by singlet-singlet energy transfer. Fur. J. Biochem. 156: 497-503. DOUTHWAITE,S., CHRISTENSEN,A. and GARRETT,R. A. (1982) Binding sites of ribosomal proteins on prokaryotic 5S ribonucleic acids: a study with ribonucleases. Biochemistry 21: 2313-2320. ECKERMANN, D. J. and SYMONS, R. H. (1978) Sequence at the site of attachment of an affinity-label derivative of puromycin on 23S ribosomal RNA of Escherichia coli ribosomes. Eur. J. Biochem. 82: 225-234. FISER, I., SCHEIT. K. H., STOFFLER,G. and KUECHLER,E. (1974) Identification of protein S1 at the messenger RNA binding site of the Escherichia coli ribosome. Biochem. Biophys. Res. Commun. 60:1112-1118. FISER, I., MAROARITELLA,P. and KUECrILZa, E. (1975a) Photoaffinity reaction between polyuridylic acid and protein S1 on the Escherichia coli ribosome. FEBS Lett. 52:281-283. FISER, I., SCHEIT.K. H., STOFFLER,G. and KUECHLER,E. (1975b) Proteins at the mRNA binding site of the Escherichia coli ribosome. FEBS Lett. 56: 226-229. FISER. I., SCHEIT,K. H. and KUECHLER,E. (1977) Poly(4-thiouridylic acid) as messenger RNA and its application for photoaffinity labeling of the ribosomal mRNA binding site. Eur. J. Biochem. 74- 447-456. GIMAtrrDINOVA,O. I., ZENKOVA,M. A, KARr'OVA,G. G. and PODtJST,L. M. (1984) Affinity modification of Escherichia coli ribosomes by photoactivatable analogs of mRNA. Molec. Biol. (Moscow) 18: 734-744. GtRI. L., HILL, W. E., WITTMANN,H. G. and WITTMANN-LIEBOLD,B. (1984) Ribosomal proteins: their structure and spatial arrangement in prokaryotic ribosomes. Adv. Protein Chem. 3 6 : 1 - 7 8 . GIRSHOVlCH, A. S., BOCHKAREVA,E. S., KRAMAROV.V. A. and OVCHINNIKOV.YU. A. (1974a) E. coli 30S and 508 ribosomal subparticle components in the localization region of the tRNA acceptor terminus. FEBS Len. 42:213-217. GmSHOVlCH, A. S., BOCHKAREVA,E. S. and POZDNYAKOV,V. A. (1974b) Affinity labelling of functional centres of Escherichia coli ribosomes. Acta Biol. Med. Germ. 33: 639-648. GOLDMAN. R. A., HASAN,T., HALL, C. C., STRYCHARZ,W. A. and COOPERMAN,B. S. (1983) Photoincorporation of tetracycline into Escherichia coli ribosomes. Identification of the major proteins photolabeled by native tetracycline and tetracycline photoproducts and implications for the inhibitory action of tetracycline on protein synthesis. Biochemistry 22: 359- 368. GORNICKI, P., NURSE, K., HELLMANN,W., BOUBLIK,M. and OFENOAND,J. (1984) High resolution localization of the tRNA anticodon interaction site on the Escherichia coli 308 ribosomal subunit. J. Biol. Chem. 259: 10493-10498. GORNICKI, P., CIESIOLKA,J. and OFENGAND,J. (1985) Cross-linking of the anticodon of P and A site bound tRNAs to the ribosome via aromatic azides of variable length: involvement of 16S rRNA at the A site. Biochemistry 24: 4924 -4930. GRANT, P. G., STRVCHARZ,W. A., JAYNES, E. N., JR. and COOPERMAN, B. S. (1979a) Antibiotic effects on the photoinduced affinity labeling of Escherichia coil ribosomes by puromycin. Biochemistry 18" 2149-2154. GRANT, P. G., COOPERMAN,B. S. arid STRYCHARZ,W. A. (1979b) On the mechanism of chloramphenicol-induced changes in the photoinduced affinity labeling of Escherichia coil ribosomes by puromycin. Evidence for puromycin and chloramphenicol sites on the 30S subunit. Biochemistry 18: 2154-2160. GUTELL, R. R., WEISER, B., WOESE, C. R. and NOLLER, H. F. (1985) Comparative anatomy of 16S-like ribosomal RNA. Prog. Nucleic Acid Res. Molec. Biol. 32: 156-216. HALL, C. C, SMITH, J. E. and COOPERMAN.B. S. (1985) Mapping labeled sites in Escherichia coli ribosomal RNA: distribution of methyl groups and identification of a photoaffinity-labeled RNA region putatively at the peptidyltransferase center. Biochemistry 24:5702-5711. HASAN, T., GOLDMAN,R. A. and COOPERMAN.B. S. (1985) Photoaffinity labeling of the tetracycline binding site of the Escherichia coil ribosome. The uses of a high intensity light source and of radioactive sancycline derivatives. Biochem. Pharmacol. 34: 1065-1071. HOWARD,G. A. and TRAUT,R. R. (1974) A modified two-dimensional gel system for the separation and radioautography of microgram amounts of ribosomal proteins. Methods Enzymol. 30: 526-539. Hsu. L. M., LIN, F.-W., NURSE,K. and OFENGAND,J. (1984) Covalent cross-linking of Escherichia coli phenylalanyltRNA and valyl-tRNA to the ribosomal A site via photoaffinity probes attached to the 4-thiouridine residue. J. Molec. Bio.. 172: 57-76. HSUING, N. and CANTOR, C. R. (1974) A new simpler photoaffinity analogue of peptidyl tRNA. Nucleic Acids Res. 1: 1753-1762. HSUING, N., REINES. S. A. and CANTOR, C. R. (1974) Investigation of ribosomal peptidyl transferse centre using a photoaffinity label. J. Molec. Biol. 08:841-855.

300

B . S . COOPERMAN

HUANG, K. H., FAIRCLOUGH,R. H. and CANTOR,C. R. (1975) Singlet energy transfer studies of the arrangement of proteins in the 30S Escherichia coli ribosome. J. Mol. Biol. 9 7 : 4 4 3 - 4 7 0 . JAYNES, E. N., JR., GRANT,P. G., GIANGRANDE,G., WILDER,R. and COOPERMAN,B. S. (1978) Pbotoinduced affinity labeling of the Escherichia coli ribosome puromycin site. Biochemistry 1 7 : 5 6 1 - 5 6 9 . KALTSCHMIDT, E. and WITTMANN, H. G. (1970) Ribosomal proteins VII. Two-dimensional polyacrylamide gel electrophoresis for fingerprinting of ribosomal proteins. Analyt. Biochem. 3 6 : 4 0 1 - 4 1 2 . KENNY, J. W., LAMBERT.J. M. and TRAUT. R. R. (1979) Cross-linking of ribosomes using 2-iminothiolane (methyl 4-mercaptobutyrimidate) and identification of cross-linked proteins by diagonal polyacrylamide/sodium dodecyl sulfate gel electropboresis. Methods EnzymoL 59: 534-550. KERLAVAGE,A. R. and COOPERMAN,B. S. (1986) Reconstitution of Escherichia coli ribosomes containing puromycinmodified $14: Functional effects of the photoaffinity labeling of a protein essential for tRNA binding. Biochemistry 25: 8002- 8010. KERLAVAGE,A. R., HASAN,T. and COOPERMAN,B. S. (1983a) Reverse phase high performance liquid chromatography of Escherichia coli ribosomal proteins: standardization of 70S, 50S and 30S protein chromatograms. J. Biol. chem. 258: 6313-6318. KERLAVAGE,A. R., WEITZMANN,C. J., HASAN,T and COOPERMAN,I . S. (1983b) Reversed-phase high-performance liquid chromatography of Escherichia coli ribosomal proteins: characteristics of the separation of a complex protein mixture. J. Chromatogr. 266: 225-237. KERLAVAGE, A. R., WEITZMANN, C. J. and COOPERMAN, B. S. (1984) Application of high-performance liquid chromatography to the reconstitution of ribosomal subunits. J. Chromatogr. 317: 201-212. KERLAVAGE,A. R., WEITZMANN,C. J., CANNON, M., HASAN,T., GIANGIACOMO,K. M., SMITH, J. and COOPERMAN, B. S. (1985) Utilization of HPLC in studies of ribosomes and their antibiotic inhibitors. Biotechniques 3" 2 6 - 3 6 . KRASSNIG, F., ERDMANN,V. A. and FASOLD. n . (1978) The synthesis of a photoreactive puromycin analogue and its application for labeling proteins in the 50S subunit of Escherichia coli ribosomes. Eur. J. Biochem. 87:439-443. KRUSE, T. A., SIBOSKA,G. E. and CLARK, B. F. C. (1982) Photosensitized cross-linking of tRNA to the P-, A- and R-sites of Escherichia coli ribosomes. Biochimie 64: 279-284. KUECHLER, E. and OFENGAND,J. (1979) Affinity labeling of transfer RNA binding sites on ribosomes. In: Transfer RNA: Structure, Properties and Recognition, pp. 431-444, SCHIMMEL,P. and St3LL, D. (eds) Cold Spring Harbor, New York. LEGOFFIC, F., CAPMAU,M.-L., CHAUSSON,L. and BONNET, D. (1980) Photo-induced affinity labeling of Escherichia coli ribosomes by chloramphenicol. Eur. J. Biochem. 106: 667-674. LEITNER, M., WILCHEK. M. and ZAMIR, A. (1982) Identification by affinity labeling of potential sites in 23-S rRNA interacting with the 3' end of tRNA. Eur. J. Biochem. 125: 4 9 - 5 5 . LESSARD,J. L. and PESTKA,S. (1972) Studies on the formation of transfer ribonucleic acid-ribosome complexes: XXIII. Chlorampbenicol, aminoacyl-oligonucleotides and Escherichia coli ribosomes. J. Biol. Chem. 247: 6909-6912. LILIAS, A. (1982) Structural studies of ribosomes. Prog. Biophys. Molec. Biol. 4 0 : 1 6 1 - 2 2 8 . LIN, F.-W., KAHAN, L. and OFENGAND,J. (1984) Cross-linking of phenylalanyl-tRNA to the ribosomal A site via a photoaffinity probe attached to the 4-thiouridine residue is exclusively to ribosomal protein S19. J. Molec. Biol. 172: 7 7 - 8 6 . LUDDY, M. A. (1982) Pbotoaffinity labeling studies of the streptomycin binding sites on the E. coli ribosome. Ph.D. Thesis, University of Pennsylvania. LUHRMANN, R., BALD, R. STOFFLER-MEILICKE,M. and STOFFLER, G. (1981) Localization of the puromycin binding site on the large ribosomal subunit of Escherichia coli by immunoelectron microscopy. Proc. Natn. Acad. Sci. U.S.A. 78: 7276-7280. MAASEN, J. A. and MOLLER. W. (1978) Elongation factor G-dependent binding of a photoreative GTP analogue to Escherichia coli ribosomes results in labeling of protein L11. J. Biol. Chem. 253: 2777-2783. MAASEN, J. A. and MOLLER, W. (1981) Photochemical cross-linking of elongation factor G to 70S ribosomes from Escherichia coli by 4-(6-formyl-3-azido-phenoxy)butyrimidate. Eur. J. Biochem. 115: 279-285. MACKEEN, L. A., KAHAN,L., WAHBA,A. J. and SCHWARTZ.I. (1980) Photochemical cross-linking of initiation factor-3 to Escherichia coli 30S ribosomal subunits. J. Biol. Chem. 255: 10526-10531. MAITRA.U., STRINGER,E, A. and CHAUDHURI.A. (1982) Initiation factors in protein biosynthesis. Ann. Rev. Biochem. 51: 869-900. MARGARITELLA,P. and KUECHLER,E. (1978) Specificity of the photo-cross-linking reaction between poly(U) and protein S1 on the Escherichia coli ribosome. FEBS Lett. 88" 131-134. MATZKE, A. J. M., BARTA,A. and KUECHLER, E. (1980) Mechanism of translocation: Relative arrangement of tRNA and mRNA on the ribosome. Proc. Nam. Acad. Sci. U.S.A. 77: 5110-5114. MOORE, P. B. CAPEL,M., KJELDGAARD,M. and ENGELMAN,D. M. (1986) Quaternary organization of the 30S ribosomal subunit of Escherichia coli. Biophys. J. 49: 13-15. NATHANS, D. (1964) Puromycin inhibition of protein synthesis: incorporation of puromycin into peptide chains. Proc. Nam. Acad. Sci. U.S.A. 51" 585-592. NICHOLSON, A. W., HALL. C. C., STRYCHARZ,W. A. and COOPERMAN, B. S. (1982a) Photoaffinity labeling of Escherichia coli ribosomes by an aryl azide analogue of puromycin. On the identification of the major covalently labeled ribosomal proteins and mechanism of photoincorporation. Biochemistry 21" 3797-3808. NICHOLSON, A. W. HALL, C. C., STRYCHARZ.W. A. and COOPERMAN, I . S. (1982b) Photoaffinity labeling of Escherichia coli ribosomes by an aryl azide analogue of puromycin. Evidence for the functional site specificity of labeling. Biochemistry 21: 3809-3817. NIELSEN, P. E., LEICK, V. and BUCHARDT, O. (1978) On photoaffinity labeling of Escherichia coli ribosomes using an azidochloramphenicol analogue. FEBS Lett. 94: 287-290. NIERHAUS, K. H. (1982) Structure, assembly and function of ribosomes. Curr. Top. Microbiol. lmmunol. 97: 82-155. NIERHAUS, K. H. (1984) New aspects of the ribosomal elongation cycle. Molec. Cell. Biol. 6 1 : 6 3 - 8 1 . NOLLER. H. F. (1984) Structure of ribosomal RNA. Ann. Rev. Biochem. 53: 119-162. NOLLER, I"I. F. and LAKE, J. A. (1984) Ribosome structure and function: localization of rRNA. In: Membrane Structure and Function, pp. 217-297, BITTAR,E. (ed.) Wiley, New York.

Escherichia coli ribosomes

301

OAKES, M. I., CLARK. M. W., HENDERSON, E. and LAKE, J. A. (1986) DNA hybridization electron microscopy: ribosomal RNA nucleotides 1392-1407 are exposed in the cleft of the small subunit. Proc. Nam. Acad. Sci. U.S.A. 83: 275-279. OFENGAND, J. (1980) The topography of tRNA binding sites on the ribosome. In: Ribosomes: Structure, Function and Genetics, pp. 497-529, CHAMBLISS,G., CRAVEN,G., DAVIES,J., DAVIS, K., KAHAN, L. and NOMORA,M. (eds) University Park Press, Baltimore. OFENGAND, J. and Llou, R. (1981) Correct c o d o n - anticodon base pairing at the 5'-anticodon position blocks covalent cross-linking between transfer ribonucleic acid and 16S RNA at the ribosomal P site. Biochemistry 20: 5 5 2 - 559. OFENGAND,J., LIOU,R., KOHUT,J., I~, SCHWARTZ,I. and ZIMMERMANN,R. A. (1979) Covalent cross-linking of transfer ribonucleic acid to the ribosomal P site. Mechanism and site of reaction in transfer ribonucleic acid. Biochemistry 18: 4322-4332. OLSON, H. M., GRANT, P. G. COOPERMAN,B. S. and GLITZ, D. G. (1982) Immunoelectron microscopic localization of puromycin binding on the large subunit of the Escherichia coli ribosome. J. Biol. Chem. 257: 2649-2656. OLSON, H. i . , NICHOLSON,A. W., COOPERMAN,B. S. and GLITZ, D. G. (1985) Localization of sites of photoaffinity labeling of the large subunit of Escherichia coli ribosomes by arylazide derivative of puromycin. J. Biol. Chem. 260: 10326-10331. PAULSEN,H. and WINTERMEYER,W. (1986) tRNA topography during translocation: steady-state and kinetic fluorescence energy-transfer studies. Biochemistry 25: 2749-2756. PAULSEN,H., ROBERTSON,J. M. and WINTERMEYER,W. (1983) Fluorescence energy transfer measurements between A and P site-bound tRNA ph¢. J. Molec. Biol. 167: 411-426. PETTERSSON, I., HARDY, S. J. S. and LILJAS, A. (1974) The ribosomal protein L8 is a complex of L7/L12 and L10. FEBS Lett. 64: 135-138. POL1TZ, S. M. and GLITZ, D. G. (1977) Ribosome structure-localization of N6,N6-dimethyladenosine by electron microscopy of a ribosome-antibody complex. Proc. Nam. Acad. Sci. U.S.A. 74: 1468-1472. POLITZ, S. M. and GLITZ, D. G. (1980) Magnesium-dependent interaction of 30S ribosomal subunits with antibodies to N6,N6-dimethyladenosine. Biochemistry 19: 3786-3791. PONGS, O., ST6FFLER, G. and LANKA,E. (1975a) The codon binding site of the Escherichia coli ribosome as studied with a chemically reactive AUG analog. J. Molec. Biol, 9 9 : 3 0 1 - 3 1 5 . PONGS, O., LANKA, E. and BALD, R. (1975b) Proteins involved in the ribosomal decoding site. Fed. Eur. Biochem. Soc. Meet., lOth, Abstract No. 447. PRINCE, J. B., TAYLOR,B. H., THORLOW,D. L., OFENGAND,1. and ZIMMERMANN,R. A. (1982) Covalent cross-linking of tRNA val to 16S RNA at the ribosomal P site: identification of cross-linked residues. Proc. Natn. Acnd. Sci. U.S.A. 79: 5450-5454. PRINCE, J. B., GUTELL, R. R. and GARRETT. R. A. (1983) A consensus model of the Escherichia coli ribosome. Trends Biochem. Sci. 8: 359-363. RAMAKRISHNAN,V., CAPEL,M., KJELDGAARD,M., ENGELMAN,D. M. and MOORE, P. B. (1984) Positions of proteins S14, S18 and $20 in the 30S ribosomal subunit of Escherichia coli. J. Molec. Biol. 174: 265-284. REIHL, N., REMY, P., EBEL, J.-P. and EHRESMANN,B. (1982) Cross-linking of N-acetyl-pbenylalanyl [S4U]tRNAPhe to protein SI0 in the ribosomal P site. Eur. J. Biochem. 128: 427-433. ROnBINS, D. and HARDESTY,B. (1983) Comparison of ribosomal entry and acceptor ribonucleic acid binding sites on Escherichia coli 70S ribosomes. Fluorescence energy transfer measurements from Phe-tRNAphe to the 3' end of 16S ribonucleic acid. Biochemistry 22: 5675-5679. RYOJI, M., KARPEN,J. W. and KAJI, A. (1981) Further characterization of ribosome release factor and evidence that it prevents ribosomes from reading through a termination codon. J. Biol. Chem. 256: 5798-5801. SCHENKMAN.M. L., WARD, D. C. and MOORE,P. B. (1974) Covalent attachment of a messenger RNA to the EscherichM coli ribosome. Biochim. Biophys. Acta 353: 503-508. SCHWARTZ, I. and OFENGAND,J. (1982) E. coli tRNA Phe modified at the 3-(3-amino-3-carboxypropyl) uridine with a photoaffinity label is fully functional for aminoacylation and for ribosomal interaction. Biochim. Biophys. Acta 697: 330-335. SCHWARTZ, I., VINCENT, i . , STRYCHARZ,W. A. and KAHAN,L. (1983) Photochemical cross-linking of translation initiation factor 3 to Escherichia coli 50S ribosomal subunits. Biochemistry 22: 1483-1488. SIEGRIST, S., MOREAU, N. and LEGOFFIC, F. (1985) Photo-induced affinity labeling of Escherichia coli ribosomes by dihydrorosaramicin, a macrolide related to erythromycin. Eur. J. Biochem. 153: 131-135. SONENBERG, N., W1LCHEK, M. and ZAMIR. A. (1975) Identification of a region in 23S rRNA located at the peptidyl transferase center. Proc. Num. Acad. Sci. U.S.A. 72: 4332-4336. SPIRIN. A, S. (1985) Ribosomal translocation: facts and models. Prog. Nucleic Acid Res. Molec. Biol. 32: 75-113. STEINER, G., LUHRMANN,R. and KUECHLER,E. (1984) cross-linking transfer RNA and messenger RNA at the ribosomal decoding region: identification of the site of reaction on the messenger RNA. Nucleic Acids Res, 12:8181 - 8191. STIEGE, W., GLOTZ, C, and BRIMACOMBE,R. (1983) Loclaization of a series of intra-RNA cross-links in the secondary and tertiary structure of 23S RNA, induced by ultraviolet irradiation of Escherichia coli 50S ribosomal subunits. Nucl. Acids Res. 11: 1687-1706. STOFFLER, G. and STOFFLER-MEIL1CKE,M. (1984) Immunoelectron microscopy of ribosomes. Ann. Rev. Biophys. Bioengng. 13: 303-330. SYMONS, R. H., HARRIS, R. J., GREENWELL,P., ECKERMANN,D. J. and VANIN, E. F. (1978) The use of puromycin analogs and related compounds to probe the active center of peptidyl transferase on Escherichia coli ribosomes. In: Bioorganic Chemistry: A treatise to Supplement Bioorganic Chemistry: An International Journal, Vol. 4, pp. 409-436, VAN TAMELEN, E. (ed.) Academic Press, New York, TANGY, F., CAPMAU, M.-L. and LEGOFFIC, F. (1983) Photo-induced labelling of Escherichia coli ribosomes by a tobramycin analog. Eur. J. Biochem. 131:581-587. TF.JEDOR, F. and BALLESTA.J. P. G. (1985a) Ribosome structure: binding site of macrolides studied by photoaffinity labeling. Biochemistry 2 4 : 4 6 7 - 4 7 2 . TEJEDOR, F. and BALLESTA,J. P. G. (1985b) Components of the macrolide binding site on the ribosome. J. Antimicrob. Chemother. 16: Suppl. A., 53-62.

302

B . S . COOPERMAN

TEJEDOR, F. and BALLESTA,J. P. G. (1985c) Protein $4 is near the elongation factor G binding site in the ribosome. FEBS Lett. 182: 253-256. TEJEDOR, F., AMILS. R. and BALLESTA, J. P. G. (1985) Photoaffinity labeling of the pactamycin binding site on eubacterial ribosomes. Biochemistry 24: 3667-3672. TINOCO, I., BORER, P. N., DENGLER,B., LEVINE,M. P., UHLENBECK,O. C., CROTHERS.O. M. and GIRALLA,J. (1973) Improved estimation of secondary structure in ribonucleic acids. Nature N e w Biol. 246: 4 0 - 4 1 . TOWBIN. H. and ELSON, D. (1978) A photoaffinity labeling study of the messenger RNA-binding region of Escherichia coli ribosomes. Nucl. Acids Res. 5: 3389-3407. TRAtrr, R. R., LAMBERT,J. M., BOILEAU,G. and KENNY,J. W. (1980) Protein topography of Escherichia coli ribosomal subunits as inferred from protein cross-linking. In: Ribosomes: Structure, Function, and Genetics, pp. 89-110. CHAMBLISS,G., CRAVEN,G. R., DAVIES,J., DAVIS,K. KAHAN, L. and NOMURA,M. (eds) University Park Press, Baltimore. TREMPE, M. R., OHCI, M. and GUTZ, D. G. (1982) Ribosome structure. Localization of 7-methyl guanosine in the small subunits of Escherichia coil and chloroplast ribosomes by immunoelectron microscopy. J. Biol. Chem. 257: 9822 -9829. VASILIEV,V. D. and SHATSKY,I. N. (1984) Structural organization of the Escherichia coli ribosome and its functional centers. Sov. Sci. Rev. D. Physiochem. Biol. 5: 141-178. VERSCHOOR, A., FRANK, J., RADERMACHER, M., WAGENKNECHT, T. and BOUBLm, M. (1984) Three-dimensional reconstruction of the 30S ribosomal subunit from randomly oriented particles. J. Molec. Biol. 178: 677-698. VERSCHOOR,A., FRANK,J. and BOUBLIK,M. (1985) Investigation of the 50S ribosomal subunit by electron microscopy and image analysis. J. Ultrastruct. Res. 92: 180-189. VERSCHOOR,m., FRANK,J., WAGENKNECHT,T. and BOUBLIK,M. (1986) Computer-averaged views of the 70S monosome from Escherichia coli. J. Molec. Biol. 187:581-590. VLADIMtROV,S. N., GRAIFER,D. M. and KARPOVA,G. G. (1981) The proteins of donor tRNA-binding site of Escherichia coli ribosomes. FEBS Lett. 135:155 - 158. WEXTZMANN,C. J. and COOPERMAN,B. S. (1985) On the structural specificity of puromycin binding to Escherichia coli ribosomes. Biochemistry 24: 2268-2274. WtNKELMANN, D. A., KAHAN, L. and LAKE, J. A. (1982) Ribosomal protein $4 is an internal protein: localization by immunoelectron microscopy on protein-deficient subribosomal particles. Proc. Nam. Acad. Sci. U.S.A. 79: 5184-5188. WXNTERMEYER,W., LmL, R., PAULSEN,H. and ROBERTSON, J. M. (1984) Transfer RNA binding to ribosomes. In: Mechanisms of Protein Synthesis, pp. 7 6 - 8 9 , BERMEK, E. (ed.) Springer, Berlin. W1TTMANN, H. G. (1983) Architecture of prokaryotic ribosomes. Ann. Rev. Biochem. 52: 35-65. WOLLENZEIN, P. L., MURPHY, R. F., CANTOR,C. R., EXPERT-BEZANCON,A. and HAYES, D. H. (1985) Structure of the Escherichia coli 16S ribosomal RNA. Psoralen cross-links and N-acetyl-N'-(p-glyoxylbenzoyl) cystamine crosslinks detected by electron microscopy. J. Molec. Biol. 184: 6 7 - 8 0 . YANG, C.-H. and SOLE, D. (1974) Studies of transfer RNA tertiary structure by singlet-singlet energy transfer. Proc. Natn. Acad. Sci. U.S.A. 71: 2838-2842. YONATH, A., SAPER, M. A., MAKOWSKI,J., MUSSIG,J., PIEFKE,J., BARTANIK,H. D., BARTELS,K. S.and WITTMANN, H. G. (1986) Characterization of single crystals of the large ribosomal particles from Bacillus stearothermophilus. J. Molec. Biol. 187: 633-636.