Comparative Biochemistry and Biophysics of Ribosomal Proteins

INTERNATIONALREVIEW OF CYTOLOGY, VOL. 124

Comparative Biochemistry and Biophysics of Ribosomal Proteins ANDERSLILJAS Department of Molecular Biophysics, Chemical Center, Lund University, S-22100 Lund, Sweden

I. Introduction The ribosome is the site of protein synthesis in all organisms. It is a complex organelle composed of a few RNA molecules and a complement of a large number of relatively small proteins. The analysis of ribosomal function and structure has been in progress for over three decades (Watson, 1964; Spirin, 1986), and the results give a complex picture of a macromolecular assembly where individual components cooperate in a large number of functional steps. In the analysis of macromolecular functions there is often a strong need to identify the groups involved and to visualize the structures responsible for a certain function. This is particularly useful with the advent of in vitro mutagenesis methods where hypotheses about structure-function relationships can tested. Thus, in enzymology one tries to determine the functional roles and the stereochemical relation of different amino acids in an active site. However, in the analysis of ribosomes it has been difficult even to clarify which components are involved in the different functional steps. This can to some extent be expected since the “substrates” or “cofactors” in protein synthesis are themselves macromolecules like messenger RNAs (mRNA), transfer RNAs (tRNA), or factor proteins. This will obviously lead to large areas of interaction where several cornponents can be involved. In addition, if the ribosome undergoes conformational changes (Moazed and Noller, 1989), components not in direct contact with the substrates can also affect the activities. In the first phase of the analysis of ribosome structure and function, interest was strongly focused on the ribosomal proteins which were expected to be the active components and more easy to study (Kaltschmidt and Wittmann, 1970; Nomura and Held, 1974; Moore and Capel, 1988). However, in recent years, observations of RNA molecules with enzymatic activity have caused the interest to turn toward the ribosomal RNAs (rRNAs), which are no longer thought to be the passive backbone of the ribosome but likely to be responsible for the core of the ribosomal functions (Woese, 1980; Cundliffe, 1986; Moore, 1988). The possibility that the ribosome is a ribozyme is being explored. What, then, is the role of the ribosomal proteins? Is it a passive one in which they participate in the folding and protection of the RNA moieties and possibly 103

~

. .

. .

.

- .

104

ANDERS LILJAS

modulate the activities of the rRNA, or are there particular functions for the ribosomal proteins which merit the extensive interest in their chemistry and structure? What is special about the structures of ribosomal proteins that permit them to fulfil these functions? This review tries to deal with these questions partly by utilizing the rapidly expanding information about protein structure and function in a large number of species. To be able to discuss the role of the ribosomal proteins we must first summarize the ribosomal functions and describe to what extent rRNA is found to participate in these activities.

STEPSIN PROTEIN SYNTHESIS A. FUNCTIONAL Protein synthesis is performed in three separate phases: initiation, elongation, and termination. Figure 1 gives a summary of the main events during these three phases for bacterial protein synthesis, which may serve as a model for protein synthesis in general. The ribosome has at least three binding sites for tRNA (Rheinberger and Nierhaus, 1980; Moazed and Noller, 1989): a site for aminoacyl-tRNA (A site), a site for peptidyl-tRNA (P site), and a site for deacylated tRNA (E site or exit site). During initiation, the mRNA binds to the small subunit (30s)which associates with the large subunit (50s).The initiator tRNA (Met) binds to the P site in complex with one of the initiation factors (IF-2), a large protein molecule ( 1 12K), and a GTP molecule (Grunberg-Manago, 1980). The elongation cycle is the central feature of Fig. 1. It consists of three main events: tRNA binding, peptidyl transfer, and translocation. The aminoacyl-tRNA binding is assisted by one of the elongation factors, EF-Tu (42K), a protein which in complex with GTP has high affinity for aminoacyl-tRNA (Kaziro, 1978). The anticodons of the tRNA in such ternary complexes are tested against the exposed codon in the A site of the ribosome. Incorrect matching is rejected through the initial selection pathway. After GTP hydrolysis and dissociation of EF-Tu*GDP, the aminoacyl-tRNA is rechecked in the proofreading pathway, which improves the fidelity of protein synthesis by about two orders of magnitude. The tRNA that has passed these two branch-points will be able to accept the peptide of the tRNA in the P site (Hopfield, 1974; Ninio, 1975; Kurland and Ehrenberg, 1984). Peptidyl transfer occurs on the 50s subunit, most probably without assistance of any factor protein. The peptide moiety on the tRNA in the P site is donated to the amino acid on the tRNA in the A site. Thus the new peptidyl-tRNA is located in the A site and the P site tRNA is now deacylated. To permit the next cycle of elongation, the peptidyl-tRNA has to be moved to the P site. Likewise, the mRNA has to be moved by one codon to expose the next one in the A site. These processes are facilitated by the elongation factor G

105

COMPARISONS OF RIBOSOMAL, PROTEINS

Peptide transfer

aa- tRNA binding

A

A

3a41

I

p,P

n.

r

88

n

Tran

FI...

I ni t i'at ion

FIG.1. A simplified representation of protein synthesis on bacterial ribosomes (Liljas, 1990). See text for further details.

(EF-G), an 81K protein which also binds in complex with a GTP molecule and dissociates after GTP hydrolysis (Kaziro, 1978). During this process the deacylated tRNA in the P site is moved to the E site, from where it can subsequently dissociate (Rheinberger and Nierhaus, 1980). This completes the elongation cycle. A careful investigation of tRNA protection of rRNA during the different steps in elongation gives a more complex picture (Moazed and Noller, 1989). Here, hybrid states for tRNA binding, such as A/P and PE, are also observed. In case a termination codon is exposed in the A site , one of two slightly different release or termination factors (RF-1 or RF-2) binds to the ribosome and induces hydrolysis of the peptide from the tRNA in the P site (see Tate, 1990, for a review). A certain error frequency is associated with these processes. Thus, erroneous amino acids can be inserted at a frequency of about 10-3-104 in a normal protein (Edelman and Gallant, 1977; Bouadloun et al., 1983). Furthermore, during translocation, the mRNA may be shifted by more or fewer than three nucleotides, which leads to reading frame errors. The release factors compete with the aminoacyl-tRNA molecules for binding to the A site . Thus, the synthesis can be prematurely terminated, or, conversely, stop codons can be read through.

106

ANDERS LILJAS

B. PARTICIPATION OF rRNA IN RIBOSOMAL FUNCTIONS For some time it has been evident that rRNA participates directly in several of the ribosomal functions, and some examples are given below. Thus in the binding of mRNA to the ribosome during the initiation phase, a region of the 3’ end of the 16s RNA forms a double helix with a small region preceding the initiation codon of the mRNA. This base-pairing (Shine and Dalgarno, 1975) has also been found in archaebacteria (Kopke er al., 1989; Shimmin er al., 1989). The same region of the 16s RNA may also be involved in interaction with the stop codons in the termination phase (Shine and Dalgarno, 1974; Lang er al., 1989) but direct evidence is lacking (Tate, 1990). It has been firmly established that the anticodon of the tRNA in the ribosomal P site comes very close to the base of nucleotide 1400 of the 16s RNA in E . coli (Prince et a/., 1982). This region is highly conserved betweeen species and is found to interact with the tRNA even in the case of eukaryotes (Ofengand er al., 1986). Many antibiotics are inhibitors of specific functional steps in protein synthesis. In many cases they probably bind at “active sites”, such as binding sites for tRNA, factors, or at the site for peptidyl transfer. Resistance against these antibiotics is not only obtained by mutations of some proteins but also by methylation of some bases or sugars of the rRNA. The modified region of the RNA can directly or indirectly, through conformational changes, be involved in ribosomal functions (see Cundliffe, 1986, for a review). In addition, several toxins function as enzymatic inhibitors of protein synthesis by modifying the structure of rRNA at some specific site (Stirpe and Barbieri, 1986; Endo er al., 1987). In cases where a particular ribosomal function is thought to be closely associated with a certain region of rRNA this can be tested by site-specific mutagenesis. The functional importance has been verified in several cases (De Stasio er a/., 1988). Thus, it is clear that the rRNA is not only the structural backbone of the ribosomal particles but also participates in the active sites of the organelle. Several of the other molecules involved in protein synthesis are also RNA molecules (mRNA, tRNA). It cannot be excluded that primordial protein synthesis required only RNA molecules (Woese, 1980). A minimal role for the proteins in todays ribosome is to add to the range and specificity of interactions between ribosomes and transiently bound molecules. Since some ribosomal proteins are highly conserved between species, one could expect that there must be specific roles for these proteins. In this article we first discuss some general aspects of ribosome structure. The structure of the ribosomal proteins must be closely related to their function and is discussed in general terms, and the structure and functional relationship for some proteins are reviewed. Within the limits of this review, we have chosen to focus mainly on the proteins involved in fidelity and factor interactions.

COMPARISONS OF RIBOSOMAL PROTEINS

107

11. Structural Organization of the Ribosome A. STRUCTURE OF THE RIBOSOMAL PARTICLES Several methods have been used to obtain information about the general shape of the ribosome. Obviously, crystallographic approaches should be able to produce a very reliable picture, provided that the resolution is good enough. Remarkable progress has been made with regard to crystallizingribosomal particles from different bacteria (Yonath and Wittmann, 1989). However, the analysis is complicated due to the size of the ribosome, the lack of symmetry, and the quality of the crystals. The improvements in crystal quality and techniques for crystal handling are very impressive and give promise for the determination of the detailed structure of the ribosome in the future (Yonath and Wittmann, 1989). So far, crystals, monolayers, and separate ribosomal particles have been analyzed using electron microscopy and image reconstruction methods (Yonath et al. 1987; van Heel and StofflerMeilicke, 1985; Radermacher et al., 1987). The results have provided additional details compared to the images of single particles. For example, channels have been observed through the 50s subunit (Yonath et al., 1987). At the moment these reconstructed images give no details of the locations of the different components. Comparisons of the shapes of ribosomal particles from different types of organisms have been made using electron microscopy. Striking homologies in shape have been observed. Larger ribosomes are observed to have additional features on the surface of the particle, whereas the main body seems to remain the same (Oakes et al., 1986). Despite the great variation in rRNA size and number of ribosomal proteins (Table I) the ribosomal and subunit structures show distinctive homologies. Thus, the differences in the rRNA and proteins between different species must in a broad sense be compensatory. TABLE I COMPONENTS IN DIFFERENT SPECIES RIBOSOMAL RNA components Species Eubacteria Archaebacteria Chloroplasts Plant mitochondria Yeast mitochondria Mammal mitochondria Yeast Rat liver ~

Protein components.

Large subunit

Small subunit

Large subunit

Small subunit

Reference

5S, 23.90 5s. 23s 5s. 4.5s. 23s 5s. 26s

16s 16s 16s 18s 15-17s 12s 18s 18s

33 33 (36?) 33

2&21 20 (25?) 20 ? 34 33 31 34

See text and Table 11 Wittmann-Liebold er 01. (1990) Wittmann (1986) Witbnann (1986) Graack er al. (1988) . Matthews er ~ 1(1982) Planta ef QI. (1986) Wool (1980)

21-24s 16s 5S, 5.8S, 26s 5S, 5.8s. 28s ~

.The numbers are in most cases approximate. 6vedberg.

? 34 52 44 50

108

ANDERS LIWAS

B. LOCATION OF RIBOSOMAL PROTEINS Details of the location of individual proteins have been studied mainly by immune electron microscopy and neutron scattering. Whereas electron microscopic work has been performed on several species, neutron scattering has so far been completed only for the Escherichia coli 30s subunit (Moore and Capel, 1988) and is in progress for the 50s subunit (Nowotny et al., 1986). As long as the comparison of the structural and functional relationship between ribosomal proteins from the different kingdoms remains fairly incomplete, it is impossible to make any general conclusions, but we must refer to the best characterized ribosomes, those from E. coli. The results and interpretations from immune electron microscopy have gradually been improved (Oakes ef al., 1986; Stoffler-Meilicke and Stoffler, 1987; Walleczek er al., 1988), and details of locations of smaller epitopes of some proteins are even beginning to emerge (Nag ef al., 1986). In general, the locations of the ribosomal proteins observed using neutron scattering and immune electron microscopy agree very well. The proteins are spread over the surface of the ribosome and the rRNA forms the core of the subunits as well as being highly exposed in surface areas devoid of proteins. A model with the location of some proteins can be seen in Fig. 2. C. STRUCTURE OF THE rRNA The structural analysis of the rRNA molecules has primarily benefited from the rapid increase of sequence data from a wide variety of species. The interest in rRNA for evolutionary relationships has greatly contributed to this (Woese et al., 1980; Glotz and Brimacombe, 1980; Stiegler el al., 1981). With the availability of sequences of rRNA from a large number of species the analysis of secondary structure becomes meaningful. It is evident that there is a pattern of base-paired, double-helical regions which can be found in all organisms. These regions of secondary structure do not show any general sequence homology between species. On the other hand, several single-stranded regions are highly homologous between all available sequences. The pattern of basepairing and single-stranded regions has also been analyzed by a number of methods such as cross-linking, chemical modification and enzymatic digestion, providing experimental evidence and fine details of the secondary and tertiary structure arrangements (Brimacornbe er al., 1988; Stem er al., 1988). The locations of binding sites on the rRNA for ribosomal proteins have been established by a variety of methods (Brimacombe et al., 1988; Stem et al., 1989). Since the neutron scattering and electron microscopic location of these proteins is known, the approximate location of the corresponding region of RNA can also be established.

COMPARISONS OF RIBOSOMAL PROTEINS

109

30 S

FIG. 2. A model of the 70s ribosome from Escherichia coli highlighting some of the proteins which are close to the factor binding site or which are related to factor binding. The subunits are pulled apart to permit a view of some of the proteins in the interface area. The model is drawn according to Oakes et al. (1986), Stoffler-Meilickeand Stoffler (1987), Walleczek et al. (1988). and Moore and Cape1 (1988). The factor binding area is the striped area between the subunits. Protein L7/L12 is represented by two dimers in different positions as suggested by Moller et al. (1983) and Olson et al. (1986).

Several regions of rRNA have also been located by more direct methods (Oakes et al., 1986). Detailed models of the complete 16s RNA in E. coli have been presented (Brimacombe et al., 1988; Nagano et al., 1988; Stern et al., 1988). It is of interest for our continued discussion to note that the core of the ribosomal particle and parts of the surface seem to belong to the regions of conserved double-stranded structure. Thus the loops and structures that differ between different species occur on the surface. Since all 30s ribosomal proteins in E. coli have accessible epitopes on the surface (Stoffler and StofflerMeilicke, 1986), it seems likely that the large species differences in loop stucture of the RNA on the surface of the ribosome must have effects on the proteins bound to these surfaces. This could be part of the reason for the differences in number, composition, and structure of some of the ribosomal proteins.

110

ANDERS LILJAS

111. General Properties of Ribosomal Proteins A. COMPARISONS OF RIBOSOMAL PROTEINS FROM DIFFERENTSPECIES

Escherichia coli has been the standard organism used in the analysis of ribosomal structure and function. Thus, the proteins from the small and large ribosomal subunits of E. coli were numbered S1-S21 and Ll-L34, respectively (Kaltschmidt and Wittmann, 1970). Some proteins have been eliminated from this list. S20 is identical to L26 (Wittmann and Wittmann-Liebold, 1974), L7 is the aminoacetylated form of L12 (Terhorst et al., 1973), and L8 is a strong complex of L7/L12 and L l 0 (Pettersson ef al., 1976). For some time, the remaining proteins seem to have been considered as the unique and complete set of proteins in E . coli. However, the continued investigations of DNA sequences and gel electrophoresis of ribosomal proteins have reminded us of the uncertainties of the methods used and a few new proteins have been added (Table 11; Wada and Sako, 1987). The analysis may still not be exhaustive. Due to the rapid increase of DNA and protein sequencing technology, we have today an amount of sequence information which is very difficult to overview in its entirety. A few generalizations can nevertheless be made. Most of the E . coli ribosomal proteins have been found in other eubacteria. Furthermore, chloroplast ribosomes also have a set of proteins which show extensive resemblance to those from eubacteria (Table 11). One exception is the largest ribosomal protein S1 which seems to be absent in many gram-positive bacteria (Schnier and Faist, 1985; Mikulik ef al., 1986). No sequence information is at this time available to indicate any homology to some of the E . coli ribosomal proteins, such as L13, L19, L25, L28, and L31. These remain the only possible proteins which can be totally unique to E . coli. The number of proteins in archaebacteria is not much greater than in eubacteria. Nevertheless, a significant number of proteins have been found for which there are no obvious counterpart in eubacteria. In several cases there are eucaryotic proteins with sequence similarities to those archebacterial proteins (Wittmann-Liebolde f al., 1989). B. CLASSES OF RIBOSOMALPROTEINS Almost all ribosomal proteins are relatively small (Table HI), the majority have less than 150 amino acids and some even less than 50 amino acids. From the early investigations of ribosomal proteins in E . coli it is evident that most of them are highly basic (Kaltschmidt and Wittmann, 1970). However, a small number of the proteins are acidic. The analysis of ribosomes from other species has shown this fact to be true in most cases (Wittmann-Lieboldet al., 1989). The simple division of the ribosomal proteins into basic and acidic is not useful since the proteins from extreme halophilic archaebacteria such as

COMPARISONS OF RIBOSOMAL PROTEINS

111

Halobacterium cutirubrum and Halobacterium marismortui are almost all acidic. The proteins that are most acidic in other species remain most acidic in extreme halophiles. The most prominent of the constantly acidic proteins in E . coli are called L7/L12 and L10, which form a strong protein complex. Another classification, partly on functional grounds, is to divide the ribosomal proteins into those that are essential for the proper folding of the rRNA and the subsequent binding of other proteins. These proteins bind to rRNA early in reconstitution of the ribosomal subunits (Table 11). The other group in this division consists of those that can be attached later during assembly to the RNA moieties as well as to other proteins. In E. coli the group of proteins which are essential for complete assembly consists of S4, S7, S8, S15, S17, and S20 for the small subunit (Nomura and Held, 1974) and L1, L2, L3, L4,L9, L20, and L24 for the large subunit (Rohl and Nierhaus, 1982).

c.

SHAPE OF RIBOSOMAL PROTEINS

The shape of the ribosomal proteins has been analyzed by several methods. It has been clearly established using circular dichroism (CD) spectra and hydrogen-1 nuclear magnetic resonance ('H-NMR) that most of the ribosomal proteins have three-dimensional structures even when isolated from the ribosome, but that it may be of importance not to denature the protein during the purification (Momson et al., 1977). In early studies using observations with immune electron microscopy and small-angle X-ray and neutron scattering, it was thought that the ribosomal proteins were highly elongated. Renewed analyses by EM and neutron scattering indicate that the ribosomal proteins have relatively compact structures (Table 111). In addition, X-ray crystallography has yielded structures at medium or low resolution that, for some proteins (S5, L6, and L30), show that their deviation from globular shape is small (White et al., 1983; Appelt et al., 1984; Wilson et al., 1986). Some proteins that deviate greatly from this pattern are S1, S2, S4, and L7/L12, for which the radii of gyration determined by scattering methods are around 60, 30, 30, and 40 A, respectively (Moore and Capel, 1988; Osterberg et al., 1976). Many proteins are divided into several domains or separate globular entities along the polypeptide chain (Rossmann and Liljas, 1974). A number of frequently found domain structures have been classified. Such well-known domains can be recognized by comparison of the amino acid sequence or predicted secondary structure with proteins of well-known structure. However, in no case has sufficient homology been found between a ribosomal protein and any characteristic sequence for known domain structures from other proteins. Other evidence for domain structure is the internal repeats of regions of homologous amino acid sequence within a protein. Ribosomal protein S1 is an example of a repeat of 87 or possibly 44 residues occurring several times in the

ANDERS LIUAS

112

TABLE I1 HOMOLoGlES BETWEEN RIBOSOMAL hOTElNS FROM DIFFERENT SPECIES"

Protein

Assemblyh

E. coli repressor'

Mutantsd Eubacteria

Chloroplasts

Archaebacteria

ss S6

s7

S8 s9

A

R

S15 S16 S17 S18

S19 s20

s21

LI L2 L3 L-2 L5 L6 19 LIO Lll

L12 L13

L14 LlS L16 L17 L18

L19 L20 L2 1 L22 L23 L24 L25 L26 = S20 L27 L2X L29 L30 L3 I L32 L33 L34 L35 L36

a

C C

a

-

a a

-

A A

R R

U

a a

a

s10 s11

s12 s13 s 14

Eukaryotes

a (partial)

S1

S2 53 s4

Mitochondria

a

C

a (I

-

i

U U

A

a

A

-,

A

R

-

A

R

-

c

a

(1

U

A A

U

A .A A

R

U

a a

i

C A A A

a

a

A

R

U

U

-

C

a a a, m a a

a

u, ni

a

a U

U

A A

A A A

a

a a

a U

a

A

a

a

COMPARISONS OF RIBOSOMAL PROTEINS

113

structure (Wittmann-Liebold et al., 1983). Furthermore, this repeat structure is homologous to a stretch of 69 amino acid residues in polynucleotide phosphorylase (Regnier et al., 1987). In the cases where the proteins have several domains the connections between the domains may be easily accessible to degradation and the domains can sometimes be separated from each other without using denaturing agents. This has been done for S1 (Suryanarayana and Subramanian, 1984) and L7/L12 (Liljas et al., 1978). Such domain arrangements can have several roles. The domains can have separate functional roles or functional sites can be located at the interface between the domains, and the flexibility which can be obtained between several separate entities may be of functional importance. The three domains of elongation factor Tu may give a good example of this type of arrangement (LaCour et al., 1985; Jurnak, 1985). It is likely that many of the small ribosomal proteins are composed of one single domain, as has been indicated by mild proteolysis experiments (Table 111), but some of the larger proteins (S 1) are probably multidomain proteins. Another structural feature has been found for histones and for many viral coat proteins (L. Liljas, 1986; Rossmann and Johnson, 1989) and is probably also of significance for many ribosomal proteins. Here, parts of the polypeptide chain are not folded on some more-or-less globular part of the protein structure but are rather protruding from the domain structure and engaged in interactions with the nucleic acid of the particle. These protruding regions are frequently highly basic, with half or more of the amino acid residues being lysyl or arginyl. Such regions occur most often at either or both of the two termini, in which case the protein has an N- or a C-terminal tail. If an unfolded region occurs in the interior of the primary structure, the protein may be bilobal and possibly equipped with a hinge region which gives it flexibility. Such tails or hinge regions are highly accessible Owhen no further reference is given, see Wittmann-Lieboldet al. (1990) or references therein. bProteinsmarked with A bind to the 16s or 23s RNA independently of other proteins (Nomura and Held, 1974; Rohl and Nierhaus, 1982). 'Proteins marked with R function as translational repressors in the synthesis of ribosomal proteins (Gourse et al., 1986). Troteins marked with - are absent in different mutant ribosomes (Dabbs, 1986). Mutants of proteins marked with C affect the accuracy of protein synthesis (Wittmannet al., 1980). S e e Ohto et al. (1988). S e e Ohyama et al. (1986). *A gene cluster from Mycoplusmu cupricolium (Ohkubo et al., 1987). hA gene cluster from Zea mays chloroplast (Larrinua and McLaughlin, 1987). 'See Nielsen et al. (1986). 'See Matheson et al. (1984). *See Marquis and Fahnestock (1978). 'See Bartsch et al. (1982). "No convincing sequence homology has been found, only functional homology.

TABLE 111 STRUCTURAL AND F’HYSICAL PARAMETERS OF Escherichia coli PROTEINS Protein

Molecular R , ( A ) Mild Basic Crystals/ weighr’ neutronb digestiond regions< structure (A) Comments

s1

61,159

55.4

15K

S2

32.8 25.4 30.2 133 13.1‘ 13.7, 21.1

Degrades 23K I8K 4K

58

26,hI 1 25,852 23,137 17.515 15.704 17.131 13.996

s9 s10

1 1,736

14,569

20.8 12.6 12.5 12.6 12.5 12.0 263 25.7 13.2 10.9 11.6‘ 11.3’ 243

S3

s4 SS

Sh 57

s1 I s12

S13 S14 S15 516 S17 SIR S19 S 20

s71

13.728 13,606 12.968 11,162 10,001 9.191 9,573 8,896 10,299 9.553 8,369

LI L2 L3 L4 L5 L6 L9 L10 LI 1

24.599 29.416 22.258 22.087 20.171 18,832 15.696 17,737 14,874 12.178

26

L13

16.019 13.541 14,981 15,296 14,364 12,770 13.002 13.366

21

21

L30

12.221 11.013 ll.lX5 10,964 9.553 8,993 8,875 7,274 6.411

L3 I L32 L33 L 34 L35 LJh

6.97 1 6.325 6.315 5.381 6.984 4.365

L12

LI4

L15 1.16 L17 L18 L19 L20 L? 1 L?2 L13 L24 L25 L26 L27 L28 L29

22 25

19

23

13 24 22 18

Homologous to polynucleotide phosphorylase (Regnier er a/.. 1987)

not folded‘ not foldedd

5’

6K 8K + 6K

A central basic region found for some chloroplast proteins (Ohyama ef al., 1986)

Forms a complex with S 19

8K Resistant Resistant Resistant

Forms a complex with S I3

bK 7K

Not folded”

22K Degrades 13K

Not folded’

R.9K

6x Bs LP L12, * L10’

Degrades 9K 4K + 7K

NTF‘

-

LIZ, LIO’

Forms a complex LIZ, * LIO (Petterssonet al., 1976)

-

Forms complexes L12, and L12, L10 CTF 1.7’ Structure similar to L30

13.5K I 5K 12K

11,565

15

19

5.5K 5K 8K

Yes‘ Yes-

=s20

Not folded”

Degrades 6.5K Degrade5 Resistant

Yesh 2.5”

3.5K Degrades

Not folded” Structure similar to C- term of L12 (Leijonmarck efa / , 1988) Not folded”

N + M(2)

Wada and Sako (1987) Wada andSako (1987)

COMPARISONS OF RIBOSOMAL PROTEINS

115

to proteolytic enzymes, particularly when the protein is isolated from its natural environment in a macromolecular assembly. Other methods which can detect such flexible regions include 'H-NMR methods and sequence analysis. Such clusters of basic residues have also been observed in ribosomal proteins (Wittmann-Liebold, 1973). Table I11 gives a summary of ribosomal proteins with half or more of the residues being basic for at least a stretch of 10 residues and which thus may have such unfolded regions which interact with the rRNA. To some extent, these regions may function as some type of polyamine with a single unique binding site on the rRNA. One very interesting observation has been made in this respect. Kashiwagi and Igarashi (1987) have observed that the majority of the E. coli ribosomal proteins can inhibit the enzyme ornithine decarboxylase. The same type of inhibition is also obtained by polyarginine, protamine, histones, and polylysine in order of decreasing inhibitor strength. The ribosomal proteins that do not function as inhibitors are S1, S5, S6, S8, S10, L3, L5, L6, L7L12, L9, and L10, most of which have no basic stretch (Table 111). There are thus good reasons to assume that many of the other ribosomal proteins have polyamine-like regions that can inhibit ornithine decarboxylase. These basic tails, loops, or hinge regions are likely to be important for their affinity to the rRNA, as in the case of histones or virus coat proteins. Naturally, there must be many other ways for proteins to attach to the RNA molecules, as can be judged by a comparison with the set of protein structures that bind to DNA in different ways. D. TERTIARY STRUCTUREOF RIBOSOMAL PROTEINS One line of research in the structural analysis of ribosomes is the crystallographic analysis of isolated components or complexes of components. It is highly appropriate to ask whether this work is meaningful or not, especially since ~

'T3i1-i et al. (1984). b30S proteins studied by Moore and Capel (1988); 50s proteins studied by Nowotny er al. (1986). These gyration radii were determined by constraint (Moore and Capel, 1988). Tittlechild et al. (1987). 45K means fragment of molecular weight: 45,000 was obtained. eLiljas,A,, unpublished observations. N and C, N- and C-termini; M, middle region of protein. White et al. (1983). gAppelt etal. (1984). hAppeltet al. (1981). 'Liljas and Newcomer (1981). 'Liljas et al. (1978). Teijonmarck and Liljas (1987). Wittmann (1986). "'Abdel-Mequidet al. (1983). 'Wilson et al. (1986).

116

ANDERS LILJAS

there are good crystals of subunits or whole ribosomes (Yonath and Wittmann, 1989). The resolution that can be achieved for crystals of subunits or ribosomes is not great enough to trace the polypeptide chain other than under special circumstances, and it is even less possible to analyze the details of functional sites. Thus, there is a large number of separate proteins or their complexes with other proteins or fragments of RNA of significant interest for crystallographic analysis. Domains of the ribosome for which there is some knowledge about functional interactions will get high priority. Furthermore, larger entities are obviously easier to position accurately in the particle and will yield more information provided that the resolution in the analysis is as high as possible. So far, interest has mainly been focused on proteins or protein complexes, but the 5s RNA in complex with proteins (Abdel-Meguid et al., 1983) as well as fragments of the large RNA molecules will be of interest with or without proteins. So far only two high-resolution structures have been determined, a C-termind fragment of L7/L12 from E. coli (Leijonmarck et al., 1980; Leijonmarck and Liljas, 1987) and L30 from Bacillus stearothermophilus (Wilson et al., 1986; van de Ven and Hilbers, 1986). The most remarkable feature of these two simple protein structures is the similarity in their folding (Leijonmarck et al., 1988). No other known structure has this folding, except for a helix-turn-helix which L7/L12 has in common with some DNA-binding proteins (Richards and Kundrot, 1988; Richardson and Richardson, 1988; Rice and Steitz, 1989). This similarity in tertiary structure is not related to a corresponding similarity in amino acid sequence. One obvious question is then whether there are other ribosomal proteins with the same folding. White (1990) has concluded after a detailed sequence comparison with other r i b o s o ~ ~proteins al that there are a few additional candidates with this folding. The lack of obvious sequence homology between ribosomal proteins and other proteins does not mean that the ribosomal proteins on the whole must have unique tertiary structures. The amino acid sequences are not sufficient to detect a structural similarity. Thus, to explore the three-dimensional structures at atomic level, we need high-resolution X-ray crystallography of ribosomal proteins as well as rRNA fragments to complement the crystallographic structure of the ribosomal particles analyzed at lower resolution.

IV. Functional Roles of Ribosomal Proteins The challenge of the functional role of the ribosomal proteins has been further substantiated by the fact that E. coli ribosomes can lack any one of a significant fraction of the proteins in vivo (Table 11) while still maintaining protein synthesis, even though the mutant strains grow poorly in some cases (Dabbs, 1986). Despite the fact that some of these proteins are found in all kingdoms they can hardly be of fundamental importance. It is interesting to note that this list of pro-

COMPARISONS OF RIBOSOMAL PROTEINS

117

teins contains some which have been closely related to essential functions such as assembly of 50s subunits (L24), mRNA binding (Sl), factor binding (Lll), and peptidyl transfer (L27). One current view of protein function is that they modulate the ribosomal activities which are mainly due to the rRNA. On the other hand, there are observations which are indicative of the importance of a number of proteins. Mutants of several ribosomal proteins give resistance against different antibiotics inhibiting some ribosomal function. As we have discussed above, these inhibitors often bind at functional sites. Therefore these mutations may also occur at sites that are directly or indirectly related to the function (Cundliffe, 1986). The proteins with functional mutations and the sites of these mutations are thus of considerable interest. This is particularly true when the mutations lead to a ribosome with altered functional properties, in some cases to the extent that the ribosomes are dependent on the drug for proper function. A whole range of different functions for ribosomal proteins can be distinguished, some of which have already been described. One such function is the repression of further synthesis of a number of ribosomal proteins from a polycistronic mRNA (Gourse et al., 1986). These repressor proteins are also among those that participate in the assembly of the ribosome and the proper folding of the rRNA. Some proteins provide part of the binding site for other ribosomal proteins. Yet another function is to provide part of the binding sites for mRNA, tRNA, or protein synthesis factors on the ribosome. Finally, some proteins may be related to the enzymatic functions in protein synthesis, peptidyl transfer, and GTP hydrolysis. We discuss some of these functions below in relation to the present knowledge about protein structure. One general comment may be reiterated. The substrates in protein synthesis, the tRNA, show extensive homology between species. Correspondingly, the mRNA and at least some of the factor proteins are also similar, regardless of species. The sites on the ribosomes to which they bind should, to a corresponding degree, display homology. Indeed, several such sites involving regions of rRNA show extensive species homology. The same could be expected for proteins at such binding sites. V. Structure and Function of Some Selected Ribosomal Proteins

A. REPRESSORS OF TRANSLATION A number of ribosomal proteins in bacteria act as repressors of translation of groups of proteins. The mechanism for this repression is simply that they have a binding site on one of the ribosomal RNA independent of other proteins. When all available rRNA is saturated with this repressor protein it can also bind to its own mRNA, which is polycistronic. When this binding occurs, other ribosomal

118

ANDERS LILJAS

proteins genes on this mRNA are no longer translated. The repressor binding sites on the mRNA molecules are similar to the structures of the binding sites for these proteins on the rRNA. The binding site does not necessarily occur at the beginning of the mRNA, and several of its genes are not regulated by the repressor. The proteins in E. coli known to exert such repressor functions are S4, S7, S8, S20, LI, LA, and LIO (Table 111; see Gourse et af., 1986, for a review). It is worth noting that all of these repressor proteins bind primarily to the rRNA without the prior binding of some other ribosomal protein (Nomura and Held, 1974; Rohl and Nierhaus, 1982). Of those, S4, S8, S20, and L1 have one or several clusters of basic residues. S8 is one of these repressor proteins which at the same time is an important protein for ribosome assembly. There are highly homologous proteins in all kingdoms (Table I1 and Fig. 3). In particular, the N- and C-terminal regions are the most similar, whereas the middle region has more differences between different species and is also variable in length. This region, particularly considering the chloroplast sequences, is one of the basic regions discussed above which may be involved in binding to the rRNA and mRNA. Certainly, this region must be highly exposed as long as there are no negatively charged groups to bind to. According to the proteolysis data (Littlechild et al. 1987; Table 11), it is possible that S8 is built up of two domains (8K + 6K). The variable basic region could connect the two domains, but it is equally possible that the protein forms only one domain with an exposed basic loop of variable size. +++ + t + ++ + ++ t ++t+ SMQDPIADMLTRIRh'GQAAEIKAAVTM-PSSKLKVAIANVLKEEGFIEDFKVEGDT-KPELELTLKYFQGKA---E.coli ( b i .,-q, M. capr i ( b ) r., .. V..........AEJQRYLKT.SV-....V.LE..RI.......S..T....V-.KTINIE...QGKTR---B.stearo(b1 V . T . . . . . . . .A...ANMVRHEKLEV-.A..I.RE..EI..R....R.YEYIE.NKQGI.RIF...GPNER---M. p o l y ( c ) ~ G N . .T.N.I.S. . . ANLGKIKT.QV-.ATNITRN . . KI.FQ. . . .DN.1DNKQNT.DI.I.N. . . QGK.KKS-N.tabac (c) ~ ~ G R . . .TE1I.S . . .ADMDR.RV.RI-A.TNITEN.VQI.LR . . . . . NVRKHREKN.YF.V . . .RHRRNRKRP-Z.maize ( c ) ~1GR.T . . .L S...ADMNK.GT.RV-V.TNITEN.VKI.LR.....SVRKHQESNRYF.VS..RHQR R.TRKGI M .van ( a M.LM..L.NA.NHVS.CEGS'G.NVAYLK.A...IGRVLK.MCDQ.Y.GN.EYIE.GKAGVYKVD.IGQIN.CG--H.rnaris(a) ~GGP:..F.NA.SALN.AESVGHLEQ.'/S.A.NEIGSVLE.fYDR.Y.DG.SFVD.GKAG.F.VE..GAINECG---

..

+ + t

E.coii(b) M.capri(b) B.stearo(b) M.pOly(c) N.tabac ( c ) Z.maize (c) M.van(ai H.marisia1

+

i

+

t

t

t

--'IVESIQRVSRPGLRIYRKRDE~PK~GLGIAWSTSKG~~TDR~RQAGLGGEIICYVA ;CGLKKI.K. . . .V.AQAN.I.QVLN ....SI . . . . Q.1 . .GKK ..L.NA ...VLAFI 1TGLK.I.K. . . .V.V.AH.V.RVLN . . . . . IL . . . Q . . S.DKE...K.I. . . . . A . . T -- Y1TTLR.I.K . . . . . . SNHK.1 . . . LG.M . .V I L . . .R.1 . . . .E...KKI...LL...W Y~NITTLK.1. . . . . . . . SNYQR1.RILG.M . .VIL . . .R.~....E,.LE.I....L..IW YRTRTFLK.I.. . . . . . .ANYQGI . . . LG .M IL . . .R.I . . . . E..LNRI...VL..IW ----- AVKPRYAVKYQEFE.FEKRYLPAK.F.LL1 . . . P..L..HDE..T..V..RL.S..Y - - - - - ?VKPRYSA.ADEFE.---RFLPARDY,TL..T..V.I.SHYE..EQ.V..QV.A..Y

FIG. 3. Amino acid sequences of S8 from the eubacteria (b) E . coli, Mycoplasma capricolium ( M . caprij, Bacillus stearothermophilus (B. stearo), chloroplasts (c) horn Marchantia polymorphus ( M . poly), Nicoriana tabacum ( N . tabac), Zea mays ( Z . mays), the archdebacteria (a) Methanococcus wnnielii ( M . van) and Hulohacterium marismormi ( H . maris). See Wittmann-Liebold et al. (1990) and Table I1 for references. Residues identical to the one in E . coli are indicated by a dot. Residues for which three or more residues are basic are indicated by + above the aligned sequences. A cluster of baqic residues occurs in the middle of the molecule.

COMPARISONS OF RIBOSOMAL PROTEINS

119

Thus, interesting targets for functional analysis by site-directed mutagenesis in S8 are not only the highly conserved areas but also the central variable basic region.

B. BINDINGOF tRNA AND FIDELITY A large number of proteins have been determined by cross-linking and affinity-labeling methods to be situated near the different sites for tRNA molecules. tRNA is an extended molecule with a longest dimension of around 75 A. This means that it is reasonable to expect that several ribosomal components are close to the tRNA molecules. However, far more components of the ribosome have been observed to be close to the tRNA than can ever be accommodated into a meaningful model (see critical analysis by Moore and Capel, 1988). It is necessary to divide the tRNA interactions into different subsites. Thus the anticodon-codon interactions are known to occur on the 30s subunit, whereas the peptidyl transfer site is situated on the 50s subunit, regardless of which of the tRNA sites is being considered. Obviously then, the tRNA must be located between the 50s and 30s subunits. Three-dimensional image reconstruction work from electron micrographs of ribosomes shows that there is sufficient space between the subunits for several tRNA molecules (Yonath and Wittmann, 1989). For the peptidyl transfer site, the proteins L2, L3, L4, and L16 remain as strong candidates from all that has been suggested (see Liljas, 1982, and Wallaczek et al., 1988, for summaries and references). These proteins are located in the body of the 50s subunit below the central protuberance of the 50s subunit in the vicinity of protein L27 (Fig. 2). This is also the location found by Spirin and Vasiliev (1989) using hapten-labeled methionine on tRNAfMetand immune electron microscopy techniques. In this connection, it is important to remember that the central circle of domain V of the 23s RNA has been implicated by many observations as being located at the peptide transferase site (see Cundliffe, 1986, for a review). The best characterized site of interaction between tRNA and the ribosome is the one between the anticodon in the P site and C-1400 of the 16s RNA (Prince et al., 1982). It is also well established that this site is located in the region between the platform and the body of the 30s particle (Gomicki et al., 1984). Proteins S18 and S21 have been labeled and are in the vicinity, but many other proteins have been labeled either from the 3' end of tRNA at the A or P site or using mRNA fragments (see reviews by Oakes et al., 1986; Ofengand et al., 1986; Moore and Capel, 1988). One group of proteins which are related to tRNA binding are the ones affecting translational fidelity. The results have been mainly obtained by isolating mutants resistant against antibiotics inducing ambiguities in reading the mRNA,

120

ANDERS LILJAS

such as streptomycin and the aminoglycosides. Thus mutations in protein S12 can lead to resistance to or dependence on streptomycin (Birge and Kurland, 1969). Resistance against neamine and gentamicine is obtained by mutations in S17 and L6, respectively (Bollen et al., 1975; Kiihberger et al., 1979). In these cases the mutations lead to ribosomes with increased accuracy. On the other hand, revertants from streptomycin dependence which give decreased accuracy have been found in proteins S4, S5, and L12 (Hasenbank et al., 1973; Kirsebom and Isaksson, 1985). It is remarkable that the proteins affecting the fidelity of protein synthesis in opposing ways are all located in a relatively small region (Fig. 2). Protein L6 is located near the base of the L12 stalk of the 50s subunit, and S4, S5, S12, and S17 are all located on the side of the 30s subunit away from the platform. This region of the 30s subunit is in the 70s ribosome adjacent to the L12 stalk. It is also worth noting that S12 depends on the previous binding of S17 in the assembly of the 30s subunit (Nornura and Held, 1974). It is at present difficult to realize what connection exists from this site to the decoding site known to be near the cleft between the platform and the body of the 30s subunit. The distance must be at least on the order of 60 A. On the other hand, the site for these proteins coincides with the partly overlapping binding sites for factors such as IF-2, EF-Tu, EF-G, RF-1, and RF-2 (see Moore and Capel, 1988, for a recent review). EF-Tu is the protein factor involved in decoding. It has been established that the proofreading branch is what is most affected by mutations in S12 (Ehrenberg et al., 1986). It is possible that EF-Tu has already dissociated from the ribosome after GTP hydrolysis but before the proofreading has taken place. Thus the factor may not be involved in the proofreading. In such a case it is difficult to imagine how S12 affects proofreading. However, several steps in the aminoacyltRNA binding process are perturbed by these mutations (Bohman et al., 1984; Ruusala et al., 1984). Furthermore, the location of the tRNA in the A site described above may not be correctly understood. The fact remains that all the proteins which affect fidelity are located near the factor binding site. 1. Protein S12

Several of the mutated proteins have been studied in detail. It has been found that the mutations in S12, which is one of the most highly conserved ribosomal proteins (Wittmann-Liebold et al. 1989), are located in two small regions (Fig. 4). One is residue 42 and the other is five of the seven residues between 85 and 91 (Wittmann et al., 1980). When the available sequences are analyzed and compared it is evident that S 12 has basic tails at both termini.The N-terminal tail can have basic residues in any one of 12 contiguous positions (residues 8-19 in E. coli numbering). Thus, it is unlikely to have specific nonpolar contacts with other parts of the protein but should be extended and accessible for binding to rRNA. The archaebacterial

121

COMPARISONS OF RIBOSOMAL PROTEINS +++ti

t t

+

t+

++

t

Ecol b ATVNQLVRKPRARKVAKSNVPALE-------------ACPQKRGVCTRVYTTTPBKPNSA K42R,T,N Mlute b MP.IQ . . . . . G.SP . .VNT.G. . .Q-------------GN.MR . . . . . . . . . . . .T..... Bstea b P.1 . . . . . . G.EK F . .KS . . .NKGYNSFKKEQTNV.S . . . . . . . . . . G.M.. . . . . . . Spiol c .Q..P Egra c P.LEH.T.S..KKIKR.TKS...K-------------G. . . . .A1.M . . . . . . . . . . . . . Ntab c MP.IK . . 1.NT.QPIRNVTKS. . . R-------------G . . .R..T Mpoiy c MP.IQ . . 1.NK.QPIENRTKS. . .K-------------G . . .R . . . . . . . . . . . . . . . . . . Zmaize c VTMP.IQ 1.NK.QPIENRRKS. . . K-------------G . . .R . . . W.....IN....... Mvan a MSGSKSPKGEFAGRKLLLKRKATRWQPYKYVNRELGLKVKADPLGGA.MG. . 1VVEKVLGEA.Q. . . . Tterm e MGVGKPRGIRAGRKLARHRKDQRWADNDFNKRLLGSRWR-NPFMGASHAK.LVTEKIG1ES.Q ....

..

.....

..

+t

+ +

+

+ +

t t

t

+

Ecol Mlute b Bstea Spiol Egra Ntab MPOlY Zmaize Mvan Tterm

LRKVCRVRLTNGF-EVTSYIGGEGH-NL-QEHSVILIRG-G~V~-----DL~VRYHTVRGALDCSGVK V . . . A....NG.I-...A..P....-..-....IV.V..-....-----.......KI......TQ... ..I-. . .A..P.I..-..S..-.ITA..P.I..-... . . ..K..V S.L-...A..P.I.. - . . - . . . . . V. . . . .A.....S . . - . ITA . .P.I. . - . . - . . . . . V.V V....-....----- . . . . . . . . 11 .. T..AV... S..-.ITA..P.I S. .-. ITA..P.I V.V..- ....----- . . . . . . .RII..T..AVA.. I..CVK.Q.IKNGRV..AFAP.NHAI.FID..DEW.E.I.GPSGQAKG.I V..CVR.L.RKNSKKIAAFVPMD.CL.FLA.NDEV.VA.L..-QGHAVG.I....FKWCVKGISLLAL

Ecoi

DRKQARSKYGVKRPKA N.G . . . . R..A.KE.K NMR.G . . . . .A.K...AK . .Q.G . . . . . . .K.. N . .N.... . . . .K..PK . .Q.G . . . . . . .K.. . .Q.G . . . . . . .KS. N.Q.G ..... A.K..K V.GRQEKVKR FKGKKEKR

+

Mlute b

Bstea Spiol Egra Ntab Mpoly Zmaize Mvan Tterm

+ +

R85S+P90 K87R P90L,Q G91D

+ t +

FIG.4. Some of the available amino acid sequences for S12. In addition to species represented in Fig. 3 there are Euglena gracialis (Egra), Micrococcus luteus (Mlute), Spirodela oligorhiza (Spiol), and Tetrahyrnena thermophilu (Tterm). S12 has basic clusters at both termini. A number of mutants have been characterized in E. coli for S12 (see Wittmann er al., 1980, for a review). Mutated residues are underlined and the nature of the changes is given on the right hand side.

and eukaryotic sequences are quite different from the eubacterial and chloroplast sequences. This is particularly true for the N-terminal basic tail where the general basic nature is maintained but with very poor sequence homology. Most of the eubacterial and chloroplast sequences are more than 10 residues shorter after the N-terminal tail region than the protein from an archaebacterium and a eukaryote. The central region of the molecule is very strictly maintained between the different species. The contacts between S12 and surrounding molecules are probably important and are therefore conserved. The mutations also occur in highly conserved positions. However, it is interesting to note that between positions 87 and 88 there are five residues inserted in both the archaebacterial and the eukaryotic sequences in the middle of the stretch where streptomycin-dependentmutations occur on both sides of the insertion. It is difficult to imagine that this region forms anything but an exposed loop on the surface. Such a loop can be extended by a number of residues at its apex without disturbing the conserved residues before and after the extension of the

122 :?v~G

Eco k,

Bst b Nva ii

a

ANDERS LILJAS I~~Ei(FAEKRKcl:TDSWEP~~QVGR~KEGT~S~~~?~:.~~i(GLPLLE AHXXQAGELQEKL: N R R N P N K L , , E i3'?J

.

.

PE:VDVLLPG .E.QV:,

+

+ +

+

+++

+ +

++

+

K T V K G G R I F S F T A L T W G D G N G R V G F G Y G K A R E V P A n l Q K E K A R R19G,S RLR.S . . ' I . . . . K..H....T...Q...E..R..I.D.KV20E R I 4 H . S . . RAR'IR.TV.. .NK.. Y . .V.M..SK.. GP . .R. . I A Q . K S 2 1 P

+

ECC Bsi M'J~

RNMINVALNNGT----------LOHPVKGVHTGSilVFM %.L.E.PIVGT.---------- 1P.E.I.HFGAGEIILK . . . . . . .V....PA L S S . i C . R V G C G S W E C G C G S P H S I P F T R . . T C G S V K . E L L . . P R V.LV . .NVAK . . .GL..VKDAWT

ECC

KAYGST-NP INVVRAT: DGLENMNSPEMVAAiKRGKS'V'EEILGK .SI..N-T...M....f...KQLKRA.D..KL...T...L..

t

BSt

!<"a

+

A

TT..3TRT"Y.FA~..F.A.!~.L.FVRCLPEQKA.LGLTECRVL

FIG.5. Amino acid sequences for S5. Merhanococcus vannielii (Mva) has an N-terminal extension. S5 has no cluster of more than 5 basic residues in a sequence of 10 residues. For E. coli (Eco) a number of mutants have been analyzed. The positions are underlined and the amino acid changes are described on the right-hand side (see Wittman cr a/., 1980, for a review). Bst, Bacillus sfcarothermophilus.

loop. It is a definite possibility that all the mutations in S12 that affect the cells' sensitivity to streptomycin are located in a small surface region on the protein. 2. Protein S5 As has already been mentioned, reversion from streptomycin dependence to independence can be arrived at by mutations in proteins S4 and S5. Unfortunately, only a few amino acid sequences are available for S5 (Fig. 5). In this case, it appears that parts of the N-terminal and central regions are very similar, whereas part of the central region and the C-terminal sequence shows less similarity. Again, an archaebacterial S5 has 10 extra residues in the central region. S5 has no obvious basic region. The mutations are again clustered and again most of them occur in positions with high homology. The connection between the SmD mutations in S 12 with the streptomycin independence mutations in S5 and also S4 (van Acken, 1975) may suggest that these modification are located in small region of the ribosome.

C. FACTORINTERACTIONS Several of the factors transiently interacting with the ribosome bind to partly overlapping sites (Fig. 2). Several proteins and regions of the rRNA have been found to be close to these factors by one-component deletion, cross-linking, or footprinting methods (see Table IV). As has been discussed above, most of the proteins affecting fidelity are at or near the factor binding site. Two of the other proteins which repeatedly are observed in factor interactions are L11 and L12. The factors that bind GTP go through a cycle of conformational changes (Fig. 6). Thus they bind in complex with GTP and, in the case of IF-2 and EF-

COMPARISONS OF RIBOSOMAL PROTEINS

123

aa tRNA Ribosome

EF-TS thiostrepton

+---'

7

Ribosome

Y

EF-TU

kirromycin

I

FIG. 6. A simplified scheme of the conformational states that several factors must go through (Liljas, 1990). The conformations are indicated in boxes. Interactions unique for EF-Tu are underlined and for EF-G hollow. Some antibiotics which are inhibiting at various stages in this cycle are indicated.

Tu, also in complex with a charged tRNA. This conformation could be called the GTP conformation. At some stage when bound to the ribosome they are induced to adopt a conformation that leads to GTP hydrolysis. Only in the case of EF-Tu is it known that the factor itself becomes a GTPase (Fasano and Parmeggiani, 1981). Due to the structural homology between the factors (Ovchinnikov el al., 1982; Sacerdot et al., 1984), we can assume that they all can become GTF'ases. This conformation could be called the GTPase conformation. The factors with GDP bound do not normally have affinity for the ribosome. This conformation could be called the GDP conformation. It is quite likely that the factors can have additional distinct conformational states. It has been observed that the interactions with the ribosome differ depending on the conformational state of the factor. This can be observed when the Tu and G factors remain bound to the ribosome in complexes with the antibiotics kirromycin and fusidic acid, respectively, despite the fact that the GTP molecules have been hydrolyzed (Gudkov and Bubunenko, 1989). This could be due to the conformational differences in the factor, in the ribosome, or both. 1. Protein L1I

L11 is one of the proteins that is lacking in some mutant ribosome (Dabbs, 1986). This gives the ribosomes partial resistance to thiostrepton (see Cundliffe,

124

ANDERS LILJAS

TABLE IV FACTOR-PROTEIN INTERACTIONS“ Protein

No addition IF-2

s4

s12 S18

s19 L6 L7R. I2

ND’

ND‘

(X

x X’

EF-Tu. GMPPCP

EF-Tu. kir

)

ND‘

ND’

ND’.Rh.A

D‘

D‘ ND’

NDJ D‘

L11

L14 L19 L27

YD‘ D’

EF-G. GMPPCP

X” X“ D‘ x’

X”

D’, Xd‘, Rh,A’ X”. R‘ X” NDJ

EF-G-GDP. fus

RF-I

RF-2

X*

D’ X‘ NDI, NX1

XP

X‘, AJ Ri, Mi

X*, AJ X y . R’. MI

Xd

NDf

”A. Inhibition of binding by antibodies: D. digested ND, not digested with trypsin; M, factor activity affected by chemical modification; R. factor activity affected by removal: X, cross-linked; NX, not cross-linked. ”Maassen and Moller ( 198I ). ‘Boileau e t a / (1983). “Traut rr a / . ( 1986). ‘Skold (1982). ’Gudkov and Bubunenko (1989). 5toffler cf a/. (1982). hHamelera/. (1972). ‘Nag r f 01. (1987). ‘Tate cr a/. (1989). ‘Schrier and Moller (1975). !Tate el a/. (1986).

1986 for a review). Thiostrepton is not bound to the protein, but L11 induces a conformation in a region of the 2 3 s RNA that enables it to bind the antibiotic. Ribosomes from L1 1-deficient mutants are unable to support uncoupled EF-G GTPase activity and stringent factor production of “magic spot,” (p)ppGpp. Thus, even though the bacterium can manage without L11, some of the ribosomal activities are lost. The amino acid sequence of L 11 from several organisms has been determined, including an extremely halophilic archaebacterium (Fig. 7). Again the sequences are highly homologous, to the extent that there is only one small deletion in one species. L11 binds to the same part of the 23s RNA as the L8 complex (see below) and is situated together with L10 and L6 at the base of the L12 stalk (Fig. 2). Even though there are insufficient data for detailed structural and functional arguments, one part of L11 is unusual and deserves comment. The sequence between residues 18 and 3 1 (E. coli numbering) has prolines (Ramirez el ul., 1989) or glycines every third residue with additional prolines and glycines in the other positions. This sequence resembles the sequences which form collagen, or rather, the polyproline structure. Such conformations have been found in globular proteins (Adzhubei et al., 1987), for example, the avian pancreatic

125

COMPARISONS OF RIBOSOMAL PROTEINS 1 Eco ( b )

Pvu Sma ( b ) Bst

+

10

40

+

50

60

+

.........

............................................................

MAETIEVL..G.Q.D.G..L..E..PTP.DVQAWQEI.DQ.EAFD-.TEV..T.E. pgG PgPP GP Gp G t

61

70

+

o

+

+

+

80

++

t

+

+

+

90

100

+ + + + +

110

+

+t

120

YAD-RSFTFVTKTPPAAVLLKKAAGIKSGSGKPNKDKVGKISRAQ-----LQ I..-...... . . . . . . . . . . . . . . . V E. . . . . TS..-----VR .S.- .................................... VT...----- V R . . . E... . . . . .S. FE.-. . . . .I.. E..RN..AT.T.DK-----VR...EL.MP.LNA.S DSSTKKYDIKVGV D.AHK.I.NLDLLE----QIAD..IK.KPQLSAKT ED .--.. SIEVGV .. T.A.V.DE FDT . . .E.QEHF.ADL.IE.-----.KT..EQ.KP.LLAV.

.........

.

..

121 Eco Pvu

+

AKKVQAYVKLQVAAGMANPSPPVGPALGQQGVNIMEFCKAFNAKTDSIEKGLPIPVVITV

sso HCU ( a )

Eco Pvu Sma Bst sso Hcu

30

20

+

130

+

140

150

160

IEAMTRSIEGTARSMGLWED

Sma

Bst

sso Hcu

KDPKDVIKEIDQGKYNDLLTNYEQKWNEAEG ARNAAKEVA..CA.L.VTI.GEDAATFNERVDDGDYDDVLGDELAAA

FIG.7. Available amino acid sequences for L11, which has no basic cluster. L11 sequences from Escherichia coli (Eco), Proteus vulgaris (Pvu), Serratia marcescens (Sma), Bacillus stearothermophilus (Bst), Sulfolobus solfataricus (Sso), and Halobacterium cutirubrum (Hcu) are shown. The N-terminal regions with sequence similarity to polyproline helix-forming residues are indicated. Prolines and glycines are particularly important for this conformation. Every third residue is marked since this is facing inward and should preferably be a proline.

polypeptide (Glover et al., 1982). The relation of this possible polyproline structure to the observation of two domains in L11, one of which is easily digestible and the other stable, is not established (Gin et al., 1978).

2 . The L8 Complex The stalk feature of 50s subunits is formed by the protein L12, in E. coli L7/L12 (Strycharz et al., 1978; Radermacher et al., 1987). This is the only component of the ribosome that is found in more than one copy (Hardy, 1975; Subramanian, 1975). It is bound to protein L10 as two dimers (for a review of the earlier work, see Liljas, 1982). The pentameric complex of L12 and L10 has been observed in several systems as a separate spot in two-dimensional gel electrophoresis and in E. coli it was named L8 (Pettersson et al., 1976). This complex has often been called the L8 complex. Similar complexes have also been observed in eukaryotic ribosomes (Rich and Steitz, 1987; Uchiumi et al., 1987). a. Eubacterial L8. The structure of the L7/L12 dimer from E. coli is unusually elongated as observed both by scattering methods (Osterberg et al., 1976; Serdyuk et al., 1980) and electron microscopy (Strychartz et al., 1978; Marquis

126

ANDERS LIUAS

et al., 1981; Tokimatsu et al., 1981; Moller et al., 1983). The protein is com-

posed of two domains, the N-terminal domain, which is known to be essential for dimer formation (Gudkov and Behlke, 1978) and binding to L10 (van Agthoven et al., 1975; Koteliansky et al., 1978), and the C-terminal domain (residues 51-120). the crystal structure of which is known (Leijonmarck and Liljas, 1987) and which is probably the functional region that interacts with the factors (van Agthoven et al., 1975; Koteliansky et al., 1978). These regions are connected by a flexible hinge (residues 37-50) as observed by 'H-NMR methods (Bushuev et al., 1989). Whereas the available sequences show extensive similarity for the N-terminal and C-terminal domains, the hinge region, despite the fact that it is composed mainly of five types of residues, is impossible to align in a convincing manner, partly due to the variation in length. Furthermore, the N-terminal part of the hinge is rich in glycyl, alanyl, and prolyl residues and the C-terminal part of the hinge has a high content of glutamates. Highly flexible linker regions with a similar composition have been observed in several multienzyme complexes (Perham et al., 1981; Fussey et al., 1988). The dimers of L12 are bound through their N-terminal domains to the C-terminal region of L10, which is bound through its N-terminal region to the 23s RNA (Gudkov et al., 1980).The binding is i n the region 1028-1127 to which L11 also binds (Beauclerk et al., 1984; Egebjerg et al., 1989). Several conflicting models for the arrangements of the proteins have been proposed (see Liljas, 1982 for a review; Georgalis et al., 1989). b. Archuebacterial and Eukaryotic Proteins Corresponding to L12 and

LIO. Acidic proteins from the large ribosomal subunit have been found in both

archaebacteria and eukaryotes (see Wool, 1979, and Matheson et al., 1980, for reviews of the early work). The molecular weights and the amino acid compositions resemble those of L 12 from eubacteria. The archaebacterial and eukaryotic proteins are clearly homologous to each other (Fig. 8), but neither shows anything but very partial similarity to eubacterial L12 (Liljas et al., 1986). These proteins are unquestionably related to the eubacterial protein since, among other things, they can be extracted in the same way with high salt and ethanol (Moller et al., 1975 ), several copies bind to the ribosomes (van Agthoven et al., 1978; Vidales et al., 1984), and they are essential for the proper function of the elongation factors and can, in some systems, reconstitute active ribosomes when eukaryotic acidic proteins are added to E . coli core particles that lack L7/L12 (Stoffler et al., 1974; Moller et al., 1975; Zinker and Warner, 1976). This is one example in which comparing amino acid sequences is insufficient to reveal a similarity in function. This can be true for other ribosomal proteins as well. In archaebacteria there is only one form of L12. However, in eukaryotic species there seem to be two main types, I and I1 or P1 and P2 (Tsurugi et al., 1978; van Agthoven e f al., 1978; Rich and Steitz, 1987; Mitsui and Tsurugi,

127

COMPARISONS OF RIBOSOMAL PROTEINS 1

20

10

30

40

50

60

Mva Mte Hcu Hha Sso

(a) (a) (a) (a) (a)

MEYIYAALLL-NSANKEVTEEAVKAVLVAGGIEANDAR-VALEGV-DIAEAIAKA-----AIAPV

Spo Sce Sce Dro Asa Rat Man Wge

L40 L44 L45 A1 L12 P2 p2 A

MKYLAAYLLLTVGGKDSPSASDIESVLSTVGIEAESER-IETLINELNGK-DIDELIAAGNEKL-ATVPT ..........NAA T.D.TK.KAI.ES. . . . 1.D.K-VSSVLSA.E..-SV... .TE. . . . . - .AVPA ..........VQ NAA A..KA.VAS..A.VDEA.-.NE.LSS.E..GSLE.I..E.QK.F-..... . . .V AVL . . . . . .AN L.KI S..V.VDA..-LTKV.K..A..-S..D..KE.R...S.S.. A V AALS.NAD TA . . .KI ..S....CNPSQ-LQK....K..-.LEA...E.QT..-.SM.. . . .V.S . . .AAL NSN . . .K..AKI.DS.....DD..KLNKV.S.....-N.EDV..G.VG..-GSGV . . .V.S AAL . .NS . . . .K KKI.DS . . . . .DDD .- LNKV.S N.EDV..Q.IG..-.S.. A LI . . . . .AYL . .NS . . . .A.VKDI.NA..A..NE.K-L.F.LA..K..????????????????S.GS

Sce Sce Asa Dro Man

L44'MSDS---IISFAAFIL-ADAGLEITSDNLLTITKAAGANVDNV-ADVYAKALEGK-DLKEILSGFH----NAGPV A1 ..TESAL--.Y . . L..-..SEI..S.EK...L.N..NVP.E.I.-..IF....DGQ-N..DL.VNFS----AGAAA L12'XASKDELACVY. . L L.DDVD . .TEKV N . . L R . . .VS.EPY .-PGLFT.....L-...SMITN------VGSG. p21 . .TKAELASVY.SL V.DDVAV.GEKIN. . L NVE.EPY.-PGLF.....AI-NV.DLITN------ISM.. pl .ASVSELACIYS.L..-H.DEVTV.E.KINALI....V..EPF.-PGLF....ANV-NIGSLICN------VGAGG

. . . . . . .M..-GTTG . . IN..N..S..E.A.A.VD ........................... . . .V . . . . I.-.E.DE.L..DNITG..E.A.VDVEES.-A........D.-..E..VEE.AAAP-.A.. A . . .V. . . . I.-.E.DE.L. .DNITG . .E.A.VDVEES.-A........D.-..E..VEE.AAAP-.A.. A

. . . . . . S...-HA.K..IS..NI.N..S.A..TVDEV.-L..VA...KE.-N.D.ILKT.-----TAM..

..

..... . . . ..... ... ..

..

... .. .. ..

..

...

Mva (a) HCU (a) Hha (a) Sso (a)

70 80 90 100 110 AAAAPVAAAA?+PA-----------EVKKEEKKEDTTAAAAAGLGALFM .SGSDDE...DDGDDDEEADADEAAEAEDAGDD.DEEPSGE...D..G .SGSDDE...DDGDDDEEHDADEAAEAEDAGDD.DEEPSGE...D..G V.AP.GQQTQQAA-----EKKE.K.E GPSEEEIGG..SS..G

..

....

Spo Sce Sce Dro Asa Rat Man Wge

L40 L44 L45 A1 L12 P2 p2 A

GGAASAAPAAAAGGAAPAA----EEAAKEEAK-EEEESDEDMGFGLFD A.P...GG . . . . S.........DAAAEE.KEEEAA ............. S...AG..GAA.GG------DAAEE.KEEEAK....D...... .. . .GGAV.A.D..PA..AGG--DKK. .K...K.E.S.SE.D. . . .A..E ...PA..AGG..AAP.A----EAK . .K. ..K.E.S..E... . . . . . . . . . .VAVSA.PG . A A P . AGSAPAAA-----.E.E.S..KKDE.. . . . . . . . .VAVSA.PGSAAP.AGSAPAAA.EK.D.K.E.S . . . . D . . . . . . . . . .GGAP.A. . . .PA.GG..AA------E.KKEEAK..K.?????????

Sce Sce Asa Dro Man

L44' A1 L12' p21

AGAGAASGAAAAGG--------DAAAEEEKEEEAAEESDDDMGFGLFD PAGV.GGV.GG---------EAGE.EA.KE . . . .K . . . . . . . . . . . . . GA.P..G. . . . .T-----EAPAAKEEKK.EKK.ES..E.E . . . . . . . . GA.P.GGA.P..AAAAPAA---ESKK K.K . . ESDQ . . . . . . . . . . . PAPA.GAAP.GGPAPSTAAAPA-EEKKV.AKK.ES . . . . . . . . . . . . .

pl

...

..

FIG.8. Some amino acid sequences for eukaryotic and archaebacterial (a) L12. The sequences are arranged in three groups, one for archaebacteria and one for each of the two forms found for eukaryotes I and II or P1 and P2. P2 is the middle group. As expected there are no basic clusters in this acidic protein. Species listed are Methanococcus vannielii (Mva), Methanobacterium thermoautotropicum (Mte), Halobacterium cutirubrum (Hcu), Halobacterium halobium (Hha), Sulfolobus solfataricus (Sso), Saccharomyces pombe (Spo), Saccharomyces cervisiae (Sce), Drosophila melanogaster (Dro),Artemia salina (Asa), and wheatgerm (Wge).

1988b,c), of which there sometimes can be several forms. In Saccharomyces cerevisiae there are four genes for L12, two of each type (Ramirez et al., 1989). However, only three forms have been observed in the ribosomes (Remacha et a1.,1988). For mammals and Artemia salina two forms have been found, one each of type I and I1 (Tsurugi et al., 1978; Rich and Steitz, 1987; Maassen et al., 1985).

128

ANDERS LLJAS

It was also shown that these proteins can be phosphorylated (Zinker and Warner, 1976; Reyes et al., 1977; van Agthoven et a1.,1977; Horak and Schiffmann, 1977). The proteins are monophosphorylated and can also be observed in the cytoplasm, in which case they are not phosphorylated (Zinker, 1980; Shchez-Madrid et al., 1981). The phosphorylation seems to control the affinity for the ribosomes (Vidales et al., 1984). The region of the archaebacterial and eukaryotic L12 (aL12 and eL12, respectively) which bears any sequence resemblance with eubacterial L12 (bL12) is a region with a high content of Ala, Gly, Val, and Pro followed by a region with a high content of Glu (see review by Matheson et al., 1980). This could be a'hinge region just as in the case of bL12 (Liljas et al., 1986). Rat liver ribosomes or 60s subunits display some domains with high flexibility just like L12 in E. coli (Cowgill et al., 1984). Thus, one may assume that aL12 and eL12 also have high flexibility due to a hinge region of 15-20 residues (Liljas et al., 1986). In E. coli L12 the hinge is located between residues 36 and 51. However, in aL12 and eL12 the hinge region is located approximately between residues 70 and 90, which makes the N- and C-terminal structures very different from bL12 (Liljas et al., 1986). The N-termini of aL12 and eL12 have amino acid sequences which seem normal for globular domains and are of the same size as the C-terminal domain in bL12. The C-terminal regions of aL12 and eL12 seem exceedingly small to form any significant domain structure. At present it is not known which part of the eukaryotic and archaebacterial L12s is involved in dimer formation and which part binds to the L10 corresponding molecule. However, Ballesta and co-workers (private communication) have found that removal of the C-terminal region did not prevent the binding of eL12 to the ribosome. The protein in archaebacteria and eukaryotes (PO or aLlO and eL10) that corresponds to L10 in E . coli is distinctly larger than L10 (Elkon et al., 1986; Uchiumi et al., 1987; Rich and Steitz, 1987; Mitsui and Tsurugi, 1988a; Shimmin and Dennis, 1989; Shimmin et al., 1989). PO comigrates with P1 and P2 as a complex with an apparent molecular weight of 140,000 (Elkon et al., 1986). The N-terminal region of PO is homologous with bLlO (Shimmin and Dennis, 1989). One remarkable observation is that monoclonal antibodies or systemic lupus erythematosus antibodies react with the C-termini of PO as well as with P1 and P2. The conclusion that the C-terminal region of PO (eL10 or aL10) is highly homologous or idential to P1 and P2 from the same species (Elkon et al., 1986) has been confirmed by sequence analysis (Rich and Steitz, 1987; Kopke and Wittmann-Liebold, 1989; Ramirez et al., 1989). In Sulfolohus solfataricus the identical regions contain 32 residues (Shimmin et al., 1989). If this region in aL12 and cL12 is considered to be a hinge, PO, with an almost identical sequence, must also have a hinge. The organization and function in the ribosome of a fifth nearly identical flexible polypeptide in addition to the four copies of L12 remain one unsolved problem.

COMPARISONS OF RIBOSOMAL PROTEINS

129

The organization of the molecules corresponding to L12 and L10 in archaebacterial or eukaryotic ribosomes also shows a high degree of homology to the eubacterial arrangement. First, the number of L12-related molecules in the ribosomes seems to be close to four, probably arranged as two homodimers in the case in which there are two types of L12, such as P1 and P2 (van Agthoven et al., 1978; Uchiumi et al., 1987). When there are two forms of L12, both seem to be present in the same ribosome, as judging from cross-linking data where trimers of the type Pl-PO-P2 have been observed (Uchiumi et al., 1987). However, it is not known whether eukaryotic ribosomes are homogeneous with regard to the stoichiometry of P1 and P2 and whether the two dimers have different tasks. Furthermore, the distribution and functional difference of the two different forms of P2, LA4 and L45, as found in S . cerevisiae remains unknown (Remacha et al., 1988). c. Structural and Functional Aspects of the L8 Complex. The classical picture of the two L12 dimers in the stalk protuberance of the 50s subunit has been challenged. From electron microscopic and fluorescence studies of the structure of L7/L12 Moller and co-workers have concluded that only one dimer is located in the stalk and the other one is located in the body or in the central proturberance of the 50s subunit (Moller et al., 1983; Thielen et al., 1984). This model has gained further support by using monoclonal antibodies directed against epitopes in the N- and C-terminal regions (Olson et al., 1986). Here it was observed that the Nterminal region of at least one dimer is located at the base of the stalk. The C-termind regions are located not only at the tip of the stalk but also in the body of the subunit not far from the base of the stalk. Whether this is a static arrangement or whether the two dimers alternate in these two positions is not clear. It is, however, clear from investigations using 'H-NMR that L7/L12 in the ribosome possesses an unusual flexibility (Cowgill et al., 1984). This flexibility must be due to the hinge region in the molecule (Bushuev et al., 1989) and may at least concern the dimer in the stalk. This flexibility is greatly reduced by the binding of EF-G in complex with GMPPNP (Bushuev and Gudkov, 1988). Several other observations are indicative of conformational changes in the ribosome due to factor interactions. Thus, Traut et al. (1986) have observed that the cross-linking between L7/L12 and EF-G differs depending on how the factor is bound. When EF-G is bound in complex with GMPPNP (probably like the state before GTP hydrolysis) there is a cross-link, but if the factor is bound in complex with fusidic acid there is no cross-link. In the latter case the GTP hydrolysis has taken place but the factor is unable to dissociate from the ribosome due to the antibiotic. The conformational change in the factor has probably led to a coordinated change in L7/L12. A series of elegant experiments using mild proteolysis on factors and ribosomes in different complexes gives evidences about coordinate conformational changes in the factors and L7/L12 (Gudkov and Gongadze, 1984; Gudkov and Bubunenko,

130

ANDERS LILJAS

1989). Thus, L7/L12 is resistant against trypsin in ribosomes without bound factors. When EF-GoGMPPNP binds, L7/L12 becomes susceptible to trypsin, but if the factor is bound with fusidic acid the protein is again inaccessible to trypsin. The opposite situation was found when EF-Tu was studied. The factor was bound with poly(U) and Phe-tRNAPheeither using GMPPNP or the antibiotic kirromycin. In the former case, L7/L,12 was inaccessible to trypsin, but it was rapidly degraded in the latter case. Thus, the conformational state of L7/L12 is not only sensitive to whether the GTP bound to the factor has been hydrolyzed but also to which factor is bound. It remains to be seen how the different factors interact with the two dimers of L12 to induce the conformational changes from trypsin sensitivity to trypsin resistance and how this is related to the factor-catalyzed steps in protein synthesis. It also remains to be seen whether the acidic protein L12 has any relation to the tRNA molecules in their different binding sites (Moller and Maassen, 1986; Moller, 1990; Moazed and Noller, 1989).

VI. Concluding Remarks The ribosome remains a great challenge for structural studies. In the current period of strong interest in the structural and functional roles of the rRNA the ribosomal proteins should not be neglected. Several of them have been found with highly conserved regions in all species investigated. Several of the proteins are also known to be closely associated with different functions. One important method for the exploration of structural and functional relationships is in v i m mutagenesis. Three-dimensional structural information is of great help in the application of this technique. Such information is still very limited for ribosomal proteins. Without the structural information there is nevertheless some amount of general structural knowledge which can be applied to ribosomal proteins, particularly in the cases where many homologous sequences can be compared and where the positions of functional mutants are known. There is already a wealth of such information available for the design of site-directed mutagenesis experiments and for the analysis of the structural and functional relationships of the ribosomal proteins. This article has primarily focused interest on proteins involved in factor relations. These include not only the proteins which are known to form the factor binding site, but the proteins influencing the fidelity of protein synthesis which are also found to be closely related to factor interactions. ACKNOWLEDGMENTS I am very grateful to Prof. B. Wittmann-Liebold and Dr. A. Kopke for access to unpublished material and compilations of amino acid sequence information and to Dr. M. Ehrenberg for valuable discussions. I would like to thank Dr. S. Bloomer for linguistic corrections and Mrs. G. SokjerPetersen for excellent secreterial help.

COMPARISONS OF RIBOSOMAL PROTEINS

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