mutations at nucleotides g2251 and u2585 of 23 s rrna perturb the peptidyl transferase center of the...

J. Mol. Biol. (1997) 266, 40±50

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Mutations at Nucleotides G2251 and U2585 of 23 SrRNA Perturb the Peptidyl Transferase Center ofthe Ribosome

Rachel Green, Raymond R. Samaha and Harry F. Noller*

Center for Molecular Biology ofRNA, Sinsheimer LaboratoriesUniversity of California, SantaCruz, California, 95064, USA

Present address: R. R. Samaha,460 Page Mill Road, Palo Alto, CA

0022±2836/97/060040±11 $25.00/0/m

Previous experiments have shown that the phylogenetically conservedG2252 of 23 S rRNA forms a Watson-Crick base-pair with C74 of pepti-dyl-tRNA. In the studies presented here, site-directed mutations wereintroduced at two other conserved positions in 23 S rRNA, G2251 andU2585, that were previously implicated in interaction of the CCA accep-tor end of tRNA with the 50 S subunit P site. The mutant 23 S rRNAswere characterized by determining (1) the in vivo phenotypes, (2) the abil-ity of mutant ribosomes to bind tRNA oligonucleotide fragments in vitro,using footprinting with allele-speci®c primer extension and (3) the abilityof mutant ribosomes to catalyze peptide bond formation using a chimericreconstitution approach. Mutations at either position confer a dominantlethal phenotype when the mutant 23 S rRNA is coexpressed with the en-dogenous wild-type 23 S rRNA. Mutations at 2585 disrupt binding of thewild-type (CCA) tRNA oligonucleotide fragment and cause a modestdecrease in the peptidyl transferase activity of reconstituted ribosomes.By contrast, mutations at 2251 abolish both binding of the wild-type(CCA) tRNA fragment and peptidyl transferase activity using the wild-type tRNA fragment. In neither case was the loss of binding or peptidyltransferase activity suppressed by mutations in the tRNA oligonucleotidefragment. Chemical modi®cation analysis revealed that mutations at 2251perturb the reactivity of bases 2584 to 2586, providing further evidencethat the 2250 loop of 23 S rRNA interacts, either directly or indirectly,with the 2585 region in the central loop of domain V of 23 S rRNA.

# 1997 Academic Press Limited

Keywords: ribosome; peptidyl transferase; tRNA binding; chimericreconstitution; rRNA mutants
*Corresponding author
Introduction

A central function of the large subunit of the ribo-some is to catalyze the acyl transfer of activatedamino acids to form peptide bonds (peptidyl trans-ferase). In performing this function, the ribosomemust bind and orient the appropriate templatedaminoacylated tRNAs for catalysis. A variety ofcrosslinking and chemical footprinting studieshave suggested that, in addition to its base-pairingwith the mRNA codons, important contacts aremade by tRNA with the 16 S and 23 S ribosomalRNAs (Prince et al., 1982; Barta et al., 1984; Moazed& Noller, 1986, 1989; Wower et al., 1989; Mitchellet al., 1993; von Ahsen & Noller, 1995). Chemical

Array Technologies,94306, USA.

b960780

footprinting data focused attention on several uni-versally conserved nucleotides in domain V of 23 SrRNA as candidates for direct interactions with thesimilarly conserved CCA end of tRNA. Two invar-iant guanosine residues in 23 S RNA, G2252 andG2253, were identi®ed whose protection fromkethoxal modi®cation by P-site (peptidyl or donor)bound tRNA was dependent on the presence ofthe CCA 30 terminus of tRNA (Moazed & Noller,1989); a third invariant guanosine located in the2250 loop, G2251, may be similarly protected bythe CCA 30 terminus of P-site bound tRNA (R.G.and H.F.N., unpublished results). Three invarianturidine residues in 23 S rRNA, U2506, U2584 andU2585, were identi®ed whose protection fromCMCT (carbodiimide) modi®cation by P-site-bound tRNA was dependent on the presence ofthe 30 terminal A of the invariant CCA sequence of

# 1997 Academic Press Limited

Figure 1. A schematic of the sec-ondary structure of domains V andVI of E. coli 23 S rRNA (Noller et al.,1981) showing the locations ofnucleotides G2251 and U2585. Theapproximate cleavage positions at2300 and 2530 used in the chimericreconstitution experiments and thesilent mutations introduced atallele-speci®c priming sites, I andII, that were used to monitor tRNAor oligonucleotide interactions atpositions G2252-2253 or U2585, re-spectively, are indicated (Aagaardet al., 1991; Powers & Noller, 1993;Samaha et al., 1995).

Mutations at G2251 and U2585 of 23 S rRNA 41

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tRNA (Moazed & Noller, 1989). These ®ndingssuggested possible Watson-Crick pairing relation-ships between the protected bases in 23 S rRNAand the respective bases in tRNA. The physiologi-cal importance of G2252 and G2253 was examinedby site-directed mutagenesis. Mutations at G2252resulted in a dominant lethal phenotype in cells ex-pressing the mutant 23 S rRNA. By contrast, twosite-directed mutations at the 30 adjacent G2253(A2253 and U2253) conferred only a recessiveslow-growth phenotype, and ribosomes bearing

these mutations at position 2253 retain their abilityto bind wild-type tRNA oligonucleotide fragmentsin vitro (Samaha et al., 1995). Parallel studiesshowed that the mutation C2253 has a signi®cantlystronger, dominant slow-growth phenotype(Gregory et al., 1994). Base-pairing between theCCA sequence of tRNA and these two conservedguanine residues was tested by in vitro genetic ex-periments that established that a Watson-Crickinteraction between G2252 of 23 S RNA and C74 oftRNA is indeed critical for binding the tRNA sub-

42 Mutations at G2251 and U2585 of 23 S rRNA

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strate in the P site of the 50 S subunit and for pepti-dyl transferase activity (Samaha et al., 1995). Nosuch relationship was found for G2253, however.

In this study, we have investigated the effects ofbase mutations at G2251 and U2585, two of theother bases in 23 S rRNA whose protections de-pend on the CCA sequence of tRNA (Figure 1).Each set of mutant 23 S rRNAs was assessed for itslevel of function in several different assays: (1) thein vivo phenotype of cells expressing mutant 23 SrRNA; (2) the ability of mutant ribosomes to bindtRNA oligonucleotide fragments in vitro; and (3)the ability of reconstituted mutant ribosomes tocatalyze peptide bond formation. Potential com-pensatory mutations in the CCA-containing oligo-nucleotide substrate (at positions C74, C75 andA76) were tested for their ability to suppress anyobserved binding or catalytic de®ciencies of themutant ribosomes. Analysis of both sets of mu-tations relied on our ability to speci®cally generateand analyze mutant ribosome populations. For theanalysis of tRNA binding to the mutant ribosomes(necessarily found in a mixed population withwild-type ribosomes), the allele-speci®c primer ex-tension strategy was utilized. Here, silent mu-tations were incorporated into the cloned, mutantcopy of 23 S rRNA allowing different DNA oligo-nucleotide primers to distinguish between the twopopulations (Aagaard et al., 1991; Powers & Noller,1993; Samaha et al., 1995). For the peptidyl trans-ferase assay, pure populations of mutant ribosomeswere prepared using a recently developed chimericin vitro reconstitution approach that allows in vitrotranscribed partial 23 S rRNA transcripts to replacecorresponding regions of Escherichia coli 23 S rRNA(Samaha et al., 1995; Green & Noller, 1996).

Results

Site-directed mutations at G2251 and U2585

Mutations were introduced at positions G2251 andU2585 (Figure 1) of E. coli 23 S rRNA genes as partof an rrnB operon-containing plasmid constructunder transcriptional control of the inducible lamb-da PL promoter. Expression of the mutant 23 SrRNA is induced by growth at 42�C. Cells expres-sing the plasmid-borne copy of 23 S rRNA containa mixture of mutant and chromosomally encodedwild-type ribosomes. Dominant phenotypes are ex-pressed on ampicillin-containing media. Recessiveeffects are observed in the presence of the anti-biotic erythromycin, which speci®cally inhibits theactivity of ribosomes bearing the chromosomal co-pies of 23 S rRNA; the mutant 23 S rRNA mol-ecules carry the G2058 mutation conferringerythromycin resistance. A comparison of 23 SrRNA expression at 42�C, with or without the en-gineered priming sites, indicates that the primingsite mutations are indeed silent (Figure 2(a) and(b), WT �/ÿ PS). By contrast, expression of 23 SrRNA containing mutations at G2251 (A2251 and

U2251) at 42�C caused a dramatic dominant inhi-bition of growth (Figure 2(b)) , whereas uninducedcells (30�C) grew normally (Figure 2(a)). A similardominant growth phenotype was observed for allthree mutations at U2585 (Figure 2(c) and (d)).

Binding of tRNA oligonucleotide fragments

Binding of full-length tRNA to 70 S ribosomes isdetermined primarily by its interactions with the30 S subunit. To study interaction of the CCA ac-ceptor end of the tRNA with the 50 S subunit P site,minimal tRNA substrates were substituted for in-tact tRNAs in the chemical footprinting analysis ofthe wild-type and mutant ribosomes. Early studiesby Monro and colleagues demonstrated that N-blocked aminoacylated tRNA oligonucleotide sub-strates such as CACCA-F-Met could substitute inthe P site for intact peptidyl tRNAs (Monro et al.,1968). These tRNA fragments are bound stably tothe 50 S ribosomal P site in the presence of metha-nol or ethanol and the antibiotic sparsomycin, andare reactive with minimal A-site substrates such aspuromycin in what is termed the fragment reaction(Monro et al., 1969). Furthermore, the set of 23 SrRNA nucleotides protected from chemical modi®-cation by these minimal P-site substrates is vir-tually identical with those protected by intacttRNAs (Moazed & Noller, 1991). The footprintingpatterns of the different tRNA oligonucleotide frag-ments on wild-type and mutant ribosomes are dis-tinguished by taking advantage of silent mutationsthat have been introduced into the cloned copy of23 S rRNA (the mutant version) to allow for allele-speci®c primer extension (Aagaard et al., 1991;Powers & Noller, 1993; Samaha et al., 1995: prim-ing sites I and II are shown in Figure 1). As in ourprevious analysis of G2252 and G2253 (Samahaet al., 1995), carbodiimide (CMCT) modi®cationwas used to monitor the reactivity of U2585, anucleotide whose protection by the CCA end oftRNA is known to be dependent on the presenceof the 30-terminal A76 (present in the wild-typeand mutant tRNAs tested). By monitoring tRNAbinding at a site distinct from the site of mutationin 23 S rRNA, two potential ambiguities areavoided: ®rst, as the identity of the base of interestis changed, different chemical probes would beneeded to study the protection pattern (e.g. whenG2251 is changed to U, it would be necessary toswitch from kethoxal to CMCT modi®cation), andsecond, any localized structural changes resultingfrom the mutation could perturb the primer exten-sion patterns, making direct comparisons dif®cult.Either mutation at G2251 (A2251 or U2251) of 23 SrRNA abolishes binding of wild-type tRNA frag-ment CACCA-N-Ac-Phe. Mutation of either C74 orC75 caused loss of binding of tRNA fragments towild-type ribosomes. Potential Watson-Crick com-pensatory mutations C74 and C75 (CAUCA-N-Ac-Phe and CACUA-N-Ac-Phe for A2251 andCAACA-N-Ac-Phe and CACAA-N-Ac-Phe forU2251) fail to suppress loss of binding (Figure 3(a)).

Figure 2. Expression of rRNA genes carrying mutations at positions G2251 and U2585 in 23 S rRNA. (a) and (b) DH1cells transformed with wild-type plasmid pLK45 (WT(ÿPS)), wild-type plasmid containing allele-speci®c primingsites I and II (WT(�PS)), and plasmid containing the priming sites in addition to mutations at G2251 (2251A and2251U) were plated on solid medium at 30�C or at 42�C, respectively. (c) and (d) DH1 cells transformed with wild-type plasmid pLK45 containing allele-speci®c priming sites (WT(�PS)) or plasmid containing allele-speci®c primingsites in addition to mutations at U2585 (2585A, 2585G and 2585C) were plated on solid medium at 30�C or 42�C,respectively.


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Thus, while G2251 is universally conserved and itsidentity is clearly essential to a functionally intactP site, the function of ribosomes containing mu-tations at this position is not rescued by nucleotidesubstitutions at positions C74 or C75 in the tRNAoligonucleotide substrate.

Next, we examined the effect of mutations atU2585 (A2585, C2585 and G2585) on tRNA frag-ment binding. As above, to assay binding we mon-itored the reactivity of a tRNA-protected sitedistinct from the site of mutation; in this case,kethoxal protection of G2252-G2253 was used as adiagnostic probe. In this experiment, tRNA oligo-nucleotide fragments bearing mutations at positionA76 were tested for their ability to suppress ob-served losses in binding ef®ciency. Wild-type(U2585) ribosomes were footprinted by the wild-type tRNA fragment CCACCA-N-Ac-Met but not

by the A76 mutant fragments CCACCC-N-Ac-Metor CCACCU-N-Ac-Met; the mutant (A2585) ribo-somes were not footprinted by either wild-type ormutant fragments (Figure 3(b)). The dominantnegative in vivo phenotype conferred by the A2585mutation may result from a failure to bind thetRNA fragment appropriately. Elongator methion-ine tRNA was used for the construction of A76mutant tRNAs because of the dif®culties encoun-tered in aminoacylating A76 mutant phenylalaninetRNAs; in the case of the CCG and CCU tRNAmutants, essentially all of the input tRNA was con-verted to a product that comigrated with CACCA-N-Ac-Phe by paper electrophoresis at pH 3.5. Thisconversion is likely to be the result of CCA repairenzymes present in the S100 extract (Deutscher,1983) and the phenylalanyl tRNA synthetase prep-aration used.

Figure 3. Binding of wild-type and mutant aminoacyl-oligonucleotides to the P site of 50 S ribosomal subunitscontaining wild-type or mutant 23 S rRNA, assayed byprotection of U2585 from attack by CMCT or by protec-tion of G2252/G2253 from attack by kethoxal, respect-ively. WT indicates wild-type 23 S rRNA, and A2251,U2251 and A2585 are the corresponding mutant 23 SrRNAs. A and G indicate sequencing lanes, K is unmo-di®ed 23 S rRNA, sp indicates the presence (�) orabsence (ÿ) of sparsomycin. CCA indicates the wild-type tRNA oligonucleotide fragment CACCA-(N-Ac-Phe), and ACA, CAA, UCA, CUA the correspondingmutant versions of the oligonucleotide in (a), andCCACCA-(N-Ac-Met), and CCC, CCU the correspond-ing mutant versions of the oligonucleotide in (b).Chemical reactivity of U2585 was monitored by primerextension from priming site II and reactivity of G2252/G2253 was monitored from priming site I, respectively,as described for Figure 1.

Figure 4. (a) and (b) Chimeric reconstitution approachfor analysis of mutations at G2251 and U2585, respect-ively. Activity refers to the peptidyl transferase activityof the chimeric reconstitution reaction using wild-type23 S rRNAs; � � , high activity; �, 10 to 30-fold loweractivity; ÿ, no detectable activity.


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Peptidyl transferase activity

The peptidyl transferase activity of the mutant ri-bosomes was assayed using a recently developed

chimeric reconstitution approach (Samaha et al.,1995; Green & Noller, 1996). This is necessary be-cause pure mutant rRNA cannot be expressedin vivo due to the existence of multiple chromoso-mal copies of 23 S rRNA, and in vitro transcribed23 S rRNA is not functional in in vitro reconstitu-tion reactions. However, because only a small seg-ment of natural 23 S rRNA (extending frompositions ca 2445 to 2523) is required for in vitro re-constitution, fragments of natural 23 S rRNA (con-taining the critical segment) can be complementedby in vitro transcripts of the remaining regions of23 S rRNA to form functional particles (Green &Noller, 1996). Mutations are thus readily incorpor-ated into the partial in vitro 23 S rRNA fragment.

Another important feature of our approach is theuse of a highly sensitive peptidyl transferase assaythat is compatible with the low ef®ciency of thechimeric reconstitution procedure. This assay usesthe standard fragment reaction, in which puromy-cin, an aminoacyl-tRNA analog, acts as a nucleo-phile to attack the N-blocked aminoacylatedfragment of tRNA in the P site, in this case the 30terminus of elongator tRNA methionine, CCAC-CA-N-Ac-[35S]Met, to form as the product N-Ac-

Figure 5. Peptidyl transferase assay. Phosphorimager ex-posure of paper electrophoresis analysis of the peptidyltransferase reaction. (a) Catalyzed by the wild-type(G2251-natural (nat) or in vitro transcribed (T7)) andmutant (A2251-T7 and U2251-T7) 23 S rRNA chimericreconstitution products using the wild-type (CCACCA-N-Ac-[35S]Met) and mutant (CCACAA-N-Ac-[35S]Metand CCACUA-N-Ac-[35S]Met) tRNA oligonucleotidesubstrates. (b) Catalyzed by the wild-type (U2585) andmutant (A2585, C2585 and G2585) 23 S rRNA chimericreconstitution products using the wild-type (CCACCA-N-Ac-[35S]Met) and mutant (CCACCC-N-Ac-[35S]Met,CCACCG-N-Ac-[35S]Met and CCACCU-N-Ac-[35S]Met)tRNA oligonucleotide substrates. Spots represent theproduct N-Ac-[35S]Met-puromycin.


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[35S]Met-puromycin (NMP). By resolving the ribo-some-catalyzed product (NMP) from an excess ofnon-enzymatic hydrolysis and methanolysis pro-ducts, the signal-to-noise ratio has been optimizedto allow detection of peptidyl transferase activities®ve to six orders of magnitude below that of native50 S subunits (Green & Noller, 1996). This sensi-tivity is suf®cient for analysis of the chimeric re-constitutions presented below.

Mutations at G2251 were characterized using thechimeric reconstitution system described pre-viously for the G2252 mutations (Samaha et al.,1995: and see Figure 4(a)). Brie¯y, 50 and 30 RNaseH fragments of natural 23 S rRNA (extending fromnucleotides 1 to 2294 and 2315 to 2904) were gener-ated by targeting a complementary DNA oligonu-cleotide to positions 2295 to 2314 of 23 S rRNA.When the two natural, puri®ed fragments of 23 SrRNA are combined in a standard in vitro reconsti-tution reaction with 5 S rRNA and proteins fromthe large subunit (TP50), catalytically active 50 S

subunits are obtained. The peptidyl transferase ac-tivity of the crude reconstitution mixture ismeasured to assess the ef®ciency of the reconstitu-tion reaction. Overall, the ef®ciency of the fragmen-ted 23 S rRNA reconstitution reaction (both piecesnatural) is low (ca 1%) when compared with thatof native 50 S subunits. This loss in activity is onlypartially the result of fragmentation; much of theloss results from the denaturing treatments that thenatural RNAs must undergo in order to be separ-ated from one another on sucrose gradients (Green& Noller, 1996). Importantly, the background ac-tivity observed for the 30 natural fragment reconsti-tuted alone is below the limits of detection (datanot shown).

For the analysis of mutant 23 S rRNAs, the 50 frag-ment of 23 S rRNA extending from nucleotides 1 to2314 was substituted with wild-type or mutantin vitro partial transcripts. When the wild-type 50in vitro transcript is combined with the wild-type 30natural fragment of 23 S rRNA in a reconstitutionreaction, the peptidyl transferase activity of the re-sulting mixture is 30% of that of the analogousreconstitution with two natural fragments(Figure 5(a)). Chimeric reconstitution reactions con-taining the mutant 50 in vitro transcripts (A2251and U2251) were severely de®cient in peptidyltransferase activity; wild-type tRNA oligonucleo-tide fragments were utilized at least 200-fold lessef®ciently by both mutant ribosomes (A2251 andU2251) than by the corresponding wild-type(G2251) ribosomes (Fig. 5a). No suppression of thisdecrease in peptidyl transferase activity was ob-served using the C75 mutant tRNA fragments. Theobserved loss in peptidyl transferase activity isconsistent with the severe dominant lethal pheno-type conferred by these mutations in vivo, the de-creased representation of the mutant ribosomes inpolysomes (data not shown), and with the loss of atRNA fragment footprint observed for A2251 andU2251 mutant 50 S subunits. Because the mutantribosomes are well represented in the populationof tight-couple 70 S ribosomes in the cell (data notshown), it is unlikely that the observed de®ciencyin peptidyl transferase activity is the result of poorin vitro reconstitution by these mutant rRNAs.

To test the peptidyl transferase activity of 50 S sub-units containing mutations at U2585, a similarstrategy was employed (Figure 4(b)). Use of adifferent RNase H target was necessary because inthe previously described chimeric reconstitutionU2585 was contained within the 30 natural frag-ment. Using a complementary DNA oligonucleo-tide targeted to positions 2524 to 2538, 50 and 30natural fragments of 23 S rRNA were generatedand puri®ed on sucrose gradients as above. Be-cause of a tendency for these two natural frag-ments to remain tightly associated, two successivesucrose-gradient puri®cations were required to ob-tain 50 natural fragment that was suf®ciently freeof 30 natural fragment to give low backgroundlevels of peptidyl transferase activity when recon-stituted alone (<1% relative to the two-piece natu-

Figure 6. Modi®cation of 2584 to 2586 in 23 S rRNA byCMCT in wild-type (WT) and mutant (A2251 andU2251) ribosomes. A and G indicate sequencing lanes, Kis unmodi®ed 23 S rRNA and the time of modi®cation(in minutes) is indicated.


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ral reconstitution). As above, the two natural 23 SrRNA fragments are combined in a reconstitutionreaction with 5 S rRNA and TP50, and the peptidyltransferase activity of the resulting reconstitutiondetermined. The ef®ciency of this two-piece naturalreconstitution containing a break at position 2530is relatively high (5% of the activity of native 50 Ssubunits) when compared with a number of differ-ent fragmentation sites that have been tested (datanot shown).

Next, wild-type and mutant 30 in vitro transcriptswere prepared. In this two-piece system, when the30 natural fragment is substituted with the corre-sponding 30 in vitro transcript, the peptidyl trans-ferase activity is 66% of that of the correspondingreaction with both natural fragments (Green &Noller, 1996). To test the activities of the U2585mutant ribosomes, the different mutant 30 in vitrofragments were combined in a reconstitution reac-tion with the natural 50 fragment and the peptidyltransferase activity was measured using wild-typeand A76 mutant tRNA oligonucleotide fragments.Each of the U2585 mutations has some effect onpeptidyl transferase activity with wild-type frag-ment; A2585 is 10(�/ÿ1)% as active as U2585 (wt),C2585 is 30(�/ÿ15)% as active and G2585 is30(�/ÿ5)% as active (Figure 5(b)). While these aresigni®cant decreases in activity, the severity doesnot compare with that of the peptidyl transferasede®ciency observed with mutations at 2251 (or2252). No compensatory activity is observed withthe A76 mutant tRNA fragments and the observeddecreased levels of peptidyl transferase activity areconsistent with the inability of A2585 mutant ribo-somes to bind wild-type tRNA fragment(Figure 3(b)).

Chemical probing analysis of 2251mutant ribosomes

Because of the severe effect of the 2251 mutationson both fragment binding and on peptidyl transfer-ase activity, we tested for possible structural per-turbations by examining the reactivities of uridineresidues in the peptidyl transferase center usingthe carbodiimide reagent, CMCT. Primer extensionfrom the two allele-speci®c priming sites (I and II)located at positions ca 2310 and 2700, respectively,was used to monitor the modi®cation pattern oftwo different segments of 23 S rRNA, from pos-itions 2100 to 2250 and 2500 to 2650, which com-prise much of the peptidyl transferase-associatedregion of 23 S rRNA. This analysis showed that theRNA in mutant (A2251 and U2251) ribosomes hasa pattern of reactivity that is generally indistin-guishable from that of wild-type ribosomes. How-ever, there are signi®cant differences speci®cally inthe reactivities of uridine residues 2584 to 2586 be-tween mutant and wild-type ribosomes (Figure 6).In both the A2251 and U2251 mutant ribosomes,the reactivity of U2586 increases substantially, andthe normally unreactive U2584 becomes reactivetoward CMCT. Thus, mutations at G2251 affect the

chemical accessibility of nucleotides 2584 to 2586,located more than 300 nucleotides away in the pri-mary sequence. These results provide evidence forinteraction between the 2250 loop and the 2585 re-gion of the central loop of domain V. We cannotdetermine whether this interaction is direct or in-direct.

Discussion

In this study we examined the effects of single-base changes at two universally conserved bases,G2251 and U2585, which have been localized tothe peptidyl transferase center of 23 S rRNA in the50 S subunit of the ribosome. G2251 is of interestfor a number of reasons: (1) it is universally con-served; (2) it is the site of a conserved post-tran-scriptional 20-O-methyl modi®cation implicated in50 S subunit assembly in yeast mitochondria(Sirum-Connolly & Mason, 1993); (3) it is protectedfrom modi®cation by P-site bound tRNA (Moazed& Noller, 1989); and (4) the adjacent base, G2252,has recently been shown to form a Watson-Crickbase-pair with C74 in the CCA acceptor end of P-site bound tRNA (Samaha et al., 1995).

Because of its proximity to G2252, we tested thepossibility that base 2251 might interact with pos-ition C75 of tRNA in a Watson-Crick manner. Twomutations (A2251 and U2251) were introduced atposition 2251 and their effects were examinedin vivo and in vitro. In an in vivo expression systemwhere mutant and wild-type ribosomes are coex-pressed, both mutations at 2251 exhibit a dominantlethal phenotype. In this system, such phenotypesare most easily explained by the interference of im-paired mutant ribosomes with functional wild-typeribosomes on shared polysomes; indeed, A2251


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and U2251 mutant ribosomes were underrepre-sented on polysomes relative to the wild-type plas-mid-encoded version (data not shown). Consistentwith this explanation, two different in vitro assaysshowed that the A2251 and U2251 mutant ribo-somes were de®cient in peptidyl transferase-relatedfunctions. Although the dominant lethal in vivophenotype of the 2251 mutations could formally beexplained as a temperature-sensitive conditionallethality at 42�C, the observed biochemical de-®ciencies in two different in vitro assays performedat 4�C suggests that this is not the case. First,chemical protection analysis using allele-speci®cprimer extension showed that the A2251 andU2251 mutant ribosomes are unable to bind thewild-type tRNA oligonucleotide fragment in the Psite. Potential compensatory mutations at positionsC74 and C75 in the CCA acceptor end of tRNAfailed to suppress the observed binding de®ciency.Second, a peptidyl transferase assay using wild-type and mutant (C75) tRNA oligonucleotide frag-ments indicated that the mutant ribosomes (A2251and U2251) are de®cient in catalyzing peptidebond formation. The peptidyl transferase assay,performed under subsaturating conditions for theP-site substrate, is unable to distinguish the relativecontributions of kcat and KM to this de®ciency; how-ever, it is likely that loss of activity is at least partlythe result of impaired binding of the wild-type andmutant fragments to the mutant ribosomes (i.e. aKM effect). Whether it is also the result of a dis-turbed catalytic center (kcat) is unknown.

Chemical probing experiments using the carbodii-mide reagent CMCT indicate that the conformationof at least one region of 23 S rRNA in the peptidyltransferase center is perturbed in the 2251 mutantribosomes. Elsewhere, uridine reactivities of mu-tant ribosomes were indistinguishable from thoseof wild-type ribosomes, indicating that the overallstructure of the 50 S subunits was probably unaf-fected by the mutations at G2251. Consistent withthis observation is the fact that the mutant ribo-somes used for the footprinting experiments wereisolated as tight-couple 70 S ribosomes, indicatingthat the mutant 50 S subunits are structurally intactand able to associate with 30 S subunits. Interest-ingly, however, the CMCT reactivities of uridineresidues 2584 to 2586 are signi®cantly enhanced inthe A2251 and U2251 mutant ribosomes (Figure 6).In a previous study, it was shown that mutationsat G2252 also affected CMCT modi®cation atU2585; in that study, the rate and extent of modi®-cation of U2585, as well as its protection by tRNA,were diminished in A2252 mutant ribosomes(Samaha et al., 1995). The observation of such loca-lized and speci®c changes in the chemical accessi-bility of nucleotides located more than 300nucleotides from the sites of these mutations pro-vides strong evidence for interaction, either director indirect, between these two regions.

The affected nucleotides around position 2585 havethemselves been placed in proximity to the 30 endof peptidyl-tRNA by two separate lines of evi-

dence. U2585 is protected from CMCT modi®-cation by tRNA bound in the 50 S P site. Itsprotection is speci®cally dependent on the presenceof the 30-terminal A76 of tRNA (Moazed & Noller,1989, 1991). Crosslinking studies have indicatedthat U2585 is in close proximity to the 30-linkedacyl moiety of peptidyl-tRNA (Barta et al., 1984).The experiments presented here provide evidencefor a structural link between this region, likelyproximal to the 30 end of the tRNA, and the 2250loop, known to interact directly with C74 of theCCA end of tRNA (Samaha et al., 1995).

Unlike our ®ndings for C74 and G2252 (Samahaet al., 1995), base mutations at C74 and C75 fail tosuppress the de®ciencies of G2251 mutant ribo-somes. Thus, of three universally conserved guano-sine residues in the 2250 loop, whose P-site tRNAprotections depend on the presence of the terminalCA sequence of tRNA, only one, G2252, has beenfound to interact directly with tRNA. Clearly, how-ever, the identity of G2251 is critical to the functionof the ribosome. The absence of Watson-Crick sup-pression does not, of course, exclude the possibilitythat other moieties such as the phosphate or riboseof the RNA backbone are involved in direct inter-actions between 23 S rRNA and tRNA. Indeed,binding the acceptor end of P-site tRNA is likely tobe much more complex than simple Watson-Crickbase-pairing. Not only are the bases of C74 andC75 protected from dimethyl sulfate modi®cationwhen bound to the P site of the 70 S ribosome(Peattie & Herr, 1981; Douthwaite et al., 1983), butin addition, the CCA backbone is protected fromhydroxyl radicals when bound in the same pos-ition (Huttenhofer & Noller, 1992). The data havebeen interpreted to mean that the CCA end of thetRNA is buried at the subunit interface, inaccess-ible to aqueous contact, a feature consistent withthe role of the ribosome in excluding water fromthe activated tRNA substrates as they undergopeptide bond formation.

Rather than providing a contact for tRNA bindingto the P site of the ribosome, it is possible thatG2251 is involved in a long-range tertiary inter-action, drawing together two important regions of23 S rRNA; disruption of this crucial contact (i.e. inthe 2251 mutants) would thus result in loss of sub-strate binding and catalysis. The altered chemicalmodi®cation pattern observed at positions 2584 to2586 could be a manifestation of such a disruption.Alternatively, the identity of G2251 could be essen-tial for maintaining the local conformation of the2250 loop, and in particular for correct orientationof G2252, to promote its interaction with C74 oftRNA (Samaha et al., 1995).

Mutations were introduced at nucleotide U2585 totest whether the A76-dependent P-site tRNA pro-tection of U2585 from CMCT modi®cation was theresult of A76-U2585 base-pairing. In the in vivo ex-pression system, all three mutations exhibited anunambiguous dominant lethal phenotype. In anin vitro assay for tRNA oligonucleotide fragmentbinding, A2585 mutant ribosomes did not bind


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wild-type CCA tRNA fragment and A76 mutations(CCC and CCU) failed to compensate for this bind-ing de®ciency. In peptidyl transferase assays usingchimerically reconstituted ribosomes, activity wasdiminished, but not abolished, in each mutant(A2585 10%, C2585 - 30% and G2585 30%). Nosuppression of the observed decreases in activitywas observed when mutant tRNA fragments weresupplied as P-site substrates, suggesting that pro-tection of U2585 is not the result of simple base-pairing with A76 of tRNA. Again, the data do notexclude the possibility of interactions between thesugar-phosphate backbones of the respectiveRNAs. Alternatively, and keeping in mind that theassays used in this study focus on very limited as-pects of ribosomal function, the strong in vivo phe-notype of the U2585 mutants may be the result ofa failure at some other step in the complex processof translation. A number of potential pairing candi-dates for A76 of tRNA remain and are currentlybeing tested using approaches similar to thoseused in this study. As explained above, the possi-bility that the dominant lethal in vivo phenotypesof U2585 mutations are the result of ts conditionallethality at 42�C seems unlikely in view of the ob-served de®ciencies of mutant ribosomes in in vitroassays performed at 4�C.

It is clear that the regions of 23 S rRNA targetedhere are of fundamental importance to catalysis ofpeptide bond formation by the ribosome. Indeed, arecent study focusing on the lower half (positions2493 to 2606) of the central loop of domain V re-ported that 13 of 21 randomly chosen mutations inthis region exhibited compromised peptidyl trans-ferase activity (Porse & Garrett, 1995). We arehopeful that a systematic structural and functionalanalysis of site-directed mutants in 23 S rRNA willprovide further insight into the nature of this cata-lytic function of the ribosome.

Materials and Methods

Construction of mutants

Oligonucleotide-directed mutations were constructed inpBS23S as described (Samaha et al., 1995), using the fol-lowing mutagenic primers: 2251(A/T), 50-GGG-TAG-TTT-GAC-TG(T/A)-GGC-GGT-CTC-C-30; 2585(A/C/G),50-CAC-GAC-GTT-CTA-(C/G/T)AC-CCA-GCT-CGC-G-30. Expression plasmids were constructed by digestion ofmutant derivatives of pBS23S with Asp718 and ligationto BamHI linkers. The resulting linear plasmid was di-gested with BamHI and SphI and introduced into plas-mid pLK45 containing the rrnB operon under control ofphage lambda PL promoter (Samaha et al., 1995).

Growth phenotypes

Mutant plasmids were transformed into E. coli strainDH1 containing plasmid pcI857, which encodes a ther-molabile allele of the lambda repressor. Resulting pLKplasmids containing the respective mutations and engin-eered priming sites, �PS, or lacking engineered primingsites, ÿPS, were selected on plates containing ampicillin(40 mg/l) and kanamycin (50 mg/l). Overnight cultures

were grown at 30�C, diluted to 10ÿ4, 10ÿ5 and 10ÿ6 in LBmedium, and 12 ml of each dilution spotted on Amp40-

Kan50 plates and grown overnight at either 30�C or 42�C.

tRNA oligonucleotide fragment footprinting

Templates for transcription of mutant and wild-typetRNAs were prepared by polymerase chain reaction(PCR) ampli®cation either of plasmid CF23 containingthe gene for tRNAPhe from E. coli (a gift from O. Uhlen-beck) or of E. coli genomic DNA for elongator methion-ine tRNA (Samaha et al., 1995). Ampli®ed DNA wastranscribed in vitro with T7 RNA polymerase (Milliganet al., 1987). tRNA transcripts were puri®ed on a dena-turing 10%(w/v) polyacrylamide gel and eluted over-night in 0.3 M sodium acetate at 4�C; eluted tRNAs wererecovered by precipitation in ethanol and resuspendedin water. Before use, tRNAs were renatured by heatingat 95�C for two minutes followed by slow cooling toroom temperature; MgCl2 was then added to a ®nal con-centration of 10 mM. Charging and N-acetylation wereperformed as described (Breitmeyer & Noller, 1976;Moazed & Noller, 1989). tRNA oligonucleotide frag-ments containing 2030-linked N-acetyl-Phe were preparedby RNase T1 digestion as described (Moazed & Noller,1991). CMCT and kethoxal probing, isolation of themodi®ed RNA, and allele-speci®c primer extension wereperformed essentially as described (Samaha et al., 1995);in Figure 3(a), tight-couple 70 S were at a concentrationof 0.2 mM and tRNA oligonucleotide fragments at 2 mM,whereas in Figure 3(b), 70 S were at a concentration of0.2 mM and tRNA fragments were at 0.4 mM.

Peptidyl transferase assay

Aminoacylated wild-type and mutant rRNA fragmentswere prepared essentially as described (Moazed &Noller, 1991) except that elongator tRNAs were tran-scribed in vitro by phage T7 RNA polymerase from PCRDNA ampli®ed from E. coli genomic DNA (Milliganet al., 1987). Peptidyl transferase activity was measuredessentially as described (Samaha et al., 1995). The 50 Ssubunits (10 pmol), or an equivalent amount of an in vitroreconstitution reaction, were incubated with CCAC-CA(N-Ac-[35S]Met) or a mutant version (2.5 pmol) in100 ml of 0.4 M potassium acetate, 50 mM Tris (pH 8.3)and 60 mM MgCl2 on ice for ®ve minutes. The reactionwas initiated on ice by the addition of puromycin (®nalconcentration 1 mM) and 50 ml of methanol. Aliquots of35 ml were removed at various times and placed in 10 mlof 2 M KOH and quick-frozen. At completion of thetime-course, aliquots were incubated at 37�C for 20 min-utes, then 150 ml of 0.3 M sodium acetate (pH 5.5) satu-rated with MgSO4 (and containing 0.02% (w/v) xylenecyanol) were added. The product, N-acetyl-methionine-[35S]puromycin (NMP), was selectively removed by ex-traction with 1 ml ethyl acetate. The dried organicsample was spotted on Whatman 3 MM paper, and sub-jected to high-voltage paper electrophoresis in 0.5 M for-mic acid (pH 2.0) at 3000 V for 40 minutes. Radioactivitywas visualized using a Molecular Dynamics Phosphori-mager.

Preparation of natural and in vitro 23 S rRNApartial transcripts

RNase H cleavage was performed by incubating equi-molar amounts of 23 S rRNA and cDNA oligonucleotide


JMB MS 2874 [24/1/97]

(either 2295 (50-TGA-TGT-CCG-ACC-AGG-ATT-AG-30)or 2524 (50-GAC-CTA-CTT-CAG-CCC-30)) in a volume ofwater such that ®nal concentrations are approximately4 mM. The 23 S rRNA and DNA were incubated on icefor ®ve minutes and then at 42�C for ®ve minutes. Next,the volume was adjusted such that the ®nal RNA con-centration was approximately 1 mM, and the salts ad-justed to 40 mM Tris (pH 7.9), 10 mM MgCl2, 60 mMKCl, and 1 mM DTT (Tapprich & Hill, 1986). Cleavagewas initiated by the addition of 0.02 units of RNaseH/ml reaction (Wako Pharmaceuticals) and allowed toproceed for 20 minutes at 42�C. Reaction was stoppedby extracting with neutralized phenol, and precipitatingwith two volumes of ethanol. The digested RNA was re-suspended in 10 mM Tris (pH 7.6), 50 mM EDTA, heatedat 80�C for four minutes, and loaded directly onto 5% to25% (w/v) sucrose gradients in 10 mM Tris (pH 7.6),50 mM KCl. SW41 gradients were spun at 30000 rpm for16 hours, fractionated, and the appropriate peaks col-lected and ethanol-precipitated for use in reconstitutionreactions. It is worth noting that the natural 23 S rRNAfragments obtained by RNase H digestion have hetero-geneous edges resulting from the enzymatic treatmentand that, moreover, the two natural pieces combinedlack a total of ca 20 nucleotides covered by the cDNAoligonucleotide (data not shown). Notably, the in vitrotranscribed partial rRNA 50 fragment includes the ca 20nucleotides removed from natural 23 S rRNA during theRNase H cleavage reaction; the inclusion of thesenucleotides increases the activity of the two-piece T7/natural reconstitution ca twofold over the equivalent T7fragment missing those 20 nucleotides (data not shown).In vitro transcripts were transcribed by T7 RNA polymer-ase using PCR DNA templates (Milligan et al., 1987;Saiki et al., 1988) obtained by amplifying wild-type andmutant rRNA sequences found on plasmids;pLK45(�PS), pLK45(ÿPS), pLK45-A2251(ÿPS), pLK45-U2251(ÿPS), pLK45-A2585(�PS), pLK45-C2585(�PS)and pLK45-G2585(�PS). The primers used for ampli®ca-tion of the 50 partial in vitro transcript at the 2300 frag-mentation site were as follows: 39.3, 50-TAA-TAC-GAC-TCA-CTA-TAG-GTT-AAG-CGA-CTA-AGC-GTA-CAC-30 and 2395, 50-TGA-TGT-CCG-ACC-AGG-ATT-AG-30.By using 23 S plasmid constructs not containing engin-eered priming sites (in particular the engineered primingsite located at 2300), the 2310 region was compatiblewith the 30 natural fragment of 23 S rRNA. The primersused for ampli®cation of the 30 partial in vitro transcriptat the 2530 fragmentation site were as follows: 35.3, 50-TAA-TAC-GAC-TCA-CTA-TAG-GGC-TGA-AGT-AGG-TCC-CA-3 0 and 21.1, 5 0-AAG-GTT-AAG-CCT-CAC-GGT-TCA-30. Transcripts were separated from unincor-porated nucleotides on a G50-Sephadex (Pharmacia) gel®ltration column (1 cm � 20 cm) run in 10 mM Tris (pH7.5), 4 mM magnesium acetate. Recovered RNA was pre-cipitated with 0.5 M ammonium acetate (pH 6.0) and re-suspended in 10 mM Tris (pH 7.5).

In vitro reconstitution reactions

In vitro reconstitution was performed essentially as de-scribed (Nierhaus, 1990). Brie¯y, in a total volume of20 ml, 0.5 A260 unit of full-length 23 S rRNA (or 0.4 A260

unit of the 50 domain and 0.1 A260 unit of the 30 domainresulting from cleavage at 2310, or 0.43 A260 unit of the 50domain and 0.07 A260 unit of the 30 domain resultingfrom cleavage at 2530) and 0.02 A260 unit of 5 S rRNA(Boehringer Mannheim) were incubated with 1.2 equiva-lents of TP50 (total protein from 50 S subunits) in 20 mM

Tris (pH 7.4), 4 mM magnesium acetate, 0.4 M NH4Cl,0.2 mM EDTA and 5 mM 2-mercaptoethanol at 44�C for30 minutes. Next, the concentration of magnesium acet-ate was raised to 20 mM and a second incubation stepwas carried out at 50�C for 90 minutes. The resultingmixture was added directly to a peptidyl transferase re-action and the products analyzed as described above.

CMCT probing of 2251 mutant ribosomes

Wild-type or mutant E. coli 70 S tight-couple ribosomes(100 pmol) were incubated in 500 ml 70 mM potassiumborate, (pH 8.0), 100 mM NH4Cl, 20 mM MgCl2 and6 mM DTT at 37�C for ten minutes. An aliquot (100 ml)was removed as an unmodi®ed control (K), mixed with10 ml of 3 M sodium acetate (pH 5.5) and quick-frozen.Next, an equal volume (400 ml) of CMCT at a concen-tration of 42 mg/ml in the same buffer was added to theribosome mixture at 37�C. Aliquots of 100 ml were re-moved after three minutes and nine minutes and the re-action stopped with the addition of 10 ml of 3 M sodiumacetate (pH 5.5) and quick-freezing. Isolation of themodi®ed rRNA and allele-speci®c primer extension wereperformed as described (Samaha et al., 1995).

Acknowledgements

We thank B. Cormack and G. Culver for critically read-ing the manuscript; B. Weiser for Figure 1; O. Uhlenbeckfor supplying phenylalanyl tRNA synthetase; and S.Joseph and K. Lieberman for helpful discussions. Thiswork was supported by grants from the NIH, the NSF,the Lucille P. Markey Charitable Trust to the Center forMolecular Biology of RNA, and a postdoctoral fellow-ship from the Damon Runyon-Walter Winchell Foun-dation to R.G.

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Edited by P. E. Wright

(Received 30 May 1996; received in revised form 14 November 1996; accepted 14 November 1996)

mutations at nucleotides g2251 and u2585 of 23 s rrna perturb the peptidyl transferase center of the...

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