THE JOURNAL OF BIOLWICAL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 269, No. 23, Issue of June 10, pp. 16163-16169, 1994 Printed in U.S.A.

The Importance of Conserved Nucleotides of 23 S Ribosomal RNA and Transfer RNA in Ribosome Catalyzed Peptide Bond Formation*

(Received for publication, January 31, 1994)

Kathy R. Lieberman and Albert E. DahlbergS From the Division of Biology and Medicine, Brown University, Providence, Rhode Island 02912

We have constructed the double mutant G2252Cl G2253C in Escherichia coli 23 S rRNA by site-directed mutagenesis. These phylogenetically conserved resi- dues are protected from chemical modification by the 3‘ CCA terminus of the peptidyl-tRNA site (P site)-bound tRNA. Expression of C2252lC2253 23 S rRNA in E. coli severely compromises cell growth. Mutant rRNA is as- sembled into 50 S subunits and 70 S ribosomes but is discriminated against in polysomes. Mutant ribosomes function at lower rates in peptidyltransferase assays than wild type ribosomes. To test whether this defect derives from disruption of base pairing with the 2 cyti- dines of the invariant 3‘ CCA terminus of tRNA, a mutant E. coli tRNAPhe gene was constructed, with the CCA se- quence changed to GGA. As deacylated species, mutant and wild type tRNAPhe inhibit peptidyl transfer identi- cally. Mutant tRNAPhe was aminoacylated in vitro but failed to react as a P site substrate, with either mutant or wild type ribosomes. These results support a role for 62252 and G2253 of 23 S rRNA in peptidyltransferase function and a role for the 3’ residues of peptidyl-tRNA in catalytically productive P site interaction; but they fail to provide evidence supporting canonical base pair- ing between these 23 S residues and the 3’ end of peptidyl-tRNA.

Ribosome-catalyzed peptide bond formation is among the most essential biochemical reactions, yet very little is known about the enzymic mechanism by which i t occurs. Although it was demonstrated 25 years ago with Escherichia coli ribosomes that catalytic function is integral to the large ribosomal subunit (11, the assignment of specific roles for any of the 32 proteins and two RNA molecules which comprise the large ribosomal subunit has been elusive. As evidence accumulates revealing the importance of rRNA in multiple aspects of the process of translation (2, 3), data from several lines of investigation indi- cate that rRNA participates in tRNA binding and may play a fundamental role in the catalysis of peptidyl transfer (4).

In E. coli ribosomes, tRNA molecules bound to ribosomal A’ and P sites protect a specific set of conserved 23 S rRNA resi- dues from modification by small chemical probes (5). The pro- tected residues are clustered at or near the central loop of domain V in the 23 S secondary structure model (6). Remark-

GM19756 (to A. E. D.). The costs of publication of this article were * This research was supported by National Institutes of Health Grant

defrayed in part by the payment of page charges. This article must

U.S.C. Section 1734 solely to indicate this fact. therefore be hereby marked “aduertisement” in accordance with 18

$ To whom correspondence should be addressed: Brown University, Box G-J, Providence, RI 02912. Tel.: 401-863-2223; Fax: 401-863-1201.

The abbreviations used are: A site, aminoacyl-tRNA site; P site, peptidyl-tRNA site; poly(U), polyuridylic acid; N-Ac-Phe-tRNAPhe, N- acetyl-phenylalanyl-tRNAPhe; N-Ac-Phe-puromycin, N-acetyl-phenyl- alanyl-puromycin.

ably, most of the same 23 S residues protected by P site-bound aminoacyl-tRNA are also protected by an aminoacyloligoribo- nucleotide fragment derived from the 3’ end of tRNA (7) . This fragment comprises the minimal essential substrate for cata- lytically productive P site interaction (81, in a nonprocessive model reaction using the aminoacyladenosine analogue puro- mycin as A site substrate.

Aminoacyl-tRNA and peptidyl-tRNA analogues have been cross-linked to 23 S RNA residues (9-111, including some of the same central loop residues detected in the protection studies ( 5 ) . Mutations that confer resistance to antibiotics known to inhibit peptide bond formation, such as chloramphenicol and anisomycin, have also been mapped to this region of the large ribosomal subunit rRNAs of several organisms (12-14). All of these data are dramatically underscored by the recent demon- stration by Noller and colleagues (4) that the large ribosomal subunit from Thermus aquaticus, depleted of over 90% of its protein component by proteolysis and extensive phenol extrac- tion, is still capable of catalysis of peptidyl transfer.

Two phylogenetically conserved, single-stranded guanosine residues at position 2252 and 2253 in domain V of 23 S rRNA (Fig. 1) are protected from kethoxal modification when the ribosomal P site is occupied by either an aminoacyloligonucle- otide (7) , by aminoacyl-tRNA, or by deacylated tRNA, but not by tRNA missing the 3’4erminal CA sequence (5) . Thus these residues are candidates for a direct interaction with the 3’ end of P site-bound tRNA, possibly via canonical base pairing with the two cytidines of the invariant tRNA 3’ sequence, CCA. The ability of single base changes at these two residues to disrupt translational fidelity in vivo has recently been described (15). In this study, we determined the effects of expression of the double mutation G2252CIG2253C on host cell phenotype and on the catalysis of peptide bond formation in vitro. We also report the construction and in vitro expression of a mutant E. coli tRNAPhe gene, encoding the 3’ sequence GGA, and examine its activity as a P site substrate, with both G225ZG2253 and C2252K2253 ribosomes.

EXPERIMENTAL PROCEDURES Construction and Expression of rRNA Mutants-The mutation

G2252CIG2253C was constructed by site-directed mutagenesis (16, 17) of an M13mp18 clone containing the EcoRI-BamHI fragment of E. coli operon r r B . Uracil containing single-stranded DNA was a gift from William Tapprich and was prepared in the dut- ung- strain RZ1032. Strain XL-1 Blue was used for cloning and propagation of MI3 mutants. Mutations were identified by sequencing, and the SphI- EspI fragment of the 23 S rRNA gene, containing the mutation, was subcloned into plasmid pGQ7, a derivative of pN02680 (18). This plasmid, a gift from Richard Skinner, contains the rrnB operon under control of the phage A leftward promoter pL and lacks the SphI site in the pBR322 vector backbone. Mutations were confirmed by sequencing of the double- stranded plasmid. E. coli strain DHI, containing the plasmid pcI857 encoding a thermolabile allele of the A repressor, was used as the host cell for the expression of mutant rRNA. This system allows repression of transcription of plasmid-encoded rRNA when cells are grown at 30 “C and induction by shifting cultures to 42 “C (18). Cells were cultured in

16163

16164 Conserved Residues of rRNA and tRNA in Peptidyl Dansfer

FIG. 1. Domain V of E. coli 23 S rRNA. Pictured is the secondary struc- ture model for residues 2043-2625 of 23 S rRNA (6). The boxed region, shown in de- tail at the right, contains residues G2252 and G2253.

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Luna-Bertani broth (10 goiter tryptone, 5 goiter yeast extract, 10 &iter NaCI) containing ampicillin (200 pg/ml) and neomycin (50 pg/ml) for the maintenance of pGQ7 or pGQ2252C/2253C, and pcI857, respectively. Growth rates were measured using a Klett-Summerson colorimeter.

Analysis of Mutant rRNA Distribution-Cultures of DH1 pcI857 pGQ2252CI2253C were grown for 2 h at 42 "C and then cooled quickly, pelleted, and lysed with lysozyme (150 pg/ml) followed by four freeze- thaw cycles and the addition of DNase I (Worthington Enzymes) to a final concentration of 80 pg/ml. Lysates were applied to 1040% linear sucrose gradients and centrifuged at 18,000 rpm for 18 h in an SW 28 rotor. Fractions containing 50 S subunits, 70 S ribosomes, and poly- somes were collected using an ISCO gradient fractionator, and rRNA was extracted and subjected to primer extension analysis (19) using a 5' 32P-deoxyoligonucleotide primer complementary to bases 2254-2274 of 23 S rRNA. Primer extension products were resolved on 12% polyacryl- amide, 7 M urea sequencing gels, autoradiographed, and the intensity of bands corresponding to extension products from mutant and wild type rRNA templates was quantitated by densitometry.

Peptide Bond Formation Assays-Salt-washed 70 S ribosomes (3 x 500 mM NH,Cl) were prepared from 1-liter cultures of DH1 pc1857 pGQ7 and DH1 pCI857 pGQ2252C/2253C harvested 2 h after induction at 42 "C by the method of Chlbdek et at. (20) with the modification that

cells were broken by passage through a French press at 20,000 p.s.i. followed by the addition of DNase I at a final concentration of 10 pg/ml. The ribosome concentration was determined spectrophotometrically us- ing the value 23 prnollml = 1 A,,,,. The percentage of mutant rRNA was measured for each ribosome preparation by primer extension and quan- titated by densitometry.

E. coli tRNAphe (Boehringer Mannheim) was aminoacylated in a 300-pl reaction containing 50 mM Tris-HC1, pH 7.4,15 mM MgCl,, 50 mM KCI, 1 mM dithiothreitol, 6.25 mMATP, 15 p~ tRNAphe, 62 PM tU-'4Clphe- nylalanine (DuPont NEN, specific activity 485.7 mCi/mmol), and 35 pl of an E. coli mixed aminoacyl-tRNA synthetase preparation (Sigma). The concentration of tRNA was determined spectrophotometrically, us- ing the relationship 1,750 pmol/ml = 1 A,,,,. The aminoacylation reaction was incubated for 15 min at 37 "C, followed by sequential extractions with equal volumes of water-saturated phenol and ch1oroform:isoamyl alcohol (24:l). Aminoacyl-tRNA was precipitated with ethanol, collected by centifugation, dried, and resuspended in 100 pl of 200 mM CH3C0,Na, pH 5.0, and subjected to N-acetylation according to the method of Haenni and Chapeville (21). The extent of aminoacylation, determined as the pmol of ['4Clphenylalanine precipitated by cold 10% trichlorocetic acid/pmol of tRNAPhe, was typically 70% or better.

Peptide bond formation in Assay System A was measured in 50 p1

Conserved Residues of rRNA and tRNA in Peptidyl Dansfer 16165

containing 50 m Tris-HC1, pH 7.4,lOO mM NH,Cl, 10 mM MgCl,, 1 mM dithiothreitol, 100 pg/ml poly(U), 525 m ribosomes, and N-Ac-['4ClPhe- tRNAPhe at the concentrations indicated in the text and figures. Reac- tions were initiated by the addition of puromycin to a final concentra- tion of 1 mM. For inhibition experiments, deacylated tRNAPhe, tRNAPh' transcripts (see below), or chloramphenicol was added to the assay mixtures at concentrations indicated in the text and figures. Reactions were incubated on ice for the times indicated and stopped by the addi- tion of 400 p1 of cold 1.5 M CH,CO,Na, pH 5.2, followed by the addition of 1 ml of ethyl acetate. Af'ter vortexing and phase separation, 500 p1 of the ethyl acetate phase was mixed with 5 ml of Cytoscint (ICN Bio- chemicals), and radioactivity was measured in a Beckman LS 5000TD scintillation counter. Initial rates of reaction were determined from time points during which less than 10% of substrate was consumed.

In assay system B, peptide bond formation was assayed as described for assay system A, except that poly(U) was omitted, and reactions were initiated by the addition of methanol to a final volume of 30%. In all peptide bond formation assays, background controls for hydrolysis (as- say system A) and methanolysis (assay system B), obtained in the absence of puromycin, have been subtracted from the values reported.

Construction, in Vitro Dunscription, and Aminoacylation of Mutant E. coli tRNAph-Plasmid p67CF23, a pUC18 derivative encoding the gene for E. coli tRNAPhe under the transcriptional control of a T7 RNA polymerase promoter (221, was a gift from Olke Uhlenbeck. The plasmid p67CFGGA was constructed by excising the StyI-SphI fragment of p67CF23, spanning the 3' end of the gene, and ligating the remaining gel purified DNA fragment to an annealed, double-stranded oligonucle- otide designed to restore the Sty1 site in the coding region of the tRNA, and to change the trinucleotide sequence at the 3' end of the coding sequence from CCA to GGA. This oligonucleotide duplex also creates an NsiI restriction site, directing cleavage immediately after the 3"termi- nal A of the mutant tRNA gene. This was necessary because the recog- nition sequence for BstNI, used to create a template for in vitro tran- scription in the wild type construct, is destroyed by the C74G/C75G mutation. E. coli strain DH1 was used as the host strain for both mutant and wild type tRNAPhe plasmids. Colonies harboring p67CFGGA were identified by restriction analysis and confirmed by double-stranded sequencing of the entire tRNA gene.

In vitro transcription reactions were as described by Sampson and Uhlenbeck (23), using BstNI-digested p67CF23 and NsiI-digested p67CFGGA as templates for the production of wild type and mutant tRNA transcripts, respectively. Reactions contained a 4-fold excess of GMP over GTP to prime RNA synthesis with a 5' monophosphate (23). T7 RNA polymerase was purchased from New England Biolabs. Tran- scription products and fully modified E. coli tRNAphe for use in controls were purified to single nucleotide resolution by denaturing electro- phoresis as described by Sampson and Uhlenbeck (231, and the A,,, was determined for each RNA. Prior to their use as inhibitors in peptide bond formation assays or as substrates for aminoacylation, transcripts were heated at 85 "C for 2 min and then slowly cooled to room tempera- ture to allow formation of tRNA structure (24).

Mutant and wild type tRNA transcripts were aminoacylated in 30 p1 as described above for tRNAph". After aminoacylation, 0.5 pl of acetic anhydride was added directly to the reaction mixture and incubated on ice for 1 h, followed by the addition of another 0.5 p1 and an additional 1-h incubation. An aliquot was removed for trichloroacetic acid precipi- tation to determine aminoacylation efficiency, and the transcripts were purified on TSK Toyopearl DEAE 650 S (Supelco) as described by Harrington et al. (24).

RESULTS

Expression of the 23 S rRNA Mutation G2252ClG2253C- The phylogenetically conserved 23 S rRNA residues G2252 and G2253 are located in a loop capping a conserved helix (Fig. 1) near the central loop of domain V associated with peptidyl- transferase function. To investigate the role of these residues in ribosome function, the double mutation G2252C/G2253C was constructed by site-directed mutagenesis and subcloned into the rrnB operon encoded by plasmid pGQ7 under transcrip- tional control of the phage A leftward promoter. Plasmid pGQ2252U2253C was maintained in E. coli strain DH1, along with the compatible plasmid pcI857, encoding a thermolabile A repressor. Thus expression of mutant rRNA was repressed at 30 "C and induced at 42 "C.

When cells harboring plasmid pGQ2252C/2253C were

N-ac-[l4C]Phe-tRNAPhc + puromycin

+ 70s ribosomes 70s ribosomes

30% methanol

+ Poly u (or 50s subunits)

(System A) (System B)

N-ac-[14C]Phe-puromycin + tRNAPhC

FIG. 2. Peptide bond formation assays. Standard conditions for both assay systems A and B in this study were 525 m salt-washed 70 S ribosomes (determined experimentally to be in the linear range for the reaction,), 1 mM puromycin (determined experimentally to be saturat- ing for both mutant and wild type ribosomes'), with incubations at 2 "C. Reactions were for 30 min, and N-Ac- [ '~CIP~~-~RNA'~~ was present at 100 m, unless otherwise indicated. In assay system A, poly(U) was present a t 100 pg/ml, and reactions were initiated by the addition of puromycin. In assay system B, reactions were initiated by the addition of methanol to a final concentration of 30%. The reaction product N-Ac- ['4ClPhe-puromycin was extracted into ethyl acetate (27) and quanti- tated by scintillation counting.

streaked on plates and incubated at 42 "C, there was no detect- able growth. In liquid culture, the doubling time of DH1 pcI857 pG2252C/2253C following induction a t 42 "C was 79 2 7 min, whereas that of cells harboring the plasmid containing the wild type rrnB operon, pGQ7, was 44 r 2 min. Thus expression of mutant rRNA compromises cell growth rate, and extended ex- pression is lethal.

The proportion of mutant rRNA in 50 S subunits, 70 S ribo- somes, and polysomes, isolated on sucrose gradients from ly- sates of DH1 pc1857 pGQ2252C/2253C, was determined by primer extension analysis (19). The C2252K2253 rRNA consti- tuted 64 2 4% of the 23 S rRNA in the 50 S fraction, 36 2 2% of the 70 S fraction, and only 13 2 2% of the polysome fraction. Thus the mutant rRNA is processed and assembled into 50 S subunits and 70 S ribosomes, but there is strong discrimination against the incorporation of mutant 50 S particles into poly- somes. Mutant rRNA constituted 50 2 2% of the total 23 S rRNA, as determined from ribosomes prepared without sucrose gradient fractionation.

Peptide Bond Formation by C2252/ (22253 Ribosomes-The chemical protection studies (5, 7) indicate that G2252 and G2253 may be recognition determinants for the 3' nucleotides of peptidyl-tRNA. To determine whether these rRNA residues are important for catalysis of peptidyl transfer, the mutant ribosomes were assayed in a model reaction (Fig. 2, assay sys- tem A) that employs poly(U) as mRNA template, the peptidyl- tRNA analogue N-Ac-Phe-tRNAPhe (100 m) as P site substrate, and puromycin (1 1llh.11 as A site substrate.

In experiments conducted for 30 min at 2 "C, which measure the extent of reaction, ribosomes isolated from the strain DH1 pcI857 pGQ2252C12253C (a mixture of 50% mutant C2252l C2253, and 50% chromosomally encoded, wild type G2252/ G2253 ribosomes) were consistently less active than ribosomes from DH1 pcI857 pGQ7 (100% wild type). The average activity of mutant ribosomes, determined from three separate ribosome preparations in 10 independent experiments, was 74 2 4.4% of wild type ribosomes. This difference between mutant and wild type ribosomes in both the extent and rate (see below) of pep-

16166 Conserved Residues of rRNA and tRNA in Peptidyl B-ansfer

400 I

0 ' I 0 125 250 375 500 625

N-ac-Phe-tRNAPhe, nM FIG. 3. Initial rates of peptidyl transfer. The P site substrate-

dependent rates of N-A~-['~C]Phe-puromycin formation were deter- mined for wild type G2252/G2253 (0) and mutant C2252/C2253 (0) ribosome preparations in assay system A, as described under "Experi- mental Procedures" and in the legend to Fig. 2. N - A C - [ ' ~ C ] P ~ ~ - ~ R N A ~ ~ was employed at the concentrations indicated, and rates were obtained from time points during which <lo% of the substrate was converted to product.

tide bond formation was also observed at 37 oC.2 To determine whether this defect in peptide bond formation

derives from disrupting the interaction of the mutant ribo- somes with P site substrate, we assayed the initial rates of peptidyl transfer. Fig. 3 shows the rates of formation of N-Ac- Phe-puromycin by C2252lC2253 and G22521G2253 ribosomes as a function of N-Ac-Phe-tRNAPhe concentration, with the con- centration of puromycin held constant and saturating (1 mM). Because of the high concentration of ribosomes employed in the assay (525 I") and the high affinity of the deacylated tRNAPhe reaction product for the ribosome (25,26), this assay measures a single catalytic turnover, and therefore steady-state rate ex- pressions cannot be derived. In addition, the presence of wild type ribosomes in the mutant preparations complicates the quantitative interpretation of kinetic data. Nevertheless, at all concentrations of P site substrate tested, the initial rates of reaction for the C2252lC2253 ribosome preparation were lower than that of wild type ribosomes.

The rate of peptidyl transfer by mutant and wild type ribo- some preparations appeared to saturate at or near the same N-Ac-Phe-tRNAPhe concentration (-250 nM), consistent with at least two possibilities. The first is that only the wild type ribo- somes in the mutant preparation are active in peptide bond formation, the C22521C2253 mutation resulting in ribosomes that are incapable of catalyzing peptidyl transfer under the assay conditions employed. Another possibility is that P site binding affinity has not been perturbed in the mutant ribo- somes, but the reactivity of the substrate bound to mutant ribosomes has been diminished. We favor the latter interpre- tation, for several reasons. First, the mutant preparations, which consist of 50% mutant ribosomes, display on average 74% of the activity of wild type ribosomes, not 50%, as might be expected if they were completely nonfunctional in the assays. Second, mutant ribosomes, although underrepresented, are found in polysome preparations, suggesting that they are ca- pable of peptide bond formation in vivo. Third, consistent with the phenotypes conferred by the individual G to C changes at residues 2252 and 2253 (151, we have detected small but repro- ducible effects on the suppression of nonsense and frameshift mutations in a lac Z reporter gene by C2252lC2253 ribosomes,' thus providing functional evidence that the mutant ribosomes participate in protein synthesis in vivo.

In several organisms, mutations in the central loop of do- main V confer resistance in vivo to the antibiotic chloramphen- icol, an inhibitor of peptide bond formation (12, 13). It was therefore of interest to know whether C2252lC2253 ribosomes

K. R. Lieberman and A. E. Dahlberg, unpublished results.

were altered in their sensitivity to this antibiotic in vitro. We measured the extent of peptide bond formation in assay system Ain the presence of 30 p~ chloramphenicol. In control reactions containing 0.03% ethanol, G2252lG2253 ribosomes catalyzed the production of 709 56 fmol of N-Ac-Phe-puromycin, whereas C2252lC2253 ribosome produced 483 7 fmol. In the presence of chloramphenicol, C22521C2253 ribosomes were in- hibited 74% (124 * 1 fmol of product formed), whereas wild type G2252lG2253 ribosomes were inhibited 75% (180 5 fmol formed). Thus the difference in peptide bond formation activity between mutant and wild type ribosomes persisted in the pres- ence of chloramphenicol, and the mutation C2252lC2253 did not appear to interfere with the ability of chloramphenicol to inhibit the peptidyl transfer reaction.

Activity of tRNAphe with a GGA 3' Terminus-A model con- sistent with both the chemical protection data (5, 7) and the phylogenetic conservation of G2252 and G2253 predicts that these residues participate in P site base pairing with the two cytidine residues of the sequence CCA at the 3' end of tRNA. To test this model, we constructed a mutant of a plasmid-encoded E. coli tRNAPh' gene (22) in which the invariant 3' sequence CCA was changed to GGA. Both G741G75 (mutant) and C741 C75 (wild type) tRNAPhe were prepared by in vitro transcription from a T7 RNA polymerase promoter (23). These tRNAPhe tran- scription products differ from tRNAPhe produced in vivo in that they contain no post-transcriptional modifications.

Since deacylated tRNA is known to bind with high affinity to the P site (25, 26), we reasoned that it might function as an inhibitor of peptide bond formation in the puromycin assay. If so, we could then ask whether any differences exist in the efficiency of inhibition of mutant and wild type ribosomes by the tRNAPhe transcripts with CCA or GGA 3' termini. Fig. 4A shows the effect of fully modified deacylated tRNAPhe on the extent of peptide bond formation in assay system A, using 100 n M N-Ac-Phe-tRNAPhe (fully modified) as the P site substrate and 1 mM puromycin as A site substrate. In the absence of added deacylated tRNA, ribosomes from DH1 pcI857 pGQ2252Cl2253C displayed 66% of the peptidyltransferase ac- tivity of ribosomes from the wild type DH1 pcI857 pGQ7 (547 t 1 fmol ofN-Ac-Phe-puromycin formed in 30 min versus 828 k 8). The addition of deacylated tRNAPhe did inhibit the reaction; both wild type and mutant ribosomes were inhibited 20% by the addition of 200 nM deacylated tRNA and were inhibited 50 and 80% by the addition of 400 and 800 nM deacylated tRNA, respectively.

The inhibitory activities of the wild type and mutant tRNAPhe transcripts are shown in Fig. 4, B and C . Regardless of the tRNAphe species used or the concentration employed, the rela- tive difference in activity between G2252lG2253 and C22521 C2253 ribosome preparations persisted (66-70%). The mutant tRNAPhe transcript, G74/G75, inhibited the peptidyltransferase reaction with the same efficiency for both mutant and wild type ribosomes, displaying no preferential interaction with mutant ribosomes (Fig. 4C). Conversely, no preference was observed toward inhibition of wild type ribosomes by the wild type tRNAPhe transcript (Fig. 4B). There was essentially no differ- ence in the concentration dependence of inhibition effected by fully modified tRNAPhe, C74IC75 transcript tRNAPhe, or G741 G74 transcript tRNAPhe, with either ribosome preparation. All three species of tRNAPhe inhibited the reaction by more than 90% when added a t a concentration of 2 PM.' Thus the inhibi- tory behavior of these tRNAPhe species was indistinguishable, indicating that neither post-transcriptional modifications (compare Fig. 4, A and B ) nor a wild type 3' terminus (compare Fig. 4, B and C ) is essential for this activity.

Aminoacylation of tRNAPhe with a GGA 3' Tkrminus-We wished to know whether any differences could be detected in

Conserved Residues of rRNA and tRNA in Peptidyl Pansfer 16167

IOoo :

0 0 200 400 600 800

competitor, nM

FIG. 4. Inhibition of peptidyl transfer by fully modified and in vitro transcribed (unmodified) deacylated tRNA species. The ef- fects of deacylated tRNAPhe on the extent of peptide bond formation by wild type G2252/G2253 (0) and mutant C2252K2253 (0) ribosome preparations were measured in assay system A, under conditions de- scribed in the legend to Fig. 2. The deacylated tRNAPhe species employed were: fully modified tRNAPhe (panel A), the wild type C74/C75 tRNAPhe transcript (panel B ) , and the mutant G74/G75 tRNAphe transcript (panel C ) . All values were obtained from assays conducted in triplicate, and all standard errors were 5 5%.

catalytically productive P site interaction between C2252/ C2253 and G2252lG2253 ribosomes, using the mutant G74G75 and wild type C74/C75 tRNAPhe transcripts directly as P site substrates. This requires aminoacylation of both transcripts. We therefore tested the transcripts as substrates for aminoac- ylation, using the same conditions employed to prepare fully modified Phe-tRNAPhe (see "Experimental Procedures"). As demonstrated previously (22), wild type, unmodified E. coli tRNAPhe was efficiently aminoacylated with phenylalanine. We obtained incorporation of 0.73 * 0.02 pmol of phenylalanine/ pmol of wild type tRNAPhe transcript, using an E. coli mixed synthetase preparation as a source of phenylalanyl-tRNA synthetase.

Surprisingly, the mutant transcript with a GGA 3' end was aminoacylated with nearly 30% efficiency under the same re- action conditions, accepting 0.29 * 0.02 pmol of phenylalanine1 pmol of G74lG75 tRNAPhe transcript. Aminoacylation was measured as the incorporation of [14C]phenylalanine into tri- chloroacetic acid-precipitable material and was dependent upon the addition of enzyme and ATP. The product of the reac- tion of the G741G75 transcript with synthetase, after purifica- tion on DEAE (see below), underwent hydrolysis a t pH 8.0 and 22 "C, at the same rate as both the aminoacylated wild type transcript and fully modified tRNAPh".' This indicates that phe- nylalanine was covalently attached to the G74/G75 transcript and provides evidence for an aminoacyl ester linkage with nor- mal chemical reactivity.

P Site Substrate Activity of Aminoacylated tRNAphe Dan- scripts with CCA and GGA 3' Ends-The preparation of ami- noacylated tRNAPhe transcripts for peptide bond formation as- says is complicated by the tendency of unmodified E. coli

tRNAPhe to denature. This requires that purification on DEAE, rather than phenol extraction and ethanol precipitation, be employed following aminoacylation (24). In addition, prepara- tion of P site substrate requires N-acetylation and subsequent purification. To avoid the loss of material incurred by two sepa- rate purification steps, we decided to acetylate directly follow- ing aminoacylation, followed by a single DEAE purification step.

The P site activity of fully modified tRNAphe, prepared in this fashion, was compared with substrate prepared by the stand- ard methods (Table I, first and second lines). Although a loss in activity was observed (20% with wild type ribosomes, 15% with the mutant ribosome preparation), the majority of the sub- strate reacted to form a product that extracted into ethyl ac- etate at pH 5.2, indirectly indicating that it was N-Ac-Phe- puromycin (27).

In contrast to the results of the inhibition experiments with the deacylated forms of these tRNAs, the absence of post-tran- scriptional modifications had a serious effect on catalytically productive interaction with the P site. The absence of modifi- cations reduced the reactivity of the C74lC75 tRNA"" tran- script by 63 and 67% with G2252lG2253 ribosomes and C2252/ (22253 ribosomes, respectively, when compared with the in vivo synthesized, fully modified tRNA prepared under identical con- ditions (compare Table I, third line with second line).

Further loss of activity occurred, as could be expected from the deacylated tRNA inhibition experiments (see above), when the concentration of deacylated tRNA was raised in the wild type transcript preparation (compare Table I, fourth line with third line). This addition was necessary to provide a control for the higher concentration of deacylated tRNA present in the GGA preparation, due to the lower aminoacylation efficiency of the GGA species. Despite the inhibitory influences conferred by the presence of deacylated tRNA and the absence of tRNA modifications, the difference between the wild type and mutant ribosome preparations was still detected. However, aminoacy- lated, N-acetylated G74/G75 tRNAPhe transcript displayed no activity with either mutant or wild type ribosomes (Table I, fifth line).

Assay system A may not be sufficiently sensitive to detect potentially low levels of activity with the G741G75 tRNAPh' transcript. We therefore tested the same substrates in another model system for peptidyltransferase. Assay system B (Fig. 2) does not require synthetic mRNA (poly(U)) but instead requires the presence of 30% methanol. The necessary macromolecular components are 70 S ribosomes or the 50 S subunit (11, amino- acyl-tRNA or an aminoacyloligoribonucleotide corresponding to the 3' end of tRNA as P site substrate (81, and puromycin as the A site substrate.

As in assay system A, we observed a difference in peptidyl- transferase activity between G2252lG2253 and C2252IC2253 ribosomes. With fully modified N-Ac-Phe-tRNAPhe substrate, the C2252IC2253 mutant ribosome preparation displayed 77% of the activity of wild type ribosomes (Table 11, second line). In contrast to assay system A, this assay appeared insensitive to the presence of deacylated tRNA (compare Table 11, second line with first line) and insensitive to lack of post-transcriptional modifications in the C741C75 tRNAPhe transcript (Table 11, sec- ond and third lines). However, under conditions in which the wild type tRNA"' transcript displayed significant activity, the aminoacylated, N-acetylated G74lG75 tRNAPhe transcript was again unreactive as a P site substrate (Table 11, fourth line). Thus, under two different sets of reaction conditions, N-Ac-Phe- tRNAPhe with a GGA 3' sequence was incapable of catalytically productive interaction with the P site. Furthermore, this sub- strate was unable to compensate for any portion of the reduced activity displayed by the C2252lC2253 mutant ribosomes.

16168 Conserved Residues of rRNA and tRNA in Peptidyl Dansfer

P site substrate activity of fully modified and in vitro transcribed tRNAph” species with C2252lC2253 (mutant) TABLE I

and G2252lG2253 (wild type) ribosomes in assay system A Assays were conducted for 30 min at 2 “C under the conditions for system A described in the legend to Fig. 2. The reported values and standard

errors were determined from two separate experiments, both conducted in triplicate.

tRNA species Deacylated __ tRNA

N-Ac-Phe-puromycin formed

Wild t w e ribosomes Mutant ribosomes

Fully modified“ Fully modifiedb Transcript, 3’CCAb Transcript, 3’CCAb Transcript, 3’GGAb

nM

33 33 33

233 233

~~~

fmol 536 f 16 433 f 2 430 f 18 366 f 10 159 f 3 126 2 8 106 t 3 85 f 3 -1 -c 4 -6 +. 11

Fully modified tRNAphe was aminoacylated with [14Clphenylalanine (see “Experimental Procedures”) followed by phenol extraction and ethanol

tRNAPhe species were aminoacylated and then treated with acetic anhydride prior to purification, as described under “Experimental Proce-

~~~~~~~~

precipitation. The Phe-tRNAPhe was then subjected to acetylation according to Ref. 21, followed by ethanol precipitation.

dures.“ The aminoacylated, acetylated tRNAPhe species were then purified on DEAE as described in Ref. 24.

TABLE I1 P site substrate activity of fully modified and in vitro transcribed tRNAph” species with C2252/C2253 (mutant)

and G2252lG2253 (wild type) ribosomes in assay system B Assays were conducted for 30 min at 2 “C under the conditions for system B described in the legend to Fig. 2. All values were determined from

two separate experiments, both conducted in triplicate.

tRNA species“ Deacylated N-Ac-Phe-puromycin formed tRNA Wild tvue ribosomes Mutant ribosomes

Fully modified Fully modified Transcript, 3’CCA Transcript, 3‘GGA

RM

50 494 494 494

fmol 604 f 4 464 t 36 764 f 76 706 f 66

464 e 42 568 50

9 f 8 0 f 10

All P site substrates used in this assay were purified on DEAE, following aminoacylation and N-acetylation, as described under “Experimental Procedures.”

DISCUSSION In this study we sought to test whether protection of bases

G2252 and G2253 in 23 S rRNA from kethoxal modification by P site-bound tRNA (5) represents an interaction essential for catalysis of peptide bond formation. Further, we tested whether these protections derive from Watson-Crick base pairing be- tween these 23 S rRNA residues and the 3’ end of aminoacyl- tRNA in the P site.

The deleterious effect on growth rate and lethality of long term expression of C2252lC2253 23 S rRNA, taken together with the underrepresentation of the mutant rRNA in poly- somes, are indicative of the disruption of an essential ribosomal function and are consistent with the phylogenetic conservation of these residues.

The peptidyltransferase activity of ribosomes isolated from cells expressing C2252lC2253 23 S rRNA was assayed in vitro. Although caution must be applied to the interpretation of re- sults obtained using a mixed population of mutant and wild type ribosomes, ribosome preparations containing 50% mutant C2252lC2253 rRNA consistently displayed less peptidyltrans- ferase activity than wild type preparations prepared in paral- lel. This difference was detected in both the extent and rate of reaction, at both 2 and 37 “C, and in the presence of the inhibi- tors chloramphenicol or deacylated tRNA. Furthermore, the difference in activity was detected in two different assay sys- tems, one dependent on mRNAfor activity (Fig. 2, assay system A), the other independent of mRNA and dependent on the pres- ence of alcohol (Fig. 2, assay system B). This decrease in pep- tidyltransferase activity in the mutant ribosome preparations provides evidence for the intimate involvement of these 23 S rRNA residues in peptidyltransferase function and suggests that G2252 and G2253, or a higher order structure in which they participate, is important for catalysis.

The experiments utilizing deacylated C74lC75 tRNAPhe and G74/G75 tRNAPhe transcripts as inhibitors of peptide bond for-

mation (Fig. 4) failed to distinguish any additional differences between C2252lC2253 ribosomes and G2252lG2253 ribosomes. No preferential interaction of C74lC75 tRNAph” with wild type ribosomes or of G74lG75 tRNAPhe with mutant ribosomes was observed. However, some interesting features of deacylated tRNA binding were revealed. The binding of deacylated tRNAphe, as determined by the ability to inhibit peptide bond formation, was insensitive to the identity of bases 74 and 75 of the tRNA. Under the conditions of this assay, most, if not all of the deacylated tRNAwould be expected to bind to the P site (25, 26). Thus these results extend the findings of Lill et al. (28) that modifications of the 3”terminal adenosine of deacylated tRNAPhe have little or no effect on P site binding.

Interestingly, we found that the binding of deacylated tRNAPh‘ was also unaffected by the presence or absence of post-transcriptional modifications of the tRNA. This is in marked contrast to the greater than 60% decrease in activity effected by the absence of modifications in the wild type tRNAPhe transcript, when it is aminoacylated and employed as a P site substrate in assay system A (Table I). The ability of unmodified or undermodified tRNAs to perturb translational rate and fidelity, both in vivo and in vitro, has been well docu- mented (24,29). The results obtained in this study suggest that unmodified tRNAPhe is compromised in catalytically productive P site interaction. This decrease in activity may derive from the influence of post-transcriptional modifications on the P site interaction with the 30 S subunit and mRNA, since when meas- ured in assay system B, in which methanol can relieve the requirement for these two components, the fully modified and unmodified substrates have comparable activity (Table 11). This is not surprising, given that aminoacyl derivatives of the CCA terminus are sufficient for P site reactivity in this assay (8) and that none of the modifications occurs in this region of the tRNA.

The N-acetylated, aminoacylated G741G75 tRNAPhe tran- script failed to react in two different peptide bond formation

Conserved Residues of rRNA and tRNA in Peptidyl ll-arzsfer 16169

assays, with either mutant or wild type ribosomes (Tables I and 11). Recently, mutants of E. coli tRNA?' with 3' sequences ACA and GCA have been described (30). These tRNAs participate in peptidyl transfer as P site substrates in vivo, where they have the property of inducing readthrough of nonsense codons im- mediately following valine codons. Thus, when present in the P site, these mutations in the tRNA 3' sequence affect the A site substrate specificity of peptidyltransferase. A mutant of E. coli tRNAMet with the 3' sequence UCA, prepared by bisulfite mu- tagenesis, was inactive in the puromycin reaction in vitro (31). Taken together with the above results, our inability to detect activity with G74lG75 N-Ac-Phe-tRNAPhe indicates that the 3' CCA sequence is critically important for accurate, catalytically productive P site function.

The failure of aminoacyl-tRNA bearing a 3' GGA sequence to compensate for the defect in peptide bond formation caused by the rRNA mutation C2252lC2253 does not formally disprove a model predicting canonical base pairing between these resi- dues. Efficient peptidyl transfer may require a precise geomet- rical relationship between aminoacyl-tRNA substrates and the ribosome. As such, a G-C base pair may not be equivalent to a C-G pair. It is also possible that G2252 and G2253 interact with the 3' end of peptidyl-tRNAin a noncanonical fashion, and thus the G74lG75 tRNAPhe transcript would not be expected to com- pensate for disruption of such an interaction. Alternatively, the inactivity of the G74lG75 tRNAPh' transcript as a P site sub- strate with wild type ribosomes is consistent with canonical base pairing of the peptidyl-RNA with another, as yet uniden- tified, region of the rRNA.

We have shown that mutations at conserved nucleotide resi- dues of both 23 S rRNA and tRNA compromise ribosome cata- lyzed peptide bond formation, yet we observe no restoration of function when the two mutant translational components are tested together. This leaves unanswered the question of whether the P site protections of G2252 and G2253 derive from direct rRNA contacts with tRNA or from a change in 50 S subunit structure induced by tRNA binding. Among the domain V P site protections, G2252 and G2253 are the most distal from the central loop in the secondary structure model. The central loop residues U2506, U2584, and U2585 are all protected by intact deacylated tRNA, but not by tRNA lacking the 3"termi- nal adenosine (5). If the contacts between tRNA and any of these uridine residues are direct, and the contacts between the two cytidines of the tRNA and G2252 and G2253 are direct, these elements of 23 S rRNA must be quite near to one another in the tertiary structure. With the exception of readily testable models such as Watson-Crick base pairing, distinguishing be-

tween direct rRNA-tRNA contacts and indirect protections must await elucidation of the spatial arrangement of the re- markably conserved rRNA features in this critical region of the ribosome.

single-stranded DNA and Olke Uhlenbeck for plasmid p67CF23 and Acknowledgments-We thank William Tapprich for uracil-containing

DE- Toyopearl. We gratefully acknowledge Michael O'Connor and

gory, Stephen Lodmell, Jill Thompson, and George Q. Pennabble for Matthew Firpo for critical reading of the manuscript and Steven Gre-

stimulating discussions.

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