Organization and structure of the Methanococcus transcriptional unit homologous to the Escherichia coli “spectinomycin operon”

.J. Mol.

Riol.

(1989) 209, 21-36

Organization and Structure of the Methanococcus Transcriptional Unit Homologous to the Escherichia coli “Spectinomycin Operon” Implications

for the Evolutionary Relationship of 70 S and 80 S Ribosomes

Johannes Auer, Gabriele Spicker and August B&k Maria-

Lehrstuhl fiir Mikrobiologie Ward-StraJe la, D-8000 (Received

13 March

der L~niuersitiit Miinchen 19, F.R.G. 1989)

Hy means of an immunological approach and a subsequent chromosome-walking strategy a chromosomal region encoding ribosomal proteins in the archaebacterium Methanococcus vannielii was cloned. The determination of the nucleotide sequence of the 7.8 x 103 hasr DNA fragment revealed the existence of 14 putative ribosomal protein genes and two unidentified open reading frames. They are organized in a transcriptional unit that is very similar to the Escherichia coli “spectinomycin operon” in respect of both gene composition and gene order. The Methanococcus transcriptional unit contains, in addition to those genes whose products have a homologue in the E. coli operon, three genes whose products share sequence similarity with eukaryotic 80 S but not with eubacterial ribosomal proteins. The Methan,ococcus ribosomal proteins almost exclusively exhibit a higher sequence similarity to eukaryotic 80 S ribosomal proteins than to those of eubacteria and many of them have a size intermediate between those of their eukaryotic and eubacterial homologues. These results are discussed in terms of a hypothesis that implies that the recent eubacterial ribosomr developed by a “minimization” process from a more complex organelle and that the archaebacterial ribosome has maint’ained features of this ancestor.

1. Introduction Although they catalyse the same overall biochemical reaction, ribosomes from prokaryotes and from the cytoplasm of eukaryotes display profound structural differences. Eukaryotes have a higher number of ribosomal components whose sizes, in addition, are on average larger (Wool, 1979). The difference also extends to the organization of the relevant genes. In eubacteria, most of the genes for ribosomal components are organized in transcriptional units (Nomura, 1986), whereas in eukaryotes they are, almost exclusively, located singly and distributed on different chromosomes (Planta et al., 19X6).

The first experimental evidence for a specific relationship between the two translational systems came from the comparison of the alanine-rich, acidic ribosomal “A”-protein (Matheson et al., 1980). With the completion of the determination of sequences of all the Escherichia COli ribosomal proteins (Wittmann-Liebold, 1986) and the constantly increasing collection of sequences for eukaryotic

ribosomal proteins (Planta et al., 1986; Wool, 1986), it would now appear that components of both types of ribosomes share significant similarities. This relationship then raised the question of whether the evolutionary process led from a eubacterial (70 S-like) ancestral ribosome to t.he recent 80 S system through the acquisition of new proteins and/or by gene duplication or fusion events. or whether the recent 70 S eubacterial ribosome developed by a reduction process from a more complex, 80 S-like ancestor (Wool, 1986). Similarly. the size differences between 70 S and 80 S components could have arisen by deletion. or vice versa. by fusion or insertion events. A reduction process, for example, has been post,ulated to lead to the elimination of segments of ribosomal RILA to generate the prokaryotic 16 S and 23 S rR?rTAs from larger ancestor molecules (Clark, 1987). As indicated first by the work of Matheson and co-workers (1980), sequence analysis of proteins from archaebacterial ribosomes can provide new information t,hat brings us closer to answering t,his basic

2%

J. ,~z(,PTet al.

biological question. Meanwhile, a number of additional ribosomal protein sequences from archaebacteria have been determined and their comparison with sequences from eubacterial and eukaryotic ribosomes has revealed either a higher. or in some oases exclusive, similarity with a eukaryotic homologue (Kimura et al., 1987; Kimura & Kimura, 1987: Strobe1 et al., 1988; Matheson et al., 1988; Kiipke & Wittmann-Liebold, 1988: Lechner et n6., 1989: Hatakevama & Kimura, 1988; Itoh? 1988; I’. Den&s; A. T. Matheson; A. Mankin, personal communications). As far as the available data can indicate. the relevant genes appear to be organized in transcriptional units that resemble those from eubacteria (Itoh, 1988; Lechner it al.. 1988, 1989: I’. Dennis; A. Mankin; A. ‘I’. Matheson, personal cxommunications). Tn this paper we present the analysis of t,hr ribosomal protein operon from ikfethanococcus mxnn~rlii that is homologous to the “spectinomycin operon” on the E. coli chromosome (Nomura. 1986). We summarize conclusions that can be drawn about the evolution of t’he ribosome in general.

2. Materials and Methods (a) Organims,

phages

and

plasmids

The bacteria. plasmids and phages used in this work are listed in Table 1 together with their characteristics. E. coli strains were cultured in LB medium (Miller, 1972): when required for the mainteinance of plasmids, ampicillin was added to 100 pg/ml. Methanococcus was grown under the

conditions given by Douglas it (I/. (b) f’onstruction

of E.

(1980).

cdi strain

JFMl

Strain JFM 1. which is a rrcR(’ derivative of strain Y1088. was required for the propagation of recombinant i (‘haron -CA phages. d C’haron 4A is one of the series ol lambda substitution vectors with a packaging capacity of 8 to 20 kbt of foreign DNA. Removal of the “stuffer” fragments of 1. Charon 4A results in a SspiCphenotype for the recombinant phages (Maniatis d al.. 1982: Kaiser 8r Murray. 1985). They are lacking the normal features of i. phages (red. gam) that would enable them to inhibit the host exonuclease \‘. with the result that they can replicate only slowly 1,ia “theta” structures. The supF and recR(’ genotype of ,JFMl enables recombinant i, C’haron 4A phages to replicate efficiently C/I the rolling-circle mode. JFMl was constructed by first isolating a thyA mutant of YlO88 using the trimethoprim selection strategy (Miller. 1972). A Y1088 thyA mutant was then transduced to thymine prototrophy with phage PI propagated on E. coli *JC7623 (recHC). Thy+ transdurtants were tested for the acquisition of the recB(’ lesions by screening for srnsitivity t,o u.v. light (;Maniatis et al.. 1982). (c) Preparation

of phage

1ysate.s

Extracts for in L&O packaging of recombinant 1 phages were purchased from Borhringer-Mannheim GmbH. The t Abbreviations used: kb. IO3 bases or base-pairs: u.v.. ultraviolet,; TPTG. isoI)ropYI-8-thiogalac,to-i,pyranoside: RF. replicative form: bp. base-pairs; ORF. open reading frame.

reaction was performefl as rrc~oi~~mf~iidrfl II\. the> marrufac,turers. Single-plaqur and plate Iysates \vertL preparf~fl its descsribed by Maniatis et nl. (1982). Lysates in the 1 I silts were obtained according to the protoc+ol of Silhavy p/ r/l. (1984). For preparation of total protein from plate lysates, the Iysate was treated for I h \vitlr IISi~st~ I (I ,ug/ml) and RXase A (IO pg/ml) at 37 ‘C’. follourtl I)\ lyophilization. Tnduction of transcription from tlrr Inr,% promoter on i, (‘haron 16 was achieved by adding i~ol~r~~J)YI-P-t,hiogalac,to-I)-J)4-ra.nositlr (IPTG) to the aoff ;~,~!;tt overlay to give a final concentration of I in\1 (II’ 11). pretreatment of nitrof~ellulose filters bait h ii IO ml1 XOIW tion of TPT(: (Young & I)avis. 19%).

(‘hron~oson~al l)SAI f’rotn .Wrt//rrlcoc,r,cc,rc.sculls u AS prepared according to the prodrrrr clesc*rihrtl hi .Jarsc.h rt al. (1983). Small-nfsalr preparations of plasmtfl 1)S.A and of phagr 3113 R,F 1)N.A were carried out 1)~ thcl m&hod of Birnboim & I)oly (I!)TS). For use in sequencing. revombinatit plasmids vf~rf~ isolatt~tl 1)~ following the protocol of (‘hen 81 Seehurg (1985). T,argc’scale preparation of plasmid 1)S.A \VW carried out H itlr method drscribrfl t)y (‘lf~well S: the .‘cleared lysatr” Helinski (1969) and single-stranded 11I3 1)X.\ ~.as prepared hy pre+tation wit)h polyethyk~nr g1,1co1 anti extractions with phenol. as described I)y Messing ( I!W). l)SA preparat,ion from lambda phagr partic~les \vas tlot~cj according to t,hr method of Silhavy rf (11. (1984) risirly “STEP” t)uffrr and 1 rxtrachons \vith ~)hr~nolfor I)tlriti(,ation. I)XA fragments styarattld on 10~ -milting i\gat’“st gf*Is were cut out of the grl and isolatrtl 1)~ th(l methocl (II’ Maniatis of ul. (1982). For isolatiotl of 1)X;;\ t’ragmrtlts from conventional agarose. the procedure drac*ribrd I)>, Vogelstein & Gillespie ( 197!)) was followrd. Tot#al RSA from .13uthrcnococ~/,.~ culls harvrstcstl it] t IIV exponential growth phase was ~)repared bvith thrl “hot phenol” mrthod given by Aiba rt rrl. (l!+Xl). RX.\ \V:IS used immediately or kept frozen at - 70”(‘.

Standard techniques likr degradation of’ I)SX u ith restriction rnzymrs. separation of 1)S.A fragments oti agarose gels. ligation of l)SA% fragmrnts with bactrriophage T4 l)Eu‘;z lipasr. dephosphor~latiort of I)Sh ends using calf intestinal phosphatasr. radioac*tive labelling ot the 3’ and .5’ ends of DNA fragments bvith 11X.4 pal>.merasr 1 (Klenow fragment) and T1 l)ol?;nu~leotitIr kinasr. respectiveI>,: mrthylation of I)iYX fragments with EcoRI methylase and nic~k-translation of I)XA fragments using J1iY.A polymerasr 1 were performed as drscrihrd I)\ Maniatis rt al. (1982). For I>X,4/DNA hybridization analysis 1)X;\ fragments from agarose gels wpre transferrrfl to nitrf~cellulf~st~ membranes according to the m&hod of Southern ( 1975). Hybridization with nick-translated probes was carried out at 65°C’ and in the presence of skimmed milk as described by ,Johnson pt al. (1984). Two approaches were followed for thr c*onstruc.t ioll ot Nethanococws genomic libraries. First. c,hromosomal DK;A of Methanococcrcs was &arrd with E’coRl and thr resulting DSiZ fragments were separated on an qarose gel. Fragments with a size of 2 to 3 kb were isolattld from the gel and ligated into i ( ‘haron 16. In ~ifro parkaging of’ the ligation mixture and infection of‘ E.di \‘lOXX

Ecolutiona,ry

Relationship

between

70 S and

2.3

X0 S Kibosvmes

Table 1 List of organisms

and pksmids

used in this study Source or reference

Organisms

DSB1 1221 Kushner rt al. (1971)

This work Yanish-Perron

rt cd. (l!M)

Young 8r Davis (1983)

Yanish-Perron rf 01. (19%) Yanish-Perron rt al. (198.5) LViIliams & Ulattner (1979)

\Villiams

& Blattner

(1979)

Soberon rf al. (19X0)

rrsult.ed in an amplified lysat,e of 1 x 10” recombinant phagesjml. This lpsate was used to infect E. coli YlO90 and to perform the plaque-immunoscreening experiments. The second method involved partial cleavage of chromosomal DNA from Methnnococcus with either AZuI or Ha~III. The DNA fragments were separated on an agarose gel and fragments with a size of 8 to 20 kb were isolat,ed. The Als1~1and HapI fragments were then mixed EcoRI linker and treated with EcoRI methylase. moleeulr~s (IOmer) were then attached to the methylated fragments and they were ligated into vector 1 Charon 4A. Since i. Charon 4A is a substitution vector the 2 EcoRI “stuffer” fragments had to be removed before cloning (JIaniatis ef al.. 1982). The ligation mixture was packaged and a lysate with 4500 recombinant phage clones was obtained. This Iysate was amplified by preparation of a plate lvsat,e after infection of E. coli JFMl. The amplified genomicz library in d Charon 4A (1 x 10’ plaque forming u&s/ml) was used for plaque-immunoscreening.

(f) Imn~~~nologicnl

techniques

(‘rude antisera directed against ribosomal proteins from the small and the large ribosomal subunit of Nethanococcus were gifts from G. Schmid, Miinchen. The fractionation of immunoglobulins using affinity chromatograph?; and the immunoblotting procedure including SDS/polyacrylamide gel electrophoresis were performed as described by Schmid & R&k (1984). For the plaque

immunosscreening, the prot.ocol of \~ounp C!L I)avis (1983) was followed. Typically. 500 plaque-forming units per plat,e were used. (g) Xorthrrn

blotting

nnrrl,~sis

Total RXA from Xrthanocowus cells. prepared as described above. was separated on I”,, agarose gels cbontaining formaldehyde. transferred to “GeneScreen” membranes (KEh-, DuPont) wit,h the capillary blot proc.rdure and hJ,bridized with nick-translated I)NA probes at 42°C in the presence of dextran sulphate as recommended by the manufact,urrrs. (h) Sucleuse

S, protection

and primrr

r.rtension

analysis

Surlease S, prot,ection experiments were caarried out according to Berk & Sharp (197i) with the modifications described by With rt al. (1986). For primer ext.ension analysis the 2 oliponucleotides ;5’ (‘AAA(‘(‘TTCTTTTAGTCACG 3’ and -5’ rlBGTT(I(‘(:T(:(:AXT(:(:A(‘A(: 3’. which are c~omplementary to t,he non-coding I)h’A st,rand at positions 174 to 196 and 983 t,o 1002. resprc+vely (see Fig. 2). were radioactively labelled at t,heir 5’ ends. About IO ng of the labelled oligonucleotides were hybridized to 5. 10 and 20 pg. respectively. of total RSA from Xethanococcux. Primer extension reactions were performed with avian myeloblastosis virus (AMV) reverse transcriptaw (Boehringer-Mannheim GmbH) as described by Sawers & Kick ( 1!189).

24

J. Auer et al. (i) Sequencing

and computer-awisted

analyses

B’j,. X’!, or -“0~ z” polyacrylamide gels containing 7 .M-urea M-IIWB). or in salt-gradient gels (69; polyacrylamide/H Computer-assisted analyses of sequence data were done with the aid of an Apple IT microcomputer (V(‘SD operation system) using the program package “Sequrmr analysis version 3.0”. designed by R. Larson, S. Mrxt,atl<~ and ?J. Messing (Department of Riochemistry. Cnivrrsity of Minnesota). Searching in the data base MIPSX of the Max-Planck-Institut) fiir Riorhemie. Martinsried. was done on a Mirrovax A computer using the algorithm of Lipman B Pearson (1985) wit,h a k-tuple of 2. (‘omparison of amino acid sequences was done on the same comput,er with the help of the UWGCG program package (Devereux et al., 1984). The comparisons with Jnogram “GAP” were performed using a gap weight of 26 and a length weight) of 0.1.

Roth the chemical cleavage and the chain termination methods were employed to determine the DSA sequence of the complete region. Sequencing reactions of the chemicaal cleavage method were carried out as int,roduced by Maxam & Gilbert (1980). with the exception of the mod;tied A+(: react,ion described bv Gray ef a,l. (1978). (Chain t)ermination reactions using D$A polymerase I (Klenou fragment) were performed according to Banger rt al. (1977). When double-stranded plasmid DKA was used as template the protocol was modified as recommended by (‘hen 8r Seeburg (1985). Chain termination reactions with the modified bact’eriophage T7 DiYA polgmerase (Sequrnase. United States Biochemical Corporation) were performed as described by, Tabor & Richardson (1987). Depending on the availabihty of srrit#able restriction sites. different strategies were followed for determining the nucleotide sequence of the Mrthnnococcus J>NA region depirt.ed in Fig. I. The insert of plasmid pRPI was sequenced by subcloning the 280 bp EcoRI/XbaI. the 470 bp XhnI/PatI. the 766 bp PstI/RamHI. the 490 bJ) namHI/(‘laT and the 680 bp PlaI/EcoRT fragmenm into the multic~loning sites of plasmids p1’(‘18 and pLTC19 and by using the rommon reverse-sequencing primer. In the case of the 760 bp PstI/BamHI fragment, internal sequencing primers were synthesized and used t,o join the sequences obtained with the common primer. Similarly. the inserbs of pRP3 and Charon RP2 were first subcloned into either pUC plasmids or Ml3 phages (see Fig. 1). The subclones RP2B/b. RP3A/a. RP3B/b and R,P3C/c were subsequently subjected to limit,ed degradation by- exonucoverlapping cl&es as described by lease ITT to generate Henikoff (1984). Both DPU’A strands were sryuencrd in each case and particular care was taken t,o sequence over the junctions of adjacent subclones either by using a neighbouring restriction site or by employing a suitable sequencing primer. Klectrophoresis of sequenced fragmenm was done in

3. Results (a) Cloning

and seyurncing of the Methanococcus oprron homologur

spc

A genomic library of Methanocowws DNA cloned into the expression vector jV Charon 16 was prepared

and screened in a plaque assay for reaction with antibodies directed against total ribosomal proteins from Methanococcus. From about 10,000 phage clones tested, one, i.e. (‘haron RF’1 (Fig. I), delivered a reproducibly positive reaction and was analysed in more detail. Plate lysates from Charon RPI were prepared and tested in an immunoblotting experiment for the presence of proteins reacting with the antibodies used in the plaquescreening procedure. It was found that Iysates of Charon RI’1 contained three cross-reacting proteins with apparent molecular weights of 23,000, dEi,oOO and 27;000, respectively (data not shown).

ChronRPl/pWl CharonRP2 pRP3 RP30lb

RP2Ala

RP3Ala

RP3Clc

RP2Wb

EXP

517 L14 L24 I

I

c I

L5 SU 93 I

I

L6 I

EAH

BC

d

A

E

e

I

I 1000 bp

Figure 1. Physical map of the chromosomal region of ,21. ~nni~lii encoding a transcriptional unit for genes of ribosomal proteins. Only those sites of restriction enzymes are shown that are relevant to this work. The gene organization is given in the blow up (below). Gene designations were adopted from Barhmann (1983). a. b, c. d and e in the lower part of the Figure denote genes whose DKA-derived amino acid sequences are not present in the ribosomal proteins of E. coli. Horizontal bars represent the insertions of clones. Capital or lower (>ase I&ters in the designations represent clones with the same insertion but, in different orientations. Double-headed arrows (I. 2. 3. 4. 5) indicate DNA fragments used for ?rjorthern blotting analysis (see Fig. 3). A. AccT; Af, AJ1IT; B. BamHI: C. (‘loI. E. EcoRT; H, HindID: Hr. Hinc~TT: P, PstT: S. SacT: Sa. SauSA: Sp SpeI; St, StuI: Su, Aau96T; X XbaT.

Evolutionary

Relationship

between 70 S and 80 S Ribosomes

For an analysis of the DNA insert of Charon RPl? the 2.6 kb EcoRI insert was subcloned into plasmid pBR328 to deliver plasmid pRP1 (see Fig. I). The determination of the nucleotide sequence of this insert revealed the existence of five open reading frames (ORF). The deduced amino acid sequence of four of these ORFs showed significant similarity to the E. coli ribosomal proteins L18, S5, L30 and the X-terminal portion of L15. The organization of these ORFs on the Methanococcus DNA was identical with that of the homologous genes within the spectinomgcin operon of E. coli (see below). The existence of a colinear gene organization bet’ween E. coli and M. vannielii encouraged further analysis of the DKA lying on either side of t.his region. To extend the cloned region of chromosomal a genomic library was DNA of Methanococcus, constructed in vector i Charon 4A, 6000 clones of which were immunoscreened with the anti-ribosomal protein serum as described above. One of the reacting phage clones, Charon RP2 (Fig. I), was and purified through lysates single-plaque rescreened: followed by immunoblotting analysis. One protein with an apparent molecular weight of 25,000 that reacted with the serum could be identified (data not’ shown). Charon RP2 was propagated on E. eoli JFMl in a one-litre batch culture and phage D9A was prepared. Restrict’ion analvsis revealed the existence of an insert of approximately 12 kb. Degradation of the DSA with EcoRI yielded four fragments, one of which was identical with the insert of Charon R,Pl. This it1entit.y was proven by hybridization analysis and by determination of the DNA sequence from both ends. Hybridization analysis showed that (Sharon RP2 extended the cloned region of chromosomal DNA from Methanococcus in a 3’ direction relative to the Charon RPl insert. Restriction fragments of Clharon RP2 were isolated and subcloned into pU:(’ and (or) Ml3 vectors (Fig. 1 and Materials and Methods). The site of insertion and the orientation of the restriction fragment relative to the vec%or were selected in a manner such that both strands were suitable for creating overlapping deletion clones with the exonuclease ITT technique (Henikoff, 1984). The nested set of clones of RP2B and RP2b generated were subsequently sequenced by the chain termination method using the common sequencing primers. Th e analysis of the DNA sequence of RJ’2K/b revealed the presence of the C-t,erminal part of the gene for L15 and of the gene coding for a protein homologous to E. co& set 1 (results to be published elsewhere). However, in this genomic region of Methanococcus no indication could be found for the existence of an equivalent to the “alphaoperon”. which follows the sJ)ectinomycin operon in the E. coli chromosome (Post et al., 1980). For extension of the cloned region of the Nethanococcus genome in the 5’ direction relative to the insert of Charon RPl, a chromosome walking step was employed. A 8.1 kb BamHJ fragment that overlapped with the insert of pRP1 but not with

25

RP2B/b was ident’ified. This fragment was cloned into vector pUC18 to yield the recombinant plasmid pRP3 (Fig. 1). Plasmid pRP3 was further subcloned and the subclones RP3A/a, RP3B/b and RP3C/c were obtained and were sequenced aft’er generation of nested deletions with exonuclease III. The total DNA sequence bordered by the A$IJ and Accl sites is given in Figure 2 together with t’he deduced amino acid sequence. It comprises 16 open reading frames: the gene products of 11 of them display significant sequence similarit’y wit’h ribosomal proteins from E. coli; they are designated in the E. coli nomenclature below (see Fig. 1, bottom). The gene products of five additional open reading frames, a t,o e, bear no similarity t.o the sequences of any known eubacterial ribosomal prot)ein. Evidence for the conbinuity of the sequence shown in Figure 2 was provided by sequencing across the junctions of the subclones and by backhybridization of the labelled inserts of the original and of the subclones against chromosomal DI\‘A restricted with the appropriate enzymes. (b) The genes are organized

in c1 tmnscriptional

unit

The coding regions of the DNA sequenced are separated by only short intergenic regions, which strongly suggests that at least some of the genes are co-t’ranscribed. Northern blotting analysis of total RNA from exponentially growing cells of M. cannielii was performed usmg the radioactive DXA probes shown in Figure 1. Using a probe specific for ORFa and ORFb. two transcripts were detected (Fig. 3: lane 2) with approximate sizes of 0.8 and 8 kb. Jn contrast, probes comprising the genes for S17 (Fig. 3; lane 3) and L15 (Fig. 3: lane 4), respectively, yielded two transcripts with lengths of about 7 and 8 kb. Additional probes that were located between probes 3 and 4 (Fig. 1) yielded t,he same result (data not shown). l+‘ith a probe upstream from ORFa and ORFb (Fig. 3: lane 1) and a probe lying downstream from the gene for the I,15 homologue (Fig. 3: lane 5) no signals in the range 7 to 8 kb could be detected. Probe 5> however, gave a signal equivalent to a transcript wit,h a. size of 1.5 kb. These results favour the conclusion that the two longer transcripts ha,ve a site or sites for transcriptional termination downstream from the gene for L15 between the sites for EcoRI and f/indTTJ and different 5’ ends. The transcriptional initiation and termination site wa.s det,ermined using nuclrase S, protection and primer extension analyses. X 1410 bp HincJJ/ Sau3A fragment (Fig. 1) was used to determine the approximate location of the 5’ ends of the transcripts. The 1410 bp fragment was 5’ labelled at the Sa/c3A site and hybridized to total JXSr\ of M. vanm&i. After exposure to nuclease S, two major signals could be detect,ed. which indicated that the transcripts have different 5’ ends (data not’ shown). The identification of t)he precise 5’ ends of the transcript species was achieved by primer extension. Two appropriate oligonucleotides were synthesized,

Fig. 2.

Evolutionary

Relationship

between 70 8 and 80 S Kibosomes

27

J. Aurr

28

0 kb

‘7kb

23s

-

16 S-

Fig. 3.

-

1.5 kb

-

08kb

et al.

5’ end-labelled and hybridized to a t)otal I
Evolutionary

Relationship

between 70 S and 80 S Ribosomes

29

(b)

0)

5’

5’ 3’

3’

TA CG

TA

TA

AT

AT

AT

TA

TA CG TA

AT

CC GC TA

GC

AT

GC

TA

AT

AT GC

AT

AT

TA

CG TA

TA TA

\

TA

I

I

3’ 5’

3’ 5’

Figure 4. Autoradiograms showing primer extension analysis to determine the 5’ ends of the 8 kh (a) and of the 7 kb (b) transcripts. Lanes G, A, T and C represent the sequence reactions determined using the chain termination method. Lanes 1. 2 and 3 show primer extension experiments using 5, 10 and 20 pg of total RXA from M. ~~nnl:~Zii, respectively.

AT G&l

A CGC

T 12

Figure 5. Autoradiograms showing S, nuclease protection analyses of the 3’ ends of mRPu’As terminating downstream from ORFb (a) and downstream from the gene for the L15 equivalent (b). Lanes G, A+G, T+ C and C denote MaxamGilbert sequence reactions of the original DNA fragment. Lane 1 in A shows the result of S, mapping analysis aft,er hybridization at 44°C; and lanes 1 and 2, in B, at’ 41 “C and 38”C, respect’ively

J. Auw et al. Table 2 Ribosomal

Mrthanococcu
proteins

from

vnrious

Homologue

organ&as that proteins

arc homologous

Lengtht (amino acids)

Kef’errnce

Hm Ec Mc

Tanaka et al. (1985) Kimura & Kimura (1987) Yaguchi & Rittmann (1978) Ohkubo rf d. (1987)

sc!$ EC I&t Mc Mpch Ntch

Leer Pf rtl. (1984) Morinaga et al. (1978) Kimura rf al. (1985) Ohkuho rf al. (1987) Ohgama rt al. (1986) Shinozaki rt al. (1986)

EC l&t MC-

Wittmann-Liebold (19i9) Kimura et al. (1985) Ohkubo d al. (1987)

SC

Ot.aka rf al. (1982)

SC EC MC

Teem rf d. (1984) Chen &, Ehrke (1976) Ohkubo rt al. (1987)

Ec MC

Yagurhi et al. (1983) Ohkubo rf al. (1987)

SC

Hm Ec MC l3st Mprh Ntch

Leer ef a/. (1985) Kimura & Kimura (1987) Allen & Wittmann-Liebold Ohkubo et al. (1987) Kimura & Kimura (1987) Ohyama et al. (1986) Shinozaki et al. (1986)

EC MC l3st

(‘hen et al. (1977) Ohkubo et nZ.(l987) Kimura et a1.(1981)

Mm HS

Dudov C Perry (1984) Young & Trowsdale (1985)

RlS

to LMethanococww

109 1.58 X3

119 103

Xl 9x 130 130 (1978)

182 176

135 135

149 196

(than et al. (1987a) (:han et al. (1987h) Brosius et al. (1975) Ohkubo et al. (1987) IS. Wittmann-Liebold & M. Kimura (personal communications) Wittmann-Licbold Q Greuer (1978) Ohkubo et al. (1987) Kimura (1984) Lin et al. (1987) Ritter & Wittmann~I,ieb~lltl Kimura (1984)

129

(19i;i)

195 296 117

225 166

154 25X 58

Evolutionary

Relationship

31

between 70 S and X0 S Ribosomes

Table 2 (continued) Nefhanocoecus

Reference

Homologue

Lengtht (amino acids)

I,15 LZ9 L15 L15 L15

Kgiufer it (II. (1983) Giorginis 8: (hen (1977) Ohkubo ~1nl. (1987) Kimura et nl. (1985)

t Number of amino acid residues in the respective ribosomal protein. : Abbreviations: Bst, Bacillus stearothermophil?rs: EP, E. coli; Hm, Halobacterium mari.smort,ti: Hs. Homo sapiens; MC. Mycoplaama eapricolum: Mm, Mus musculus: Mpch, Narchanfia pol~~morp/co (ahloroplast; Ntch. Nieotiana tabacum rhloroplast; RI, Baths norvqicz~s liver; SC. S,rcchtrrorr,y~p.s crrraisiar. $ For yeast ribosomal proteins the nomenclature of Niche1 et a/. (1983) was followed. i! The exact number of amino acid residues has not. been determined.

Methanococcus. Fig. 5(a) shows that one of the transcripts ends at a TA dinucleotide just’ downstream from ORFb (see arrows in Fig. 2). However. longer transcripts are also resolved with sizes up to the fully protected I)KA fragment. Further analysis is required in order to decide whether the smaller transcript is the result of a terminat,ion or a processing event. The 3’ end of the 7 and 8 kb transcripts downstream from the Methanococrus I,16 gene was determined using a 660 bp EcoRT/ II&d111 DNA fragment (Fig. 1) that was 3’ endlabelled at the EcwRT site. Figure 5(b) shows that two major termination sites can be resolved corresponding t’o AC and AT dinucleotides (see arrows in Fig. 2).

Discussion

4. (a)

The organiza.tion

of Methanococcus

of genes for ribosomnl proteins resembles that characteristic of eubacteria

The amino acid sequences that were deduced from the nucleic acid sequences of the open reading frames were analysed in a data-base search for homology with the primary structures of all ribosomal proteins t,ha,t are available. Table 2 gives the

results. which show that a,ll open reading frames with the exception of a and b could be identified as homologues of eubacterial and/or eukaryotic ribosomal proteins. In addition to t)lesr comparisons of tot’al amino acid sequences we were able to identif) the Methanococcus ribosomal proteins with ribosomal proteins from other archarbactteria for which the S-terminal amino acid sequences only were available: Mefhanococcus L6, I,18 and 12 are homologous to LIO, I,1 3 and IA19 of Hnlobacteriu.m cu,tirubrum. respect,ively (Smith et ~1.. 1978; Matheson et a,l., 1984). For proteins I,13 and L19 from H. cutirubrum it was shown that they are 5 S rRr\‘A binding prot)eins (Smith et a/., 197X) as is the eukaryotic eyuivalent from yeast (Sazar Pf al., 1979). Proteins I, I8 and 15 from i~Pthnlloror’c/~n, therefore. may have the same function as their (mounterparts in ot’her organisms. Figure 6 gives a comparison of the organization of the spectinomycin operon for ribosomal protrms in /C. coli (Cerretti et al., 1983) and of the genes identified as homologous genes in ~~~ethtrnoco~~zss.There is a striking similarity in both gent, c*omposition and order. with only two deviations: (1) three open reading frames (c, d and e) are quasi “inserted” into the eubacterial-type gene organizat,ion: and (2) thtl organization into a transcriptional unit is different.

E. coli TP

M. vonmeli~

b@+

Figure 6. Comparison of the gene organization of the S10 operon and the spectinomycin operon of E. roli with thr related t.ranscript,ional unit(s) of 1%‘. vannklii. The sizes of t.he genes are drawn to scale. a. I). c’. tl and e denote :WrthcLnococcu.sgenes that are not present in these operons of B. coli. P and T indicate the sitrs for transcriptional initiation

and termination.

respectively.

32

J. Auer et al.

in t,hat it starts further upstream in the Methanococcus spc operon than in the E. coli operon and in that termination occurs immediatelv downstream from the gene for the L15 protein. There are two promoters in Methanococcus: t’he first initiates transcription upstream from ORFa and delivers two transcripts, one (8 kb) reading t’hrough the whole spc operon, the other (0.8 kb) t’erminating downstream from ORFb; the second promoter is situated between ORFb and the gene for the S17 protein and gives rise to a 7 kb mRNA. Circumstantial evidence that the latter transcript is a primary transcript comes from the fact that upstream from the initiation site bhere is a sequence motif that has been identified as a promoter element in Methanococcus (With et al., 1986), and which was later demonstrated to be at the binding site of RNA polymerase (Thomm & With, 1988; Brown et ctl., 1988: Reiter et al., 1988a). However, at’ preserit the possibility cannot be excluded that the 0.8 kb and 7 kb transcripts might arise from a precise cleavage event of the 8 kb message. Analysis of the 3’ ends of the transcripts showed that termination of transcription occurs just downstream from the L15 gene between a putative secondary structure region of the message and a pyrimidine-rich sequence (Fig. 2). Similar sequence motifs were previously found at sites of transcription termination for several genes in various archaebacteria (Miiller et aZ., 1985; Dennis, 1986; Mankin & Kagramanova, 1986; With et al., 1986; Cram et al., 1987; Kjems & Garrett. 1987; Kjems et al., 1987; Bokranz et al.. 1988; Reiter et al., 19886: Lechner et al., 1989). The analysis of the transcription products has shown that the gene coding for ribosomal prot,ein S17 is a member of the spectinomycin operon in Methunococcus. In E. coli it is the promoter distal gene of the “SIO operon” (see Fig. 6). Interestingly. the promoter proximal gene of the SIO operon has been “integrated” into the streptomycin operon (Auer et al.. 1989; Lechner et al., 1989) in Methanococcus. When we extended our sequence analysis of the Methanococcus spc operon region into the 5’ direction it was found that ORFa is preceded by the genes for proteins L29. S3 and L22; the gene for protein I,16 is lacking (unpublished results). Thus, as in E. coli or in Mycoplasma capricolum (Ohkubo et al., 1987) the SIO operon and spectinomycin operon homologues of Methanococcus are immediate neighbours on the chromosome. The transcription pattern, however. has been less conserved, since in Mycoplasma the two operon homologues appear to be transcribed as a single unit (Ohkubo et al., 1987), whereas in E. coli or Methanococcus they are transcribed in several and different entities. There is a strong Shine-Dalgarno sequence present in front, of each gene. The sequence of the potent,ial ribosome binding sites is well conserved and complementary to the 3’ end of the 16 8 rRNA of M. nannieki (Steitz, 1978; Jarsch et al., 1983). The distance between the conserved “GG” dinucleotide and the ATG start codon of the respective gene is seven nucleotides for the majority of the genes.

An average distance of seven nucleotides was also reported for most E. coli genes (Gold it al.. 1981). (b) Methanococcus a higher

rihosotnal proteins

on awraye

sequence similarity to their rukasyotic their eubactwial homologurs

have

than, to

In order to analyse the sequencae similarities quantitatively. the sequences of pairs of homologous ribosomal proteins listed in Table 2 were aligned with each other using the program “GAP” of the C’WGCG program package (alignments will be presented elsewhere). As an overview, the percentage of identical amino acid residues between Methanococcus proteins and homologous ribosomal Nalobacterium marisproteins from eukaryotes, mortui and eubacteria, is given in Figurrb 7. For the eubacteria the given value of similarit,p is the arith metic average of all compared eu bact,erial-type sequences; their range of deviation is indicated in Figure 7. There is a considerable difference in the degree of similarit,y among homologous ribosomal proteins. Some proteins such as the Ll4 family arc’ well conserved, while others such as the members of’ the S14 group arc weakly conserved. The degree of similarity between the :%fethanoCOCCUB proteins and eukaryot,ic proteins varies between 2560/, (RlL7) and 47.7?<, (ScLl7A). ‘I‘hta corresponding values for the comparison wit,h eubacterial proteins range from 19.4?{, (Ntch 58) t.o 42.70/b (EcL24). A direct comparison of homologous ribosomal proteins from the three kingdoms. which could br> performed for proteins S17. L14. L5, S8. I,30 and Llfi. showed that wit,h the exceptionof I,30 Methn t/ococcus proteins arp more closely related to their eukaryotic than to their eubacteiial counterparts. From the available archaebacterial sequences? ribosomal proteins S14 and Sl6 from H. mnrismortui (Kimura &, Kimura, 1987) showed a rather high degree of sequence similarity to thr Methanococcus homologues of Sl7 (W?,,) and SX (52%). respectively. Tnterestingly. while the archarbacterial S8 sequences are qmte closely related, Nethanococcus protein S17 showed a higher degree of similarity to rat liver Sl 1 (45q,,) than to El. ,marismortui S 14 (42’?;,). Tn order to analyse t,he significan(Afs of the sirnilarity values of our sequence comparisons. \VP empirically determined the border of significance for several comparisons. For this purpose. sequences were randomized using the program “SHI’FFI,E“ and were a.ligned for a second tirnca employing t,h(b same parameters. It was found t)hat. up to P(Y),, identical amino acid residues represent the barkground noise of similarity: t’his value was also obtained for non-ribosomal proteins (I’. Palm. personal communication). All sequence’s list,cd iu Table 2 yielded similarity values of 20’?;, and higher and were, therefore, regarded as significant. An evolutionary relatjionship closer to the cukaryotic t,han t’o eubacterial homologues has also bren reported for the ribosomal A-proteins (Mathcson et

Evolutionary

Relationship

between

X0 S Ribosomes

70 A’ and

33

% 1 60

Figure 7. (Comparison of the degree of sequence similarity of rihosomal proteins from M. rnnni~lii (E. coli nomenclature. rxc*ept for proteins that have no eubarterial counterpart) to homologous ribosomal proteins from eukaryotes (hatched columns). H. naarismortui (filled columns) and eubacteria including chloroplast,s (oprn cwlumns). The degree of sequence similarity is given as the percentage of identical amino acid residues in the aligned sequences. The heights of the open cwlumns rrpresent the averages of sequence similarity of different eubacterial-type srquenws: the maximum

tirviation from the average value is indicated by vrrtical bars. al.. 19x8; Strobe1 et al.. 1988), ribosomal protein L23 (Kiipke $ Wittmann-Liebold, 1988) and elongation factors ICCand 2 from archaebacteria (Lechner & Riick, 1987: Lechner et aZ., 1988). as well as for components not belonging to the translational system, such as the subunits of RNA polymerase (Zillig et al.. 1988) or amino acid biosynthetic enzymes (Reckler $ Reeve, 1986; Morris & Reeve, 1988). At present there is no compelling argument RS to why all these archaebacterial protein sequences and also those of the 5 S rRNA (Hori $ Osawa. 1987) display a closer relationship to their rukaryotic homologues and why the sequences of the I6 S and 23 S ribosomal RNAs deviate by being more rubacterial in nature (Woese & Olsen. 1986; Leffere rt nl.. 1987). (c)

7’h.e Met hanoeoccus

ro~ztrri~~s yenrs

spectinowqein operon 1rv2h rxclusive similarity to eukaryotic homologues

LVhen the sequences of the putative gene products of ORFc. ORFd and ORFe were compared with the c*omplt%e set of eubacterial ribosomal protein sequences no homologues were detected. Surprisingly. however. ORFd and ORFe displayed significant similarity to ribosomai prot.eins I,32 from mouse and I,19 from rat liver, respectively. ORFc. on the other hand. exhibited a high degree of similarity to the sequenced N-terminal fragment of yeast S6 protein. An open reading frame whose putat’ive gene product, exhibits exclusive sequence similarit)y to a eukaryotic 80 S ribosomal protein also exists in t,he streptomycin operon of Methanococcxs (Auer et al.. 1989; Lechner et al.. 1989). There are two explanations for this intriguing fact: first. these proteins could be supernumerary component,s with no homologue at the eubacterial ribosome. It may he relevant in this connection that) the ribosome from Methanococcus appears to have a higher number of constituent proteins than the E. coli organelle (Schmid & Kiick. 1982). A second, less att.ract.ive. possibility is that. these proteins have

their homologous counterpart in the eubacterial ribosome but, due to a high sequence divergence, the similarity broke down to statistically insignificant values. Tf the latter possibility holds true the following questions arise: (1) why have these such a high degree of simiproteins “maintained” larit’y with the 80 S homologous sequence? and (2) why have these genes been excised from the spectinomycin operon and moved to some ot,her site on the chromosome? (d) The size of the Methanococcus is intevnediate

between

those from

and eukaryotic

ribosomal proteins their eubacterial

homologurs

Table 2 gives the number of amino acid residues of the Afethanococcus ribosomal proteins in comparison with those from the eubacterial and eukaryotic homologues. The Methanococcus proteins, in general, are larger than their counterparts from eubacteria and smaller than the homologues from the 80 S ribosome. The size differences (San be traced to internal deletions (or insertions) or, quite frequently to C- or S-terminal truncations (or additions). A prominent example is ribosomal protein L30, whose schematic alignment is shown in Figure 8. The eubaeterial I,30 proteins lack t.he 100 N-t,erminal as well as the about 100 C-terminal amino acid residues whereas t’he archaebacterial homologue just lacks the N-terminal domain. Similar truncat,ions or deletions have a,lso been observed for genes comprising the streptomycin operon of Methnnococcus (Auer et al., 1989; Lechner et al.. 1989) and for ribosomal prot,ein S14 from N. marismortui (Kimura KKimura, 19X7). (e) Evolutionary

considerations

Archaebacteria possess a larger number of shared properties with eubacteria and eukaryotes. respectively, than the other two lineages do. These shared features are considered to be primeval (Zillig et al., 1988). Phylogenetic trees based upon the compari-

34

J. her 0

RI L7

50

ITO

Ni

150

2Ocll K

258 aa

Mv 130

N-C

15L 00

EC L30

N-C

58 a0

BstL30

N-C

62 aa

Figure 8. Schematics alignment showing an example of the size divergence of protein I,30 homologurs (E. co/i nomenclature). The lengths of proteins are given as number of amino acid residues (aa). RI, rat liver: Mv. M. vanniellii: Ec. E. co&; Bst, RacilluCs stearothermophilus. N and C denote t’he PI’ and C termini of a protein. respectively.

son of different macromolecules show that archaebacterial sequences have evolved with a slow rate that is expressed in the smaller distance t)o the branch point that is common to all three lineages (Woese bz Olsen. 1986; Kjems et nl.. 1987; Leffers rt al., 1987; Zillig et al., 1988: Auer et al.. 1989). The! considered to be closer t’o t)hr are, therefore. common ancestor. There are four major points t,hat emerge from the results presented that are relevant to the discussion of the evolutionary relationship of t,he 70 6 and 80 S translation systems: (1) the gene organization within ribosomal protein operons in Methanococcus is almost identical with that of the respective operons on the chromosome of eubacteria: (2) the primar? structures of ribosomal proteins and of translation factors are more related to their eukaryotic than to their eubacterial homologues: (3) the Methanococws rihosomal protein operons contain open reading frames whose putative gene products show exclusive similarity only with eukaryotic homologues: (4) t’he archaebacterial proteins have a size intermediate between those of their eukaryotic and eubacterial homologues. In the light of these findings we propose that the eubacterial ribosome evolved by minimization of a more complex organelle and that the archaebacaterial system reflects t,he state of an incomplete minimization. Development of the 70 S ribosome b> “streamlining” due to selective forces for rapid duplication t’imes has already been discussed 1,~ Wool (1979). First, this selection could have forced genes together into operons to facilitate t’hr regulation of formation and of stoichiotnetry. Tt also indicates that eubacteria and archaeba&eria share a common t,ime of evolution, since it is improbable that their identical gene organizations would have developed independently from each other. Secondly, selection for rapid growth could have led to a minimization of both the numbers and sizes of ribosomal proteins with the loss of proteins. or parts thereof. whose functions were not essential or whose loss could have been compensated by other genetic events. within either t’he ribosomal RNA or some other ribosomal protein genes. Such reduction processes have also been post.ulated for the elimina,tion of segments of ribosomal R?L’A from the 18 S

et al. and 2.5 S rRXA gene products ((‘lark, 1987) and for the loss of introns from most of the prot~~i~l-t~nc~otli~~g genes (Darnell & 1)oolittlr. 1986). Our hypothesis. therefore. implies t ha.t t hfl anwstral rihosomr had a higher romplexity than the eubactwial organelle and tha,t t,he a~rchaebac~terial ribosome is closer to this ancest,ral statfk. The higher complexit,v may have been ma~intainrti or f’v(‘n augmented in the development of the eukarvot ic. 80 S organelle. e.g. by the attainment of new jbnr,tions such as attachment to the endoplasmatic reticulum (Lin et a.l., 1987) or a more highly refincbd initiat)ion reaction. The subst,antiation of these speculations requires determination of t hr sequences of a complet)e set of components from an and eukaryotic archaebacterial ribosomc to compare them with the available sequences of the E. coli ribosome. On the basis of these data it should be possible to judge the uniqueness and relationships of components and t’o delineaic t.hr genetic. events underlying this evolution. (:. Schrnitl. We at-r’ greatI> indebted t,o 0. St~rohrl. P. Buckel. X. HampI) and R. Mertz for donating total ribosomal proteins, antisera. E. (!haron 16. i. (‘haron 4.A rrsl)~&ivelv. \Z’r thank J<. and oligonurlrotidrs. Wittmann-Lieboltl for help in t,hr &nmrnt of ribosomal protein sequences. The invaluable help of J’. Palm and W. Zillig in the sequence analysis is grat,efully acknow!edged. LZ’r are very much obliged to A. T. Matheson f’ol, reading the manuscript. The editorial hrlp of M. (:eier is greatly acknowledged. This work was suJq)ortetJ by t htl Deutsche ForscBhungsgemeinschaft

References Aiha. H.. Adhya. S. & de f’rombnrgghr. I<. (I!MJ). ./. Iliol. (‘hwtr. 256. I JRO5- 11910. :illen. (:. & Cl’ittrnarrn-I.irJ~ol~l, IS. (IWX). tfol,~,r-Sr!//r~i~‘s Z. t’hysiol. c’hrru. 359. 1XN J:i”.‘,. Auer. .I.. Lerhner, K. & Mek. A, (J!M). (‘u?//I//. .I. Microhiol.. 35. 200-204.

J+rk. A. .I. & SharJ), I’. A. (1!)77). (‘r/l. 12. 7’1 7X JSirnboin. H. 1). & J)oly. .I. (1!+7!)). AYt~cl. Acids 12~ 7. 1513 -1X3. l%okranz. 11.. J-Xumet,. (i.. Alltrranst)erRt,l,. I<.. .\nkrl170. :i(iX Fuchs. I). 8: Klrin. .+\. (JWX). ./. IZncteriol. 577.

J3rosius. .l.. S&ilt,z. JC.Hr (‘hen. Ji. (1975). /‘E&S LP&VS. 56. 359 Xl. Brown. J. 1\:.. Thomnr. >I., J,. (i.. Mettrr. K. 0. & Rrtavtl. .I. S. (I%#). SW/. .-Jcids IMPS. 16. I:% 1%). (‘rrretti. I). I’.. I)taan. I).. Jjavis. ($. R.. IStvlwvll, I). 11. & Somura. Al. (I 9X3). St/cl. Acirls Krs. 11. LXW381 li. (‘hall. Y.-IA.. I,in. ‘1.. M(,NalJy. .J.. J’rlrg. I).. Meyuhas. 0. 6 LYool. I. (: (1%+7/x). J. /XC//. (‘Iuw. 262, II 11 I I I.‘,. (‘ban. Y.-L. IA. A.. McSall?~. .I. & \I’ool. I. (:. (JHXi/j). .I. Hiol. (‘hunt. 262. 128X-IP886. (‘hen. E. .J. & Srrburg. I>. H. (l!M5). ZJS.1. 4. 1% 1X). (‘hen. Ii,. X- JChrkr. (4. (JHTfi). I’EHS I,~tt~r.s, 69. PM “45 (‘hrn, It.. Arfsttan. Jr. B (‘hrrl-S~hrnrissrr. I’. (1977). Hoppr-Styler

‘k %. Ph,yysiol. ( ‘hrnr .J. .Ilol. /Cd. 25,

(‘lark. (‘. (:. (lBX7).

358. .% 1 -.5X. W1 :SNF

Relationship

Evolutionary

(‘lewell. 0. H. $ Helinski. D. (1969). f’roc. Sat. Acad. Sk., 1~..S.d. 62. 1159-l 166. (‘ram. D. C.. Sherf. R. A.. Libby. R. T.. Mattaliano. R. tJ.. Ramachandran. K. L. & Reeve. ,J. ?u’. (1987). Prof. Sat.

Acnd. Sri.,

I’.S.A.

84, 3992-3996.

Darnell. .J. E. & Doolittle. W’. F. (1986). Proc. LVat. Aca,d. Sci.. I-.S.A. 83. 1271 -1275. Dennis. P. I’. (1986). .I. Bacterial. 168. 471-478. Devereux. *J.. Haeberli, 1’. & Smithies. 0. (1984). Surl. Acids Res. 12. 387--~39.5. Douglas. c’.. Achatz. F. & B&k. A. (1980). Zhl. Rakt. Hyg., J. Abt. Orig. Cl, l-11. Dudov. K. P. 8: Perry. R. P. (1984). Cetl. 37. 457468. (iiorginis. S. & Chen. R. (1977). FEBS Letters, 84, 347-

35. 365-403.

(iray. (‘. I’., Sommer. R.. Polke. (i.. Beck, E. & Rchallrr, H. (197X). Proc. Sat. Aca,d. Sci., I’.S.A. 75. 50-53. Hatakryama. T. 8: Kimura. MM.(1988). Eur. .I. Biochem. 172. 703 711. Hrnikoff. S. (1984). fhe. 28, 351-359. Hori. H. & Osawa. S. (1987). Mol. Riol. &?ol. 4. 445-472. Ttoh. T. (1988). Eur. J. Biochem. 176. 197-303. .larsch. M.. Alt.enhuchner. ,J. & B&k. A. (1983). ;Wol. Gm. Gmet. 189. 41.-47. Johnson. D. A.. (iautsrh. ,J. ,L\., Sportsman. ,J. R. & Elder. .I. H. (1984). GPW Anal. Tech. 1. 3-X. Kgufer. X. F.. Fried. H. $1.. Schwindingrr. W’. F.. *Jasin, M. 8 \l’arner. .I. R. (1983). Xwl. Acids Res. 11. 3123~3135.

Kaiser. K. ti Murray. X. E. (1985). Tn DXA (‘/on&g, vol. 1: .-I Practicul Approach (Glover, D. M.. rd.). pp. I47. TRL Press Ltd. Oxford and Washington. DC’. (‘hewl. 262. Kimura. *J. $ Kimura. M. (1987). J. Kiol. 1~150~1%155. Kimura. J.. Arndt, E. & Kimura. M. (1987). FICRS h4ters. 224, 65~70. Kimura. 21. (1984). ,/. Biol. (!hrm. 259, 1051-1055. Kimura. M.. Rawlings. 9. 8r Appelt.. K. (1981). Flr:RS Letters. 136, 58-64. Kimura. M.. Kimura, ,J. & Ashman, K. (1985). /Cur. J. Riochum. 150, 491-497. Kjems. ,J. & (iarrett, It. A. (1987). EMBO J. 6. 3521-3530. Kjems. J.. (iarrett. R. A. & Ansorge. W’. (1987). S?ytst. Appl.

Kiipkr.

Maniatis. T.. Fritsch. E. F. & Sambrook. J. (1982). Molecular cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press. Cold Spring Harbor. KY. Mankin. A. S. & Kagramanova. V. K. (1986). Mol. Nm. Genet. 202. 15%161. Matheson, A. T.. Miiller, W.. Amons. R. & Yaguchi, M. (1980). In Ribouomes: Structure, Function and Genetics (Chambliss. G.. Craven, G. R., Davies, J. Davis, K.. Kahan, L. & h’omura. M.. eds). pp. 297-332. University Park Press. Baltimore. Matheson. X. T.. Yaguchi. $1.. Christensen. I’.. Rollin. C’. F. & Hasnain. S. (1984). (‘anad. J. Hiochem. (yell Lziol. 62, 416433. Matheson. A. T.. Louie. K. A. & I&k. A. (1988). FEES Letters.

350.

Gold, L.. Pribnow, D.. Schneider, T., Shinedling, 8.. Singer. H. S. 8 Stormo, G. (1981). Anne. Kue. Microbial.

Microbial.

9. %P-“8.

A. K. E. & Wittmann-Liebold.

35

between 70 S and X0 S Ribosomes

R. (1988). F/ZHS’

Letters. 239, 31% 318.

Kushner, S. R.. Templin, A.. Nagaishi. H. & Clark. A. J. (1971). Proc. .I:&. Acad. r\‘ci., C.9.A. 68, X24-827. Lechnrr. K. B H&-k. A. (1987). ;IZol. Qm. Genet. 208. 523.X8.

IAechner. K.. Hrller. (:. $ Biick, A. (1988). :Vwcl. Acids RPS. 16. 7817~7826. Lechnrr. K.. Hellrr. C. & B&k. A. (1989). J. ;tlol. Ecol., in the press. Leer. R. .J.. van Raamsdonk-Duin. M. M. C‘.. Hagendoorn. .\1. ,J. 11.. Mager, W. H. & Planta. R,. .J. (1984). IVz~c1. .-Irirls tlrs. 12. 6685-6700. Leer. R,. J.. van Raamsdonk-Duin, X. M. C.. Kraakman. I’.. Mayer, 12’. H. & Planta. R. J. (1985). Sucl. dcids Rcs. 13. 70-709.

Leffers. H.. Kjrms. .J.. Ostergaard. L.. Larsen, X. & (iarrrtt. R. Ai. (1987). J. Mol. Biol. 195, 43-61. Lin. .-\.. (Ihan. Y.-L.. McNally. J., Peleg, D.. Meyuhas. 0. & \Vool. I. (:. (1987). J. AWol.Biol. 262. 1X665-1267 1. Lipman. 0. .I. & Pearson. W. R. (1985). Science, 227, 1435-1441.

231. 331-335.

>Iaxam. .A M. & Gilbert. IV. (1980). Method%s Enzyme/.

65,

499-560.

Messing, ,J. (1983). Methods Enqwu)l. 101. 20-78. Michel. S.. Traut, R. R. 8: Lee. ,J. (‘. (1983). Xol. Gen. Genet. 191 . tL’,l-256. t In :VIoleculrcr Genetics. Miller. ,I. H. (1972). Experiwlrnts (loId Spring Harbor Laboratory Press. (‘old Spring Harbor. SY. Jlorinaga, T.. Funatsu. G.. Funatsu. )I.. \VittmannLiebold. I<. KTM’ittmann. H. (:. (1978). FE&S Letters. 91. 74-77. Morris. (‘. .J. &. Reeve. .J. IC. (1988). ./. Kwteriol. 170. 31”5-3130. Miiller. B.. Xllmansberger. R. &I Klein. A. (1985). ,Vucl. Acids Res. 13. 6439-6445.

Xazar. R. S.. Yaguchi, M.. Willick. (:. E.. R,ollin. (‘. F. & Roy. (‘. (1979). Eur. J. Biochem. 102. 5X-582. Xomura. >I. (1986). In ReguMion of (:enc Expression (Booth. .I. 6 Higgins. V.. eds). pp. I99-%%O. Cambridge University Press. (“ambridgr. Ohkubo. S.. Muto. A.. Kawawhi. I-.. Yamao. F. & &n, Genrt. 210. 314-322. osawa. S. (1987). Mol. Ohyama. K.. Ipukuzawa. H.. Kohchi. T.. Shirai. H.. Sano. T.. Sane. S.. LTmesono. K.. Shiki. Y.. Takrurhi. &I,. (‘hang. Z.. .%&a. S., Inokuchi. H. & Owki. H. (1986). Saturr

(London).

322. 372 57-C.

Ot,aka. E.. Hipo. K. Rr Osawa. S. (198%). f~iochrmistr,y.

21.

4545-4550.

Planta. R. .J.. Mager. ‘IV. H.. Leer. R. .I., IVoudt. L. P.. Raw?. H. A. & El Baradi. T. T. ‘1. 1,. (1986). Tn Qtruclure. Furl&on and Genetics of )libosornws (Hard&y. 13.& Kramer, G.. rds), pp. 699-758, Springer-i’erlag. iYew York. Berlin. Heidelberg. Paris and Tokyo. Post. I,. E., Arfsten. A. E.. Davis. (:. R. 8r Somura. RI. (1980). J. Kiol. (‘hem. 255. 4653. I65!). Reiter. \!‘-I).. Palm. I’. R: Zillig. \1’. (l!)XXtr) .V~r1. dcirls Rrs. 16. I-19. R,eiter. \\:.-D.. Palm. P. & Zillig. iv. (I!)#%). .VurI. .-Irin’s Revs.16. 244.5~2451). 12. (IWS). E’ISHS 1,etter.s. Ritter. E. K: \Vittmann-Liebold. 60. l5”-155.

Sanger . F.. Xicklen.,

S. & (‘oulson.

A. K. ( 1977). I’ror.

LVcrt. Acad. Sci., l:.S.A. 74. 5463-5467. Sawers. R. (:. & Biick. A. (1989). ,J. tlacteriol. 171. 1485-2489. Srhmid. (:. & Biick. A. (1982). Z/J/. Bakt. Hyg. I. Aht. Orig. C 3. 347-353. Schmid. (:. & Biick. A. (1984). S,yst. .-l)~[Jl. Microbial. 5.

I-IO. Shinozaki. K.. Ohme, %I., Ta,naka. M.. U’akasugi, T.. Hayashida. X.. Matsubayashi, T.. Zaita. S.. C’hunwongse. ?I . Obokata. J _, Yamaguchi-Shinozaki, K. & Sugiura. M. (1986). EMBO ,J. 5. %043-2048. Silhavy. T. .i.. Berman. 11. L. 8r Enyuist. I,. \V. (1!)84).

with Ornr Fusims. (‘old Spring Harbor Laboratory Press. (‘old Spring Harbor, SY. Smith. S.. Matheson. A. T.. Yaguchi. $1.. Willick, E. 8 Sazar. R’. 9. (197X). Eur. .f. Hiockrn~. 89. M-509. Soheron. X.. (‘ovarrubias. L. & Rolirar. F. (1980). Dww. 9. 287-305. Southern. JC.M. (197.5). ./. Mol. Hiol. 98. 503 ,517. Steitz. ,J. A. (1978). Suturr (London,), 273. 10. Strobel, 0.. Kiipke. A. K. E.. Kamp. R. >I.. Biick. A. & Wittmann-Lirbold. 13. (1988). .I. Hiol. C’hrm. 263. 65X-6546. Tabor. S. K: Richardson. (‘. (‘. (1987). I’roc. Srrl. .-Jccrd. Sci.. I’.S.A. 84, 4767-477 I. Tanaka. T.. Kuwano. Y.. Tshikawa. K. 8r Ogata. K. (19%). .J. Iliol. C’hrm. 260. 6329-6333. Teem. .I. L.. Abovich. 9.. KLufrr. X, F.. Svhwindingrr. 1%‘.F.. Warner. .J. R.. I,evy. :I., Woolford. .J.. Lwr. R. .J.. van Raamsdonk-JIuin. M. hl. C‘.. Mager. W. H.. J’lanta. R. .J., Schultz. I‘.. Friesen. .J. J).. Fried. H. & Rosbash. M. (1984). Swl. .-lcids Rrs. 12, 8~95-8311. Thomm. M. 8-z With. G. (1988). Sucl. Acids Rrs. 16. 151-16:~. \‘og&tein. IS. 8: (:illesJ)ir. I). (1979). I’roc. Sal. dcfrcl. Sri.. 1..S.A. 76, 615-619. IVich. (i.. Hummrl. H.. ,Jarwh. M.. 1Gir. LT. & Hiick. A. (19%). Sucl. Acids Ii~s. 14, Pl;i!f 2179. D’illiams. 1%.(:. & Hlattnrr. F. R. (1979). .J. I’irol. 29. *w-.57.5, 108. 75--M). ~2’ittmann-1,iebold. H. (1979). ZEUS httrrs. Wittmanll-T,irbolcJ, 13. (1986). In Structurr, Fvnwtion nn,d Expriwnts

(Axrtic~,s qf Kiboson~~~ ( Hardest?. K. B K ramer. ( : _. eds). pp. 326-361. Springer-Vrrlag. Pirw York. Berlin. Heidelberg. Paris. Tokyo. \2’ittman-T,irhold. I<. & (4rrurr. I<. (1978). FE/S /Atrtx 95. !)I~ !18. h’orse. (‘. R. K: Olsen. (:. .I. (1986). A’?/sI. .4j)j1/. Nic~rohiol. 7. 161 157. \z’ool. 1. (i. (IHSU). In Kihosomes. Str~cfYuru. Pctr~ction f~nr/ GwPtics ((‘hambliss. (i.. (‘raven. (:. R.. I)avirs. .J.. 1)avis. K.. K&an. I,. 6 Somura. 11.. A). ~JI. 7!)7 824. I‘nivrrsit\, I’srk I’rrss. Haltimorr. \Vool, I, (:. (I 986). In ~Vtructuw, Fwwtiou rend CA,wtics o/ tliboxonws (Hat&&g. 13.Ic: Kramw. (i.. ~1s). pp. :j!Il -Cl1. SpringevVrrlag. Xt-‘w York. I+rlin. Heid~4bryg. London. J’aris and Tokyo. Yaguchi. M. HLIl?ttmann. H. (:. (197X). r/CBS Lrtfctx. 87. 3-40. Yaguchi. %I.. Roj,. C’.. Retlithmriv. R. .I. I?.. \Vitt~mantrLirbold. fZ. $ bVittmwn. H. (:. (19X:<). FEHS Lrtlcrs. 154. dl :10. \‘anish-Prrron. C’.. \.irir;x. +I. & Messing. .J. (19X5). (;rjtr. 33, lo:4 109. lir,s. \‘oung. .J. .\. 7’. K: Tro\vstMr~. J ( I9X.5). .Vw/. .-1c~id.s 13. 8X83-X891. Young. I<, .A. h I)avis. I<. \V. (I 9X3). ScirJ/rt,c. 222. 77X 7x”. Zillig. \V.. I’alm. I’.. Rritrr. \V.-I).. (:l,opJ). F.. I’iihlrr. (:. 8 Klenk. H.-I’. (198X). F:/tr. ./. Hiorh~rn. 173. 17:1 4X”.