The Ribosomal Grip of the Peptidyl-tRNA is Critical for Reading Frame Maintenance

doi:10.1016/j.jmb.2008.10.069 J. Mol. Biol. (2009) 385, 350–367

Available online at www.sciencedirect.com

The Ribosomal Grip of the Peptidyl-tRNA is Critical forReading Frame Maintenance

S. Joakim Näsvall, Kristina Nilsson and Glenn R. Björk⁎

Department of MolecularBiology, Umeå University,S-901 87 Umeå, Sweden

Received 7 August 2008;received in revised form21 October 2008;accepted 22 October 2008Available online3 November 2008

*Corresponding author. E-mail [email protected] address: S. J. Näsvall, D

Medical Biochemistry and MicrobUniversity, S-751 23 Uppsala, SweAbbreviations used: GST, glutath

MBP, maltose-binding protein.

0022-2836/$ - see front matter © 2008 E

If a ribosome shifts to an alternative reading frame during translation, theinformation in the message is usually lost. We have selected mutants ofSalmonella typhimurium with alterations in tRNAcmo5UGG

Pro that causeincreased frameshifting when present in the ribosomal P-site. In 108 suchmutants, two parts of the tRNA molecule are altered: the anticodon stemand the D-arm, including its tertiary interactions with the variable arm.Some of these alterations in tRNAcmo5UGG

Pro are in close proximity to ribo-somal components in the P-site. The crystal structure of the 30S subunitsuggests that the C-terminal end of ribosomal protein S9 contacts nucleo-tides 32–34 of peptidyl-tRNA. We have isolated mutants with defects in theC-terminus of S9 that induce +1 frameshifting. Combinations of changes intRNAcmo5UGG

Pro and S9 suggest that an interaction occurs between position32 of the peptidyl-tRNA and the C-terminal end of S9. Together, our resultssuggest that the cause of frameshifting is an aberrant interaction betweenthe peptidyl-tRNA and the P-site environment. We suggest that the“ribosomal grip” of the peptidyl-tRNA is pivotal for maintaining thereading frame.

© 2008 Elsevier Ltd. All rights reserved.

Keywords: ribosome; translation; reading frame maintenance; peptidyl-site;frameshift
Edited by J. Karn
Introduction

The evolution of modern cells began with theonset of translation, eventually resulting in the lastuniversal common ancestor (LUCA), from whichthe three domains of life emerged.1 It is thought thatLUCA had an advanced translation apparatus ableto translate faithfully long mRNA sequences. Thus,translation, including how the ribosome maintainsthe reading frame, occurs in a similar way in allorganisms and its major features had evolved beforethe emergence of the three domains.In the process of translation, the ribosome decodes

the mRNA, incorporating one amino acid for eachsuccessive non-overlapping triplet codon.2 Thereading frame is set by positioning of the initiatormethionyl-tRNA at the start codon located in the

ess:

epartment ofiology, Uppsaladen.ione-S-transferase;

lsevier Ltd. All rights reserve

ribosomal P-site, placing the next codon triplet in theA-site. After the peptidyl transfer reaction, when thegrowing peptide is transferred to the A-site ami-noacyl-tRNA, translocation occurs by moving theacceptor stems of the P-site deacyl-tRNA and the A-site peptidyl-tRNA into the 50S E- and P-sites,respectively, while the anticodon stem–loops remainin the 30S P- and A-sites. This is followed by amovement of the anticodon stem–loops from the 30SP- and A-sites to the E- and P-sites, respectively.3,4

During this process the codon–anticodon complexesare maintained, ensuring that the next in-frametriplet ends up in the now empty A-site. Failure tomaintain the correct reading frame leads to an erro-neous peptide sequence being produced, andusually a stop codon is soon encountered in thenew frame. Errors in reading frame maintenance areusually rare and may theoretically occur in twodifferent ways. Either the translocation step size isaltered (e.g., four nucleotides instead of three for +1frameshift errors) or the ribosome in some way re-aligns on the mRNA (e.g., one nucleotide forward inthe case of +1 frameshift errors). Although we nowknow much about the structure and function of thetranslation apparatus,5–7 the mechanism that gov-erns the translocation step size and how the

d.

http://dx.doi.org/10.1016/j.jmb.2008.10.069

351Ribosomal Grip of Peptidyl-tRNA

ribosome maintains the reading frame is still notknown in detail.It was suggested earlier that the tRNA with its

three nucleotide anticodon acts as a yardstick toensure that the ribosome made a three nucleotidetranslocation. When the first extragenic suppressorsof +1 frameshift mutations were isolated,8–10 it wasthought that the length of the translocation step wasaltered; i.e. suppression of frameshift mutations wasthought to be caused by a defective translocationstep. This hypothesis was strengthened with thecharacterization of a +1 frameshift suppressor,which was shown to be a tRNA with an extranucleotide in the anticodon loop.11 From analysis ofsuch altered tRNAs, it was inferred that a +1 frame

Fig. 1. Defects in tRNA can induce frameshifting in three ddiamond) is too slow (indicated by a broken line) in enteringblue bar at the wobble position) t o decode the A-site codon. Afnear-cognate tRNA is prone to slip into an overlapping readdiamond) decodes the codon in the A-site, but once it has beeThe defective tRNA (indicated by a red diamond) is too slow (ia pause allowing the cognate P-site tRNA to slip. Adapted fromenter the +1 frame compared to the continued reading in the zprimary sequence or hypomodification of the tRNA. The occtenance,27, 28, 77 perhaps by strengthening the ribosomal grip oincreased dissociation rate from the E-site may also increase f

error induced by an inserted nucleotide in mRNAwas corrected by a tRNA having an apparent fournucleotide anticodon able to decode four bases anda quadruplet translocation moved the ribosome intothe zero frame; i.e. the altered tRNA changed thelength of the translocation. This yardstick modelwas generally accepted and is found in many textbooks,12–15 and it is still advocated.16,17 Accordingto this model, the +1 frameshift error occurs in theA-site, at which the tRNA with its oversized anti-codon in some way interacts with four nucleotidesin the mRNA, inducing a quadruplet translocation.Although the yardstick model is attractive, weshowed that it is not valid for any of the testedclassical frameshift suppressor tRNAs with an extra

ifferent ways: (a) The defective tRNA (indicated by a redthe A-site, allowing a near-cognate tRNA (depicted with ater a normal three nucleotide translocation to the P-site, theing frame. (b) The defective tRNA (indicated with a redn translocated into the P-site it may slip on the mRNA. (c)ndicated by a broken line) in entering the A-site, providingRefs 22 and 24. Broken arrows indicate slow reactions to

ero frame. “Defective” can either indicate alterations in theupancy of the E-site also improves reading frame main-f the peptidyl-tRNA. Therefore, a defective tRNAwith anrameshifting.

352 Ribosomal Grip of Peptidyl-tRNA

nucleotide in the anticodon.18,19 Several mutantsdefective in tRNA modification, with normal-sizedanticodons, also cause frameshift errors.20–22 A newmodel was proposed on the basis of these observa-tions (Fig. 1).22 According to this model, while theA-site is empty, peptidyl-tRNA may slip into ano-ther reading frame at a low frequency. The proba-bility of such realignment may be enhanced by anaberrant peptidyl-tRNA in the P-site or by slowdecoding of the A-site codon.18,22–24 This is furthersupported by the observations that both ribosomalbypassing and programmed frameshifting are sti-mulated by starvation for the aminoacyl-tRNA thatis supposed to read the A-site codon directly fol-lowing the takeoff codon.25,26 Thus, the occupancyof the A-site influences the degree of frameshiftingin the P-site. Another factor that may influence thefrequency of frameshifting is the occupancy of theE-site. An empty E-site seems to stimulate pro-grammed frameshifting in the prfB gene.27–29 AtRNA in the E-site may provide an additional gripon the mRNA, so that if the P-site tRNA dissociatesfrom mRNA, it would not land in the wrong frame.Still, an important feature of our model (Fig. 1) isthat the error in reading frame maintenance occursafter a normal three nucleotide translocation, by arealignment of the peptidyl-tRNA–codon–antico-

Fig. 2. Location of mutations in tRNAcmo5UGGPro . (a) Clove

arrows indicate the mutated positions and what they have buridine-5-oxyacetic acid (cmo5U); s4U, 4-thiouridine; D, 5methylcytidine; m1G, 1-methylguanosine; m7G, 7-methylguCrystal structure of tRNAfMet in the P-site of the 70S ribosome (the mutations found in tRNAcmo5UGG

Pro are indicated with arrowlight blue) and 50S (with C atoms in darker blue) that have atrepresentations and the rest of the protein chains are shownwww.pymol.org). The red text in b indicates ribosomal comp(H69). In both a and b, red nucleosides correspond to position(nucleotides 10–25) is coloured yellow.

don complex in the P-site and not by an alteredtranslocation step size.In crystal structures of 70S ribosomes containing

peptidyl-tRNAs in the P-site, extensive interactionsbetween peptidyl-tRNA and the rRNA and proteinresidues making up the 30S and 50S P-sites havebeen identified (Fig. 2b). In the 30S P-site, ten 16SrRNA residues and two proteins (S9 and S13) contactthe anticodon stem–loop of the peptidyl-tRNA, andonly two of these residues contact the anticodon.30 Inaddition, 24 residues of 23S rRNA and nine residuesfrom three large subunit proteins (L5, L16 and L27)are in close proximity to the peptidyl-tRNA. In fact,the interactions between ribosomal components andpeptidyl-tRNA outnumber by far the interactions ofthe codon–anticodon complex in this site. This leadsus to hypothesize that, in addition to the stability ofthe codon–anticodon complex, the interactions be-tween peptidyl-tRNA and the ribosome contributesignificantly to the “ribosomal grip” of the peptidyl-tRNA, which may be an important feature for main-taining the correct reading frame. Besides the sug-gestive structural data,5,6 experimental evidencesupporting such a hypothesis is scarce.31–33

If the ribosomal grip of the peptidyl-tRNA ispivotal in maintaining the reading frame, changesin either the structure of the peptidyl-tRNA or the

r-leaf representation of the tRNAcmo5UGGPro sequence. The

een changed to. Δ at position 47 indicates a deletion. V,,6-dihydrouridine; Um, 2′-O-methyluridine; Cm: 2′-O-anosine; m5U, 5-methyluridine, Ψ, pseudouridine. (b)PDB codes 2J00 and 2J01).5 The positions corresponding tos. Protein and rRNA residues of the 30S (with C atoms inoms within 3.8 Å of the peptidyl-tRNA are shown as stickas tubes. The image was rendered with PyMOL (http://onents; proteins S9, S13, L5, L27 and 23S rRNA helix 69s where mutations cause +1 frameshifting. In b, the D-arm


P-site environment may induce frameshifts. Salmo-nella enterica serovar Typhimurium (S. typhimurium)has three proline isoaccepting tRNAs: tRNAGGG

Pro ,tRNACGG

Pro and tRNAcmo5UGGPro (the subscript indicates

the anticodon sequence 5′→3′, cmo5U is uridine-5-oxyacetic acid). tRNAcmo5UGG

Pro can, in addition toreading A- and G-ending codons, as predicted bythewobble hypothesis, also read the U- and C-endingcodons.34,35 Mutations that cause an increased levelof +1 frameshifting at CCC codons have been iden-tified in the proL gene, which encodes tRNAGGG

Pro .19 Inmany of these mutants, frameshifting occurs whentRNAcmo5UGG

Pro resides on a CCC codon in the ribo-somal P-site.18 Apparently, the interaction betweentRNAcmo5UGG

Pro and a CCC codon is accepted ascognate in theA-site, but its interaction in the P-site isnot optimized for preventing errors in reading framemaintenance. In order to further improve ourunderstanding of how the ribosome maintains thecorrect reading frame, we have isolated over 100mutants of tRNAcmo5UGG

Pro with an increased rate offrameshifting at CCC codons located in the P-site.Our results support the existence of a ribosomal gripof the peptidyl-tRNA, since many of the alterationsof the peptidyl-tRNAcmo5UGG

Pro are in regions that arein close proximity of specific components of theribosomal P-site and thus may weaken the interac-tion between the peptidyl-tRNA and the ribosome.As an alternative to changing the structure of the

peptidyl-tRNA we can introduce changes in the P-site environment by altering ribosomal components.In the 30S P-site, ribosomal protein S9 penetrates theribosome and contacts the anticodon loop of thepeptidyl-tRNA (Figs. 2b and 3a). The length of theC-terminus of S9 is phylogenetically conserved andalways ends with an arginine that, in the 3Dstructure, appears to contact the 5′-phosphate ofnucleotide 32 in the anticodon loop of peptidyl-

Fig. 3. Interactions between peptidyl-tRNA and the ribosomloop (positions 26 –44) of tRNAi

Met is shown as spacefill. The an(b) The “elbow” region of peptidyl-tRNA. The nucleotides G1indicated with “H69”. Residues of rRNA and ribosomal proteistick representations, and the rest of the proteins are shown awww.pymol.org) from the crystal structure of the Thermus the

tRNA. The penultimate amino acid of eubacterial S9is a conserved lysine (tyrosine in archeal andeukaryal S9 homologues), which interacts with the5′-phosphate groups of positions 33 and 34 of pep-tidyl-tRNA.5 If ribosomal protein S9 participates increating a good grip of the peptidyl-tRNA by theribosome and, thereby, participating in mainte-nance of the correct reading frame, one wouldpredict that certain alterations of this protein willinduce errors in reading frame maintenance. Here,we address this prediction and present datasupporting it.Thus, by either altering the tRNA moiety of the

peptidyl-tRNA or changing the environment of theribosomal P-site by altering ribosomal protein S9,known to be close to the anticodon loop of peptidyl-tRNA, we have obtained results supporting thehypothesis that the ribosomal grip of the peptidyl-tRNA is pivotal for maintaining the reading frame.Our results are also consistent with the idea thattranslocation and reading frame maintenance aretwo distinct features of the ribosome.

Results

Isolation of novel +1 frameshift suppressors

To generate mutants in the gene encodingtRNAcmo5UGG

Pro , we mutagenized the proM genewith either hydroxylamine or by PCR. Mutantswere selected that were able to suppress the +1frameshift mutations hisD3749 or hisD10122, bothof which contain a CCC (Pro) codon in theframeshift window (Fig. 4a). Among 108 suchmutants, alterations at 12 different positions ofproM tRNAcmo5UGG

Pro were obtained (Table 1). One

e in the 70S P-site. (a) The 30S P-site. The anticodon stem–ticodon is blue and positions mutated in this study are red.907 –C1909 and G1922 –C1924 in helix 69 of 23S rRNA arens that have atoms within 3.8 Å of the tRNA are shown ass tubes. The images were rendered with PyMOL (http://rmophilus 70S ribosome (PDB codes 2J00 and 2J01).5

Fig. 4. Frameshift windows used in this study. (a) Sequences of wild type hisD and the +1 frameshift mutationshisD3749 and hisD10122. (b) Sequences of the “slippage junctions” between the gst and malE genes in plasmids pUST290,pUST292 and pUST310. First line, mRNA sequences. Second line, peptide sequence encoded in the zero frame. Third line,peptide sequence encoded by the +1 frame. Bold triplets, downstream stop codons in the zero frame. Bold italic triplets,upstream stop codons in the +1 frame. Triplets on grey background, proline (or phenylalanine in pUST292) codonspresent within the frameshift windows. Black box, frameshift window. A shift to the +1 frame when any of the codonswithin the frameshift window is present in the P-site will result in production of full-length HisD or GST-MBP proteins(but some of the possible frameshift products may result in an inactive protein). Earlier shifts will result in termination atthe upstream stop codon in the +1 frame.


mutant, ΔU47, was initially isolated on the basisof a slow growth phenotype and was later testedfor frameshift suppression. All mutants, exceptthose with alterations A38G or U17C+A38G,caused increased suppression of both hisD3749(CCC-UGA) and hisD10122 (CCC-CAA) mutationscompared to a proM+ strain. The only alteration oftRNAcmo5UGG

Pro that caused enough frameshifting tosuppress hisD3749 (CCC-UGA) without the co-suppressor ΔproL was G31A (Table 1, hisD3749proL+). Alteration at both positions 17 and 38(U17C+A38G) did not show any difference in thelevel of suppression compared to the A38G singlealteration, indicating that the U17C alteration doesnot contribute to frameshifting. We quantified theframeshift frequencies in β-galactosidase assays onstrains containing hisD2504∷MudK, which placeslacZ in the original zero frame of hisD but down-stream of the frameshift mutation in hisD10122(Table 1). The strong suppressor G31A showedmoreframeshifting in this assay, both in the presence andin the absence of the ΔproL allele, as expected fromthe relative growth rates on plates lacking histidine.The level of frameshifting in the G31A mutant issimilar to that of another strong suppressor, theclassical sufB2 suppressor with an extra nucleotidein the anticodon loop (Table 1). However, combiningΔproL and G31A caused even higher levels of frame-shifting (N8%), as expected from the very efficientsuppression seen on plates. As the frequenciesobtained from the weaker mutants were within therange of the variation of the wild type, we conclude

that the β-galactosidase assay was not sensitiveenough to quantify the expected small increases inframeshifting from these mutants. Thus, comparinggrowth on plates is much more sensitive than anenzymatic assay to detect small differences in thelevel of frameshifting.

The shift in frame is caused by the alteredtRNAcmo5UGG

Pro

To determine which amino acid is the last to beinserted in the zero frame, and thus which tRNAshifts frame, we purified the frameshift productsexpressed from plasmids pUST290 and pUST310(Fig. 4b). These plasmids carry the gene encodingglutathione-S-transferase (GST) fused to the malEgene (encoding maltose-binding protein (MBP),36,37

with a slippage junction containing a +1 frameshiftsite between the fused genes (the slippage junctionsin these plasmids both contain the sequence CCC-CAA, but differ in the surrounding sequences, seeFig. 4b). The sequence of the purified peptide frompUST290 in the wild type indicated a background ofpeptides resulting from +1 frame shifts at both theCCC (Pro) codon and the CAA (Gln) codon (thepeptide sequence was GPLGILNP[K/Q]ANN,where the amino acids in bold are consistent witha +1 frameshift by tRNAPro, resulting in P as the lastamino acid inserted in the zero frame and the resi-dues in brackets indicate that a mix of lysine (K) andglutamine (Q) was found in one position. Q is theresult of a +1 frameshift by tRNAGln at the CAA

Table 1. Isolated proM frameshift suppressor mutants

proM allele (mutation) No. isolates

SuppressionCCC-UGA (hisD3749)

SuppressionCCC-CAA (hisD10122)

FS%aproL+ ΔproL proL+ ΔproL

proM+ (WT) - - (-) (-) +++ 0.74±0.27(1.49±0.36)

proM2219 (G31A) 35 + +++++ +++++ ++++++ 2.16±0.11(8.38±0.43)

proM2220 (C48U) 31 - ++ ++ +++++ N/Db

proM2221 (C40U) 18 - + + +++++ 0.95±0.06proM2222 (G15A) 6 - + +++ +++++ 1.09±0.07proM2223 (C13U) 2 - + + +++++ N/Db

proM2224 (C23A) 2 - + ++ ++++ 0.85±0.07proM2225 (Um32Cm)c 1 - + + ++++ 0.85±0.14

(1.36±0.05)proM2226 (A38G) 4 - (-) + +++++ 0.75±0.09

(2.16±0.16)proM2227 (U43C) 5 - + + ++++ N/Dc

proM2228 (U43A) 2 - + (+) ++++ N/Dc

proM2229 (U17C A38G) 1 - (-) + +++++ N/Dc

proM2230 (ΔU47) 1 - ++ (+) +++++ N/Dc

proL2240 (sufB2)d d d d +++d d 2.33±0.20

++++++, Colonies larger than about 0.1 mm in diameter were visible after one day at 37°C. Thereafter, growth was scored with one less +for every day it took for the colony diameter to reach 0.1 mm ormore. (+) Colonies larger than about 0.1 mm in diameter were visible afterseven days. (–) Traces of growth, but either no colony was visible or the diameter of colonies was smaller than 0.1 mm after seven days. –,No detectable growth after seven days.

a Frameshift frequencies were measured in β-galactosidase assays. Numbers within parentheses are from strains containing the ΔproLdeletion, other numbers are from proL+ strains.

b N/D, Not determined.c Analysis of nucleosides from tRNAcmo5UGG

Pro from this mutant show that the T→C mutation results in the presence of 2′-O-methylcytidine (Cm) instead of 2′-O-methyl uridine (Um), which is present at position 32 of the wild type tRNA.

d The proL2240mutant, found in the same genome-wide selection as the rpsI2 and rpsI3mutants, is identical with the “classical” sufB2mutant (an extra G inserted between m1G37 and U38 in the anticodon loop of tRNAGGG

Pro 18). Some of the column headers (proM allele,proL+, ΔproL and No. isolates) are not relevant to this mutant.


codon (see Fig. 4b). The result from the C23Amutantwas similar to the wild type, while in the G15A andG31A mutants most or all of the peptides containedproline as the last amino acid encoded in the zeroframe. When using plasmid pUST310, the sequencewas GPLGILI[C/V]PNDK, where the aminoacids in bold are consistent with a +1 frameshift bytRNAPro, resulting in P as the last amino acid in-serted in the zero frame. In one position both theexpected cysteine (C) (or a derivative of it, seeMaterials and Methods and Supplementary DataTable III) and valine (V) was present in all samples.In the wild type, the peptide contained more valinethan cysteine at this position, while the mutantsincreased the amount of cysteine relative to valine.Valine may be the result of a +1 frameshift by atRNAIle at the AUA (Ile) codon (see Fig. 4b). If that isthe case, tRNAIle would need to shift from the AUA(Ile) codon to a UAU (Tyr) codon. The tRNA cognateto the AUA codon is the minor tRNAk2CAC

Ile , whichwould not form a single base pair with the UAUcodon. Even if such an event appears unlikely, it isnot impossible, as demonstrated by Herr et al.,38who detected landing in a stop codon bypass assayat a codon where the peptidyl-tRNA could not forma single base pair. The high-level expression of thereporter mRNA (expressed from a high copy num-ber plasmid) could lead to the AUA codon being readby the more abundant near-cognate tRNAGAC

Ile . Thiswould result in a wobble position mismatch (G-A),which is known to be stimulatory for +1 frameshift-

ing, and would result in a G-U wobble base pairafter the shift to the UAU codon. In the TY3 pro-grammed frameshift site, it has been demonstratedthat peptidyl-tRNAs on near-cognate codons (wob-ble position mismatches) induce very efficient +1frameshifting, even if they cannot form a single basepair in the +1 frame.39 Although we observed abackground of frameshift products other than theexpected, the altered tRNAcmo5UGG

Pro s caused accu-mulation of only the expected products. Thus, in themutants, the major frameshift product containedproline as the last amino acid in the zero frame,suggesting that it was the altered tRNAcmo5UGG

Pro thatcaused the frameshift event.

The shift in frame occurs whentRNAcmo5UGG

Pro occupies the P-site

If frameshifting occurs while tRNAcmo5UGGPro is in

the P-site and the A-site is empty, over-expression ofthe tRNA reading the next in-frame codon willdecrease the length of time when the A-site is empty,leading to inhibition of frameshifting according toour model (Fig. 1). In the case of the hisD10122 allele,a CAA (Gln) codon is in the A-site when the zeroframe CCC (Pro) codon is in the P-site. PlasmidpUST136 carries the S. typhimurium metT operoncontaining seven tRNA genes of which the glnU andglnW genes both encode tRNAcmnm5s2UUG

Gln , whichreads CAA and CAG codons.40 Introduction of thisplasmid caused complete inhibition of growth on


plates lacking histidine for the proM wild type andall but one of the suppressor mutants. Suppressionby the most efficient suppressor mutant, G31A, wasreduced to the level seen in the wild type (proM+)(data not shown). We conclude that frameshiftinginduced by these altered tRNAcmo5UGG

Pro s occurswhen the CAA (Gln) codon is in the A-site and,consequently, tRNAcmo5UGG

Pro is located at the CCC(Pro) codon in the P-site.

Frameshifting induced by the mutant proMtRNAcmo5UGG

Pro is not caused byhypo-modification of the altered tRNA

Poor reading of proline codons or changes of themutated tRNA other than the indicated basesubstitutions might in some way induce frameshift-ing. In a wild type strain, tRNAcmo5UGG

Pro competeswith tRNAGGG

Pro for reading CCU and CCC codons,and with tRNACGG

Pro for reading CCG codons. Themutant tRNAcmo5UGG

Pro s are all able to compete withtRNAGGG

Pro for the CCC codon (Table 1, proL+) andthey can also support growth of a mutant lackingtRNAGGG

Pro (Supplementary Data Table I, ΔproLproK+), with no major effect on growth rate (theG31A and U43C mutants showed a small butnoticeable synergism with ΔproL). However, somemutants (G31A, C40U, C13U, C23A, Um32Cm,U43C and ΔU47) show severe reduction in growthrate when combined with lack of tRNACGG

Pro (Sup-plementary Data Table I, proL+ proKbNfrt), indicatinga possible reduction in the efficiency of reading CCGcodons. Since CCG is the proline codon used mostfrequently,41 a strain lacking tRNACGG

Pro may be par-ticularly sensitive to perturbations in the function oftRNAcmo5UGG

Pro . One reason for poor reading of pro-line codons could be hypo-modification of themutant tRNA. Lack of m1G37, which is present inall proline tRNAs, is known to induce frameshiftingat proline codons21 as well as causing a reduced rateof A-site selection at all four proline codons.42 Hypo-modification of cmo5U34 causes a reduced rate of A-

Table 2. Alterations of the C-terminal end of ribosomal prote

StrainAmino acid sequence of the C-terminal end o

wild type and mutant S9a

rpsI+ MEYDESLRGELRKAGFVTRDARQ[-15aa-]QFrpsI2 MEYDESLRGELRKAGFVTRDART(20 aa shorrpsI3 MEYAAWRTA (33 aa shorter)rpsI4 (R130A) MEYDESLRGELRKAGFVTRDARQ[-15aa-]QFrpsI5 (K129A) MEYDESLRGELRKAGFVTRDARQ[-15aa-]QFrpsI6bNkan The entire rpsI gene is replaced with a kanam

resistance cassette. No S9 in the cell.a The frameshift mutations rpsI2 and rpsI3 shorten the C-terminal en

acid sequence after the mutations, bold letters indicate amino acid subthe indicated mutations in the rpsI gene and the hisD10122 mutation.

b Cells, grown on rich medium, were applied to agar plates containdicated. The agar plates were incubated at 37 °C for two days in the pNumbers marked with an asterisk (⁎) were incubated for six days. Cmagnifying glass.

c Frameshift frequencies were measured with β-galactosidase assayd –, No growth.e Heterogeneous colony sizes.f N/D, not determined.

site selection at CCG codons.35 To test whether hypo-modification is the cause of slow growth and possiblyinduction of a frameshift, tRNAcmo5UGG

Pro was purifiedfrom a proM2219 (G31A) strain and analyzed byHPLC. All modifications were present at the samelevels as in tRNAcmo5UGG

Pro prepared from a wild typestrain. Similar results were obtained with a Um32Cmmutant, except this mutant had 2′-O-methylcytidine(Cm) instead of 2′-O-methyluridine (Um), which ispresent at position 32 of wild type tRNAcmo5UGG

Pro .Thus, we refer to this mutant as Um32Cm to indicatethat the 2′-O-methyl group on the ribosyl moiety atposition 32 is still present, even though the base isdifferent from wild type (data not shown).

Poor prolylation of some frameshift suppressorderivatives of tRNAcmo5UGG

Pro does not affect theirframeshift suppressor activity, consistent with aP-site frameshifting event

Another reason for the observed synergistic effects(Supplementary Data Table I) when combiningmutations in proM with lack of tRNACGG

Pro is thatthe altered tRNAcmo5UGG

Pro could be a poor substratefor the prolyl-tRNA synthetase (ProRS), rendering areduced coding capacity and perhaps in some wayinfluencing frameshifting. Over-expression of ProRSfrom plasmid pNTR-SD-proS43 suppressed thegrowth defects of these proM proK double mutants,suggesting some of the defective tRNAcmo5UGG

Pro swere indeed poor substrates for ProRS (Supplemen-tary Data Table II). To test if ProRS had any effect onframeshifting in these mutants, the pNTR-SD-proSplasmid was introduced into hisD10122 proL+ proK+

and hisD10122 ΔproL proK+ strains carrying eitherthe wild type, G31A, Um32Cm, A38G, U43C orU43A proM alleles. Growth of these strains was com-pared on minimal medium with or without IPTG,and with or without histidine (data not shown).Over-expression of ProRS had no significant effect onframeshifting, except perhaps a very slight stimula-tion of the proM2219 (G31A) proL+ mutant. Growth in

in S9 mediates +1 frameshifting

f the+Hisb (2d)

No Hisb

(4d or 6d⁎) FS(%)c

SKR 3.8 –d 0.74±0.27ter) 1.8 1.0 2.86±0.56

2.9 0.5 3.64±0.11SKA 0.6–2.1e 0.1⁎ 2.26±0.18SAR 3.5 b0.1⁎ 0.91±0.11ycin 0.1–0.5e 0.2 N/Df

d by 20 and 33 amino acids, respectively, and changes the aminostitutions compared to the wild-type sequence. All strains containThe listed sequences start from methionine 88 of S9.ining medium E and glucose, with or without histidine (His) asresence of histidine, or four or six days in the absence of histidine.olony size (in mm) was measured with digital calipers under a

s.


the presence of histidine was slightly improved forthe mildly slow growing proM2227 (U43C) ΔproLstrain, but no apparent difference was seen for any ofthe other strains. As negative control for these expe-riments we used plasmid pNTR-SD-leuL43 with theORF leuL, encoding the leu-operon leader peptide,which did not cause any visible difference in growthat any of the conditions tested.We conclude that evenif some of the proM mutants appear to have a defectin aminoacylation, frameshifting is not dependent onpoor aminoacylation.

Alterations in the C-terminal end of ribosomalprotein S9 mediate +1 frameshifts

The C-terminal end of ribosomal protein S9 pene-trates the ribosome like a tentacle, and its two lastamino acids make contact with the 5′-phosphate ofnucleotide 32 (R130) and the 5′-phosphates of posi-tions 33 and 34 (K129) of peptidyl-tRNA5 (Fig. 3a).Accordingly, ribosomal protein S9 may be a func-tional part of the ribosomal grip of the peptidyl-tRNA to maintain the reading frame. If so, defects inthe C-terminal tail of S9 may induce errors inreading frame maintenance, which can be detectedas suppression of a frameshift mutation. Amongmore than 300 randomly isolated histidine-indepen-dent clones of the hisD10122 mutant, not countingthose with additional mutations in hisD, two (rpsI2and rpsI3) had a mutation in the structural gene(rpsI) for ribosomal protein S9. Both mutants containa frameshift mutation resulting in a truncated pro-tein S9 lacking the last 20 (rpsI2) or 34 (rpsI3) C-terminal amino acids. In addition to these mutants,we constructed rpsI4 (R130A), where the last aminoacid (arginine 130) is changed to alanine, rpsI5(K129A), where the penultimate amino acid (lysine129) is changed to alanine. Escherichia coli mutantscompletely lacking protein S9 are viable.44–46 To testif complete absence of S9 leads to increased frame-shifting, we generated an rpsI6bNkan mutant, whichlacks the entire rpsI gene. All these alterations ofprotein S9 suppressed the +1 frameshift mutationhisD10122 (Table 2). Introduction of the wild typeallele of rpsI+ on a plasmid restored growth to that ofthe wild type and inhibited the suppression com-pletely (data not shown). Thus, suppression of thehisD10122 frameshift mutation and thereby growthin the absence of histidine is caused by an alteredribosomal protein S9.To determine which tRNA is causing the frame-

shift, the frameshift products expressed from plas-mid pUST290 (CCC-CAA) and plasmid pUST292(identical with pUST290, but UUU-CAA instead ofCCC-CAA) were determined. From pUST290 (CCC-CAA, see Fig. 4b) the first 12 amino acids of thepeptide from the rpsI2 mutant were determined tobe mainly GPLGILNP-KANN, although a minorpart was GPLGILNPQ-ANN, where P (proline) or Q(glutamine) was the last amino acid inserted in thezero frame. Using pUST292 (UUU-CAA), all frame-shift products from the rpsI2 mutant had Phe as thelast amino acid inserted in the zero frame. Thus, at

the CCC-CAA sequence, frameshifting was causedmainly by tRNAPro and, to a lesser extent, bytRNAGln, whereas when UUU preceded the CAAcodon, all detectable frameshift events were causedby tRNAPhe.

Ribosomal assembly and protein content in 30Ssubunits from rpsI2, I4, and I5 mutants aresimilar to that of the wild type

It is known that S9 is critical for the assembly ofthe 30S subunit.47 We therefore analyzed ribosomeprofiles by sucrose gradient centrifugations to revealany immature 30S particles, indicative of anaberrant assembly process. All mutants, except therpsI6bNkan mutant, showed a ribosome profilesimilar to that of the wild type, suggesting thatprecursor particles were not accumulating (Fig. 6).The 30S ribosomal proteins were analyzed by one-and two-dimensional gel electrophoresis (Figs. 7and 8). The rpsI2, rpsI4, and rpsI5 mutants had thesame protein profiles as the wild type, whereas 30Sparticles from an rpsI3 mutant lacked ribosomalprotein S14, and 30S particles from the rpsI6bNkanmutant were missing several proteins. As expected,protein S9 from the point mutants rpsI4 and rpsI5migrates like the wild type protein, while the frame-shift mutants rpsI2 and rpsI3 contain truncatedvariants. We conclude that the only differenceobserved between the 30S subunits in the rpsI2,rpsI4, and rpsI5 mutants compared to wild type isthe altered ribosomal protein S9, suggesting that it isthis alteration and not some secondary defect of theribosome that causes the ribosome to shift frame.

The occupancy of the A-site influencesframeshifting, suggesting that the frameshiftoccurs in the P-site

The C-terminal end of ribosomal protein S9 is partof the P-site environment and, accordingly, weexpect that the absence or alterations of its C-terminus should induce frameshifts in the P-site. Ifso, the reading frame errors should be sensitive tothe occupancy of the A-site. The short frameshiftwindow (see Fig. 4a) created by the hisD10122mutation contains the sequence CCC-CAA-UGA. Toproduce a full-length HisD enzyme, a +1 frameshiftmust occur between the upstream +1 frame stopcodon and the downstream zero frame stop codon.The CAA codon present within the frameshift win-dow is read by tRNAcmnm5s2UUG

Gln . The presence of aplasmid over-expressing tRNAcmnm5s2UUC

Gln inhi-bited the suppression of the hisD10122 mutation(data not shown). Lack of the cmnm5 moiety of thewobble nucleoside cmnm5s2U34, which is presentin tRNAcmnm5s2UUC

Gln , as in the gidA mutant, reducesthe A-site selection rate22 without affecting thegrowth rate very much (80% of that of the wildtype). Also, the sufY219 (ybbB) mutant has a reducedrate of A-site selection of tRNAcmnm5s2UUG

Gln andgrows as well as a wild type strain.48 None of thetRNAs reading the other codons within the frame-

Table 3. Reduced A-site selection increases P-siteframeshifting mediated by defective ribosomal protein S9

Genotypea + His (2d)b – His (3d, 5d⁎ or 6d⁎⁎)b

wt 3.4 –sufY219 2.8 0.1rpsI2 1.4 0.3sufY219 rpsI2 1.2 1.5sufY219 2.8 0.1rpsI3 2.4 0.1sufY219 rpsI3 2.2 1.2gidA1 2.1 b0.1⁎rpsI2 1.5 1.8⁎gidA1 rpsI2 b0.1 3.2⁎gidA1 3.0 0.3⁎⁎rpsI4 2.2 0.6⁎⁎gidA1 rpsI4 1.4 3.0⁎⁎gidA1 3.0 0.3⁎⁎rpsI5 3.2 0.1⁎⁎gidA1 rpsI5 2.5 1.8⁎⁎

a All strains contain the hisD10122 allele -CCC-CAA-UGA).The “gain of function”mutation sufY219 (G67R in ybbB) results inaddition of a C10H17 fragment to the 2-sulphur of (c)mnm5s2U34present in tRNAcmnm5s2UUC

Gln , causing a reduced rate of entry of thistRNA into the A-site48. The gidA1mutation results in a deficiencyfor the cmnm5-moiety of the same wobble nucleoside, alsoleading to a reduced rate of A-site entry of tRNAcmnm5s2UUC

Gln 22.b Growth was scored as colony diameters (in millimetres) after

2 days on plates containing histidine (+ His) and 3, 5 (indicatedwith an asterisk) or 6 days (indicated with two asterisks) on plateslacking histidine (– His).


shift window in hisD10122 is affected by the gidA orsufY (ybbB) mutations. We combined each of thesemutations with the various rpsImutations and, in allcases, the suppression of the hisD10122 mutationwas much more efficient in the double mutants thanin any of the single mutants (Table 3). These twokinds of experiments suggest, as expected, that theframeshift occurs in the P-site at the CCC codonupstream of the CAA codon.We conclude that an altered C-terminal of ribo-

somal protein S9 induces frameshift in the P-site,consistent with our suggestion that the C-terminalend of this ribosomal protein is a functional part ofthe P-site environment critical for maintaining thereading frame.

Combinatory effects upon combining alteredproM tRNAcmo5UGG

Pro and defective ribosomalprotein S9

As shown above, changes of either the peptidyl-tRNAmoiety or protein S9 in the P-site environment

Table 4. Additive effects of mutant tRNA and S9

rpsI+ (6) rpsI2 (3)

proM+ N/A 0.1proM2225 (Um32Cm) 0.3 0.1proM2226 (A38G) 0.3 1.0proM2221 (C40U) 0.3 1.2

Approximate colony sizes (in mm) after the number of days (three,column. The strains were grown on medium E + glucose (no histidinehisD10122mutation better than one of the single mutations (rpsI or procompared to both the single rpsImutant (top row, proM+) and the singlesix days the wild type (proM+ rpsI+) strain did not form any visible co

induce errors in reading frame maintenance. To testhow the combination of changes in both tRNA andprotein S9 affects reading frame maintenance, weintroduced rpsImutations into strains carrying proMmutations and monitored the effect on suppressionof the hisD10122 allele (Table 4). If our suggestionthat frameshifting in these mutants (both rpsI andproM) is caused by a weaker interaction betweenpeptidyl-tRNA and the ribosome is correct, weexpected that such combinations would be additiveif the alterations affect different interactions betweenthe peptidyl-tRNAcmo5UGG

Pro and ribosomal proteinS9. However, if alterations of tRNAcmo5UGG

Pro and ofprotein S9 affect the same interaction, we expect noadditive effect by such combinations. Indeed, anadditive effect was observed for all the combinationstested, except for the combinations between theUm32Cm alteration in proM tRNAcmo5UGG

Pro and therpsI2 and rpsI3 alterations of S9 (Table 4). Since thelatter combinations were indistinguishable from thestronger of the single mutations, the Um32Cm alte-rations and the rpsI2 and I3mutations may affect thesame interaction. In addition to the combinationslisted in Table 4, the rpsI3 mutant was combinedwith the rest of the proM mutants listed in Table 1,except the very slow growing ΔU47 mutant, and allmutants other than Um32Cm caused an improve-ment of suppression compared to the rpsI3 singlemutant (data not shown). The crystal structure of theribosomal P-site suggests that the C-terminal end ofS9 makes a bond with the 5′-phosphate of position32 of peptidyl-tRNA.5 Therefore, the reason forframeshifting in the Um32Cm mutant may be thatthe change in position 32 causes a weaker interactionwith protein S9. As the rpsI2 and rpsI3 mutantsalready lack this interaction completely, the changein the tRNA at this position has no further effect.

Discussion

The isolated +1 frameshift suppressor mutantsof the proM tRNAcmo5UGG

Pro affect two differentparts of it; the anticodon stem (G31A, Um32Cm,A38G, C40U, U43A and U43C) and part of the“elbow region”, which is composed of the D-armand its interactions with the variable arm (Figs. 2and 3). The shift of reading frame by wild typeor mutant tRNAcmo5UGG

Pro s occurs after peptidyl-tRNAcmo5UGG

Pro has translocated successfully into

rpsI3 (4) rpsI4 (6) rpsI5 (6)

0.5 0.1 b0.1–0.30.5 0.9 0.51.7 1.5 0.42.0 2.0 1.5

four or six) indicated within parentheses in the header for each) at 37°C. Double mutations (rpsI, proM) that do not suppress theM) are indicated in bold. Values for each double mutant should beproMmutant (left-hand column, rpsI+). N/A, Not applicable; afterlony.


the P-site. Since these alterations of the peptidyl-tRNAcmo5UGG

Pro are suggested to be in close proxi-mity of various components of the ribosomal P-site (Fig. 3b), they may weaken the ribosomal gripof the peptidyl-tRNA and thereby induce errors inreading frame maintenance. Accordingly, changes inthe P-site environment of the ribosome, such asalterations of the C-terminal end of ribosomal proteinS9, which is suggested to be close to the anticodonloop of the peptidyl-tRNA, also induces shifts in thereading frame (Table 2). These two kinds of reciprocaldata are consistentwith our proposal that a ribosomalgrip of the peptidyl-tRNA is critical for maintainingthe reading frame. The results given in Table 4 suggesta direct interaction between the C-terminal end of S9and position 32 of peptidyl-tRNA.In the 30S P-site, several 16S rRNA nucleosides as

well as amino acid residues of S9 and S13 are withinonly 3.8 Å of the amino acid stem–loop of peptidyltRNA (Fig. 3a).5,30 Except for the stacking of C1400and G966 against the wobble nucleoside and, in onestructure,5 an interaction between lysine 129 of pro-tein S9 and the 5′-phosphate of the wobble position,all of these suggested interactions are in the lowerhalf of the anticodon stem, around base pairs 29–41,30–40, 31–39 and 32–38. If these interactions areimportant for reading frame maintenance, onewould expect some of our isolated +1 frameshiftsuppressors to have alterations to the tRNA thataffect any of these four base pairs. This is indeed thecase, since four of the mutations we found in proM(G31A, Um32Cm, A38G and C40U) did change therelevant base pairs. Base substitutions of C74 to A74or G74 of tRNAcmo5UAC

Val induce –1 frameshifting,33

and this part of the peptidyl-tRNA base pairs withG2252 and G2251 present in the peptidyl centre ofthe 23S rRNA.5,49 Alterations of the ribosomal resi-dues involved in the suggested interactions betweenthe peptidyl-tRNA and the ribosome should alsoresult in increased frameshifting.5,6 This is true, asshown by our isolation of rpsI mutants as frameshiftsuppressors (Table 2) as well as by base substitutionsof G2252 in 23S rRNA abolishing base pairs with theCCA end of tRNA thus inducing frameshifts.32

Moreover, a deletion of C1400 in 16S rRNA results ina dominant lethal phenotype and increased frame-shifting (both +1 and –1) if the mutant rRNA istransiently expressed.31 Thus, our results and earlierobservations support our suggestion that a riboso-mal grip of the peptidyl-tRNA is an important para-meter for reading frame maintenance.The strongest by far of the suppressors isolated

was the G31A mutant. It was the only mutant thatcaused stronger suppression than ΔproL (Table 1,compare proM+ ΔproL with G31A proL+). Thismutation changes the last canonical base pair(G31-C39) in the anticodon stem into an A-C mis-match. This may induce destabilization or structuralalteration in the lower part of the anticodon stem,which may affect the above-mentioned interactionswith rRNA or protein residues in the P-site. Muta-tions affecting this base pair in other tRNAs havebeen characterized as frameshift suppressors. A

G39A mutant of E. coli tRNAcmo5UACVal induces +2

hops and the bypass of stop codons50, and severaldifferent mutants with mismatches between posi-tions 31 and 39 in yeast tRNAncm5UGG

Pro induce +1frameshifts at proline codons.51 The C40Umutant oftRNAcmo5UGG

Pro (Table 1 and Fig. 2) changes the nextbase pair “above” the G31-C39 pair from a G-CWatson–Crick base pair to a G-U wobble pair. In the70S ribosome with tRNAi

Met in the P-site, A1339 of16S rRNA reaches into the minor groove of theanticodon stem, forming a type I A-minor interac-tion with the G30-C40 base pair.5 This type of inter-action is stronger for a G-C pair than for an A-U pair,and may require that the base pair is in Watson–Crick geometry.52 It is therefore likely that the pre-sence of a G-U wobble instead of a G-C base pair atthis position results in weaker binding to the P-site.The identity of the 32-38 base pair affects the

wobble capacity of tRNA,53 and appears to haveevolved with the anticodons, ensuring uniformbinding of all tRNAs to the A-site.54 The ribose ofnucleotide 38 of the peptidyl-tRNA stacks againstA790 in 16S rRNA, and the backbone of position 32is close to protein S9 and the backbone of 16S rRNAresidues 1340 and 1341.5, 30 A change of any of thebases (Um32 toCmorA38 toG) aswell as alterationsof S9 led to increased frameshifting (Table 1). Thecrystal structure of the 70S P-site suggests extensiveinteraction between the acceptor-stem and the CCAend of peptidyl-tRNAwith 23S rRNA and ribosomalproteins in and around the peptidyl transferasecentre.5,30 In addition to these interactions, proteinL5 interacts with the “elbow” of tRNA and severalresidues of 23S rRNA helix 69 interacts with the D-stem (positions 11–12 and 24–25) (Fig. 3b).5 We didnot isolate any mutant with changes in the tip ofthe elbow or the acceptor stem, but we did findbase substitutions at position 13 (C13U) and 23(C23A) in the D-stem. We suggest it is likely thatthe C13U and C23A mutations affect the local con-formation in the D-stem in such a way that theinteractions with helix 69 (Fig. 3) become weaker.Frameshifting in these mutants may be explainedby weaker ribosomal grip of the peptidyl-tRNA.Five of the mutants isolated in this study have

alterations in tRNA positions that are not very closeto any ribosomal residue in the P-site (G15A, ΔU47,C48U, U43A and U43C). It is possible that the G15Aand C48U mutations affect the flexibility or overallstructure of the elbow region in such a way thatsome interaction with the ribosomal P-site becomesweaker. Since the great majority (96.5%) of alltRNAs55 contain either a Watson–Crick base pairor a wobble (U-G or G-U) pair at positions 27–43, thetwo different mutations at position 43 (U43A andU43C), both of which are likely to disrupt the A27-U43 base pair at the top of the anticodon stem, maychange the structure of the tRNA in such a way thatit does not fit perfectly into the P-site. If so, themechanism by which these mutant tRNAs causeframeshifting may be similar to those alterations inthe tRNA discussed above, which are within 3.8 Å ofsome P-site ribosomal components.


The structure of the ribosome suggests a criticalposition of the C-terminal end of ribosomal proteinS9 in relation to the peptidyl-tRNA (Figs. 2b and 3a)and alterations of its C-terminal end induce +1frameshifts (Table 2). These observations suggestthat such a defect of the P-site environment mayinduce frameshift errors generally, and thus be ableto suppress many, if not all, frameshift mutations.However, the way the C-terminal end of this proteinis positioned relative to the peptidyl-tRNA may besensitive to which tRNA is in the P-site; i.e. altera-tions of S9 would affect only some specific pep-tidyl-tRNA. In line with this suggestion, an alteredC-terminal end of protein S9 selectively affects theP-site binding to the 30S subunit of tRNAs thathave sequences that are more divergent from thatof the initiator tRNA.46 The initiator tRNACAU

fMet hasthe G29-C41 and G30-C40 base pairs (see Fig. 2a) inthe anticodon stem, whereas the tRNAs that aremost affected in their binding by a defective proteinS9, have a different base pair in at least one of thesepositions.46 The ribosomal grip of such tRNAswould be weaker compared to the tRNAs that aremore similar to the initiator tRNA. Interestingly, atthe frameshift site used in this study (-CCC-CAA-UGA), the frameshifting tRNAcmo5UGG

Pro has onlyone of these two critical base pairs, suggesting thatits P-site binding to a 30S subunit should be lessefficient with a defective S9, and thus prone to shiftframe.Combining the Um32Cm mutation in proM with

the rpsI2 and rpsI3 mutations revealed that thesedouble mutants did not increase the frameshiftingrelative to the frameshifting induced by the indivi-dual mutations (Table 4). These results may indicatethat the cause of frameshifting in the position 32mutant of tRNAcmo5UGG

Pro is a weaker interaction withprotein S9, giving a weaker binding of the defectivepeptidyl-tRNAcmo5UGG

Pro to the P-site. As the rpsI2 andrpsI3 mutants already lack this interaction comple-tely, the change in the tRNA at this position wouldhave no further effect. On the other hand, it is hardto predict the effect in the rpsI4 and rpsI5 mutants,which may have one of the two suggested interac-tions with position 32 (K129/3′-phosphate or R130/5′-phosphate) still operating. These results are con-sistent with a functional interaction between thepeptidyl-tRNA and ribosomal protein S9 to main-tain the reading frame.As stated in Introduction, none of the bacterial or

yeast classical frameshift suppressors with an extranucleotide in the anticodon loop mediate suppres-sion by quadruplet translocation, but instead act bycausing realignment in the P-site.18,19 The frameshiftsuppressor sufD, which is a derivative of S. typhi-murium tRNACCC

Gly , has an extra nucleotide in theanticodon loop.11 This tRNA is, besides suppressing+1 frameshift mutations, able to suppress –1 frame-shift mutations.56 The latter ability is not easilyexplained by a quadruplet translocation. Also,tRNAcmo5UAC

Val (hopR) mutants with oversized antic-odon loops can suppress –1 frameshift mutations byhopping two bases forward (+2 hopping) and by-

pass in-frame stop codons by slipping six basesforward (stop-hopping).50,57 Such suppressor activ-ity is not consistent with an increased translocationstep-size but may be explained by P-site realign-ment. These observations suggest that even for thosemutant tRNAs that appear to have extra large anti-codons, the actual frameshift is induced because of adisturbed interaction between the peptidyl-tRNAand the ribosomal P-site, and not through anaberrant translocation. It is likely that some of theribosomal residues that make contact with the anti-codon stem–loop in the 30S P-site have to realign toaccommodate the extra nucleoside in the anticodonloops of these tRNAs, and thereby the binding ofthese peptidyl-tRNAs to the P-site may be severelyaffected.The stability of the codon–anticodon interaction

in the P-site was proposed to be pivotal for readingframe maintenance by ensuring that the next in-frame codon is positioned properly in the A-site.36

We suggest that the interactions between the pep-tidyl-tRNA and the ribosomal P-site are morecritical than the codon–anticodon interaction formaintaining the correct frame. Indeed, the sug-gested interactions between the body of the pepti-dyl-tRNA and the ribosome greatly outnumber theanticodon–codon interactions.30 Still, there aresuggestive interactions between the wobble basepair and residues of 16S rRNA,5 which probablycontribute to the ribosomal grip. This may providean explanation of why many of the characterizedframeshift suppressors and programmed frameshiftsites act by allowing a near-cognate tRNA to beaccommodated in the A-site, leading to a frameshiftonce it has been translocated into the P-site.18,24,39

The interaction between the wobble base pair and16S rRNA may be unstable for near-cognateinteractions. The mutants presented here affect atRNA that may be seen as near-cognate to the codonit shifts at, and it is possible that some or all of themutants cause frameshifting by weakening theinteraction between the peptidyl-tRNA and theribosomal P-site.

Materials and Methods

Bacteria and growth conditions

The strains and plasmids used in this study are givenin Table 5; strains that are not listed were constructed byP22 transductions, using the listed strains as donorsand recipients. All strains are derivatives of Salmonellaenterica serovar Typhimurium strain LT2. LAwas used assolid rich medium (per litre: 10 g of Trypticase peptone,5 g of yeast extract, 5 g of NaCl, and 15 g of agar).Medium E (E+Glu) was used as solid minimal medium(per litre: 15 g of agar and 2 g of glucose),58supplementedwith 0.1 mM histidine when needed (E+Glu+His).Cultures for analysis of ribosomal proteins were grownin liquid medium E containing glucose and histidine. Forβ-galactosidase assays, cultures were grown in Mopsminimal medium59 containing 0.2 mM histidine and 0.4%(w/v) sodium acetate as carbon source (Mops+acetate+

Table 5. Salmonella enterica serovar Typhimurium strains and plasmids

Strain Genotype Source/Reference

GT853 hisO1242 hisC3737 Laboratory collectionGT1696 hisD2504∷MudK hisO1242 leuA414 73GT6315 pKD46/LT2 Laboratory collectionGT6787 ΔproL hisO1242 hisD3749 34GT6877 proKbNfrt 34GT6879 ΔproL 34GT6902 ΔproL proKbNfrt 34GT7128 pKD46/hisD10132∷tetRA hisO1242 zdd-2532∷cat This studyGT7298 proM+ zhe-2533∷cat hisO1242 hisD3749 This studyGT7321 hisD10122 hisO1242 zdd-2532∷cat This studyGT7476 pKD46/hisD10131∷tetRA hisO1242 This studyGT7478 gidA1(58 bp internal deletion in gidA) zie-2536∷Tn10dTc

(between genes yieE and yidZ) hisD10122 hisO1242 zdd-2532∷catLaboratory collection

GT7484 sufY219 (G67R in ybbB) sfbA-2537∷MudSac hisD10122 hisO1242 zdd-2532∷cat Laboratory collectionGT7586 hisD10122 zdd-2532∷frt hisO1242 This studyGT7588 proM2219 (G31A) zhe-2533∷cat ΔproL hisO1242 hisD3749 This studyGT7589 proM2220 (C48U) zhe-2533∷cat ΔproL hisO1242 hisD3749 This studyGT7590 proM2221 (C40U) zhe-2533∷cat ΔproL hisO1242 hisD3749 This studyGT7625 zhd-2534∷kan (between genes yhcM and yhcB) rpsI-tetRA/pKD46, pMK76 This studyGT7629 rpsI2 zhd-2534∷kan (between genes yhcM and yhcB) hisD10122 hisO1242

zdd-2532∷catThis study

GT7700 proM2222 (G15A) zhe-2533∷cat hisD10122 zdd-2532∷frt hisO1242 This studyGT7701 proM2223 (C13U) zhe-2533∷cat hisD10122 zdd-2532∷frt hisO1242 This studyGT7743 rpsI4 (R130A) zhd-2534∷kan (between genes yhcM and yhcB) hisD10122

hisO1242 zdd-2532∷catThis study

GT7747 rpsI3 yhcM-2535∷Tn10dTc hisD10122 hisO1242 zdd-2532∷cat This studyGT7764 proM2224 (C23A) zhe-2533∷cat hisD10122 zdd-2532∷frt hisO1242 This studyGT7765 proM2225 (U32C) zhe-2533∷cat hisD10122 zdd-2532∷frt hisO1242 This studyGT7766 proM2226 (A38G) zhe-2533∷cat hisD10122 zdd-2532∷frt hisO1242 This studyGT7767 proM2227 (U43C) zhe-2533∷cat hisD10122 zdd-2532∷frt hisO1242 This studyGT7768 proM2228 (U43A) zhe-2533∷cat hisD10122 zdd-2532∷frt hisO1242 This studyGT7769 proM2229 (U17C, A38G) zhe-2533∷cat hisD10122 zdd-2532∷frt hisO1242 This studyGT7771 rpsI5 (K129A) zhd-2534∷kan (between genes yhcM and yhcB) hisD10122

hisO1242 zdd-2532∷catThis study

GT7772 proM2230 (ΔU47) zhe-2533∷cat hisD10122 zdd-2532∷frt hisO1242 This studyGT7775 proL2240 hisO1242 hisD10122 STM2235-2538∷Tn10 zdd-2532∷cat This studyGT7900 rpsI6bNkan hisD10122 hisO1242 zdd-2532∷cat This studypLG339 Low copy cloning vector, TetR KmR 78pUST136 S. typhimurium metT operon in pLG339, KmR 40pKD46 λ red recombinase under araBAD promoter, CbR 61pKD3 Template plasmid for chloramphenicol resistance cassette, CmR CbR 61pSMP24 dinB (DNA pol. IV) under araBAD promoter. 69pMK76 E. coli rplM+ rpsI+ operon in pSU19 (CmR) 79pNTR-SD-leuL Clone 109#4 from the mobile plasmid library; leuL under IPTG-inducible

promoter, CbR43

pGHM57 GST-MBP-His6 fusion vector (CbR) 37pUST290 CCC-CAA-A +1 frameshift site in pGHM57 (Fig. 4B) 48pUST292 UUU-CAA-A +1 frameshift site in pGHM57 (Fig. 4B) This studypUST310 CCC-CAA-U +1 frameshift site in pGHM57 (Fig. 4B) This studypNTR-SD-proS Clone 123#10 from the mobile plasmid library; proS under IPTG-inducible

promoter, CbR43

Not all of the strains used in this study are given here, but all of the strains not given here were constructed by P22 transductions using thelisted strains as donors and recipients.


histidine). Antibiotics were used at the following con-centrations: carbenicillin (Cb), 50 mg/L; kanamycin(Km), 100 mg/L; chloramphenicol (Cm), 12.5 mg/L;tetracycline (Tet), 15 mg/L.The frameshift mutation hisD3749 is an insertion of an

extra C in codon 14, changing the sequence -UGU-AGC-CCU-GAA- (codons 12–15, encoding the peptide sequence-Cys–Ser-Pro-Glu-) to -UGU-AGC-CCC-UGA-A- (enco-ding -Cys–Ser-Pro-stop).60 The frameshift mutationhisD10122 was constructed as follows: First, the tetracy-cline resistance genes tetA and tetR from Tn10dTc wereinserted into hisD in strain GT6315 (pKD46), replacingcodons 12–15, using λ-red recombineering as described.61A strain carrying this insertion GT7128 (pKD46/hisD10132∷tetRA, zdd-2532∷cat, hisO1242) was trans-

formed with a 60 nt DNA oligonucleotide designed toreplace the tetracycline resistance cassette with thedesigned mutation, selecting tetracycline-sensitiverecombinants.62 The end result of this was that codons13-14 (AGC-CCU) of hisD were replaced by the sequenceCCC-CAA-U. The complete frameshift windows aregiven in Fig. 4a. In order to produce a functional HisDprotein from these mutant mRNAs, the ribosome has toshift to the +1 reading frame before the UGA (stop)codons in the new zero frames are decoded by RF2. InhisD3749 a +1 shift when tRNAcmo5UGG

Pro is in the ribo-somal P-site at the CCC codon will result in a peptidewith the wild type sequence (-Cys-Ser-Pro-Glu-), while asimilar event in hisD10122 will result in a mutant peptidesequence (-Cys-Pro-Asn-Glu-).


Genetic procedures

To transfer chromosomal markers or plasmids betweenSalmonella strains, transductions were performed asdescribed,63 with a derivative of phage P22 containingthe mutations HT105/I64 and int-201.65

Localized mutagenesis of proM and selection forframeshift suppressors

zhe-2533∷cat is an insertion of the chloramphenicolresistance (CmR) cassette from plasmid pKD3, generatedas described.61 Its location only 56 bp downstream fromthe proM gene makes it almost 100% linked to proM inphage P22 transductions (Fig. 5). A phage P22 lysate pre-pared from strain GT7298 (proM+ zhe-2533∷cat ΔproLhisD3749 hisO1242) was treated with hydroxylamine asdescribed,66 until approximately 0.1% phage P22 infec-tious particles remained. This lysate was used to transducestrains GT6787 (ΔproL, hisD3749, hisO1242) and GT7586(hisD10122 hisO1242), selecting His+, CmR mutants on E+Glu+Cm+EGTA plates. Colonies that grew faster thanthe background of proM+ transductants were picked untilthe sixth or seventh day of incubation. When strainGT6787 was used as recipient, a total of approximately100,000 transductants yielded 88 His+ clones; 32 of thesewere linked to zhe-2533∷cat and in all of these, proM wassequenced. Each of these mutants was found to containone of three different mutations in proM; G31A, C40U andC48U (Table 1). When strain GT7586 was used asrecipient, 234 His+ clones were picked among approxi-mately 300,000 transductants. Of these, 171 were linked tothe zhe-2533∷cat insertion. To simplify further character-ization of these, they were divided into three groupsaccording to how efficiently they suppressed the frame-shift mutation in hisD, and approximately one-third of allmutants in each group were sequenced. This led toidentification of multiple isolates of the same three muta-tions already found in the previous screening (above) aswell as two more mutations; G15A and C13U (Table 1).A 1.9 kb PCR fragment containing proM and zhe-

2533∷cat but no other gene was amplified in an error-prone PCR reaction as described (Fig. 5).67 This PCRfragment was used to transform GT7476 (pKD46/hisD10131∷tetRA hisO1242) to CmR. Approximately 1230CmR colonies were pooled in 13 pools. Twelve slow-growing mutants also identified were tested separately.Four more pools were prepared from approximately80,000 colonies obtained from a similar transformationusing a PCR fragment amplified using Taq DNA poly-merase under standard conditions. P22 lysates preparedfrom these pools were used to transduce GT7586(hisD10122 hisO1242), selecting His+ and CmR. Fromeach transduction, between 10 and 100 larger coloniesappeared among a total of approximately 1000 – 2000

Fig. 5. Organization of the hisR operon. argX, hisR, leuTand proM encode tRNACCG

Arg , tRNAQUGHis , tRNACAG

Leu andtRNAcmo5UGG

Pro , respectively. The triangle containing theword cat indicates the location of the zhe-2533∷catinsertion used as selectable marker for the mutagenesis.The two grey arrows indicate the PCR primers used in themutagenesis.

transductants for the pools obtained from the mutagenicPCR or 10,000 – 20,000 transductants from the poolsfrom the standard PCR reaction. Growth of 10 – 20mutants from each pool was compared on E+Glumedium plates, and mutants that appeared to growsimilarly were considered to be siblings. One of the 12slow-growing mutants (proM2230 (ΔU47)) was also His+.Inall, 20 independent mutants were sequenced, resultingin identification of all mutations listed in Table 1 exceptthe C13U mutation.

Isolation of rpsI mutants able to suppress thehisD10122 (CCC-CAA) mutation

Phage P22 was grown on several cultures of strainGT6588, a strain that overproduces DinB (an error-proneDNA polymerase).68,69 Such phage stocks were used toinfect strains GT7321 (hisD10122) or GT853 (hisC3737),and His+ transductants were selected at 37 °C. More than300 mutants were characterized (unpublished results) asbeing extragenic suppressors to these two +1 frameshiftmutations in the his-operon. Two of these mutants, rpsI2and rpsI3, were shown by DNA sequencing to haveframeshift mutations in the rpsI gene. In the rpsI2 mutant,the C-terminal end of S9 is shortened by 20 amino acids,and the last amino acid is altered compared to the wild-type sequence as shown in Table 2. Likewise for the rpsI3mutation, the C terminus is shortened by 33 amino acidsand the six last amino acids are changed as shown inTable 2.To alter the last and the penultimate amino acid of

ribosomal protein S9, a PCR fragment containing the tetAand tetR genes from Tn10dTc was inserted by lineartransformation directly after the stop codon of the rpsIgene.61 A strain containing this insertion (GT7625;pKD46/pMK76 (rplM+ rpsI+)/rpsI-tetRA STM3346/47∷kan) was transformed with 78 nt long DNA oligonu-cleotides, which were designed to replace the tetracyclineresistance cassette and replace either the arginine 130codon or the lysine 129 codon with an alanine codon. TherplM+ rpsI+ plasmid (pMK76) was included to comple-ment a severe growth phenotype caused by the insertionof the tetracycline resistance cassette. The rpsI4 (R130A)and rpsI5 (K129A) mutants were obtained by selection fortetracycline-sensitive clones, and verified by DNAsequencing.62

tRNA preparation and HPLC analysis

For determination of the modification status oftRNAcmo5UGG

Pro , cultures were grown to late exponentialphase at 37 °C in LB medium. Total tRNA was pre-pared as described,34 and tRNAcmo5UGG

Pro was affinitypurified using a 5′-biotinylated DNA oligonucleotidecomplementary to nucleotides 38 – 67, as described.70

Purified tRNAcmo5UGGPro was degraded to nucleosides

using nuclease P1 and bacterial alkaline phosphatase.71

HPLC analysis was done essentially as described,72 butwith a slight modification of the protocol to separatecmo5U from other compounds.34

β-Galactosidase assays and calculation of frameshiftfrequencies

A hisD2504∷MudK insertion73 places lacZ after codon71 in hisD, creating a translational fusion in the zero frame.Amutant containing both hisD10122 and hisD2504∷MudK

Fig. 6. Sucrose gradient of washed ribosomes fromwild type cells (rpsI+) and different rpsImutants. About 10A260 units of washed ribosomes was applied on thegradient (the top of the gradient is to the left). The left-hand peak (30S subunits) or peaks A and B in panelrpsI6bNkan were pooled and their protein content wasanalyzed by one- or two-dimensional gel electrophoresis(Figs. 7 and 8).

Fig. 7. One-dimensional gradient gel electrophoresis of30S proteins. To the left, proteins from peaks A and Bobtained from rpsI6bNkanmutant (see the sucrose gradientin Fig. 6). A band that was missing from the samples of therpsI3 and rpsI6mutants was identified as protein S14 byN-terminal sequencing of the peptide from the rpsI+ strain.The migration of protein S9 is similar to that of anotherprotein, since a protein band is still present in therpsI6bNkan mutant as well as in the rpsI2 and rpsI3mutants, which both contain a truncated form of S9(denoted S9Δ20 and S9Δ34, respectively).


produces β-galactosidase only when a +1 frameshiftoccurs in the frameshift window of the hisD10122 muta-tion (see Fig. 4a). To produce such double mutants, a strain(GT1696) carrying hisD2504∷MudK was used as donor inP22 transductions with hisD10122 strains as recipients.Blue-white screening on X-gal plates identified recombi-nants that had recombined between the frameshift muta-tion and the Mud insertion as a few white or light bluecolonies among thousands of blue colonies. Cultures for β-galactosidase assays were grown to A600 of 0.4 –0.5 inMops + acetate + histidine medium, as we found thatexpression from the his promoter was too low in any richermedium (data not shown). Assays were done as de-scribed,74 using chloroform and SDS to permeabilize thecells, with the modification that the cultures were pelletedand resuspended in a volume of Z-buffer suitable formeasuring very low activities (cultures containing the

hisD10122 mutation (0.3–5.2 Miller units) were concen-trated 10-fold to get more concentrated extracts, whilecultures without the hisD10122 mutation (47–95 Millerunits in different strains and different experiments) werediluted two or threefold). The frameshift frequencies inTable 1 and 2 are expressed as:

FS = ðShiftÞ=ðIn frameÞ � 100%

where Shift is the activity of a strain containing thehisD10122 mutation and In frame is the activity of anisogenic strain without the hisD10122 mutation. As thetranslational fusion is present on the chromosome, FS % isan estimate of which proportion of the total number ofribosomes that reaches the frameshift window in hisDshifts into the +1 frame. The error ranges for the FS%values in Table 1 are expressed as:

Error = FS%� ½ðs:d: of Shift=ShiftÞ + ðs:d: of In frame=In frameÞ�:

Determination of the amino acid sequence of theslippage junction

The full-length GST-MBP-His6 fusion proteins ex-pressed from plasmids pUST290, pUST292 and pUST310were purified essentially as described,37 except the Ni-NTA purification step was omitted and the MBP-His6 partof the fusion was released by digestion with PreScissionProtease (GE Healthcare), while the GST part was stillbound to glutathione-Sepharose. The MBP-His6 peptideswere separated by SDS/15% (w/v) PAGE and electro-blotted onto a Sequi-Blot PVDF membrane (Bio-Rad). Thebands corresponding to the MBP-His6 peptides wereexcised from the membrane and subjected to N-terminalsequence analysis by Edman degradation. A +1 reading


frame shift when tRNAPro is in the P-site at the CCC codonin pUST310 would result in the sequence GPLGI-LICPNDK. Unmodified cysteine is too reactive duringN-terminal sequencing and is usually seen only indirectlyas the absence of an amino acid in one cycle. Instead ofcysteine, an unknown compound was observed. Toconfirm the presence of cysteine in the peptides purifiedfrom pUST310, peptides purified from the wild type andthe G31A mutant were reduced with dithiothreitol (DTT)and alkylated with iodoacetic acid to modify cysteine.75This resulted in reduced levels of the unknown com-pound and, as expected, the appearance of S-carboxy-methyl cysteine as a major peak in the G31A mutant anda minor peak in the wild type.

Ribosome preparations, sucrose gradient separation,and electrophoresis

Cells were grown in 200 ml of medium E containing0.2% (w/v) glucose and 0.1 mM histidine to a cell densityof A600=0.7. Cells were harvested by centrifugation andsuspended in 1 ml of 20 mM Tris–HCl, 1 mM MgCl2, pH7.5. Lysozyme (1 mg) was added and the cell suspensionwas frozen and thawed three times before DNase I (4 μg)and deoxycholate (30 μl of a 10% solution) were added.

Fig. 8. Two-dimensional gel electrophoresis analysis ofribosomal proteins from sucrose gradient-purified 30Ssubunits from wild type and different rpsI mutants asindicated. In panels rpsI2 and rpsI3, S9Δ20 and S9Δ34denote the 20 and 34 amino acids truncated forms ofribosomal protein S9, respectively. An aberrant matura-tion of ribosomes from the rpsI6bNkan mutants is alsoapparent from the sucrose gradient analysis (Fig. 6) andthe one-dimensional gel electrophoreses (Fig. 7).

The lysate was centrifuged for 30 min at 15,000 rpm in aBeckman JA-25.50 rotor. About 100 μl of this cleared lysatewas applied directly as unwashed ribosomes to a sucrosegradient (5%–30% (w/v) sucrose in R-buffer (10 mM Tris–HCl, pH 7.5, 50 mM KCl, 10 mM MgCl2, 6 mM 2-mercaptoethanol) and centrifuged at 20,500 rpm for 15 h at4°C in a Beckman SW41Ti rotor. To wash the ribosomes,6 ml of R-buffer was added to the cleared lysate and thesolution was made 1 M in NH4Cl. After incubation on icefor 2 h, the ribosomes were recovered by centrifugation for80 min at 50,000 rpm using a Beckman 70.1 Ti rotor. Thepellet was suspended in 7 ml of R-buffer containing 1 MNH4Cl and the ribosomes were recovered as before. Thewashed ribosomes were dissolved in 0.5 –1 ml of R-bufferand about 50 A260 units were applied onto a sucrosegradient (5%–30% sucrose in R-buffer) and run asdescribed above.The fractions containing the 30S subunits (Fig. 6) were

pooled and the material was concentrated in a CentriconYM3 ultrafiltration unit (2×2–3 h at 7500g). The concen-trated solution was made 150 mM in magnesium acetateand two volumes of concentrated acetic acid were addedto precipitate the 16S rRNA. Following centrifugation at25,000g for 10 min, the supernatant, which contains thebasic ribosomal proteins, was concentrated using aCentricon YM3 ultrafiltration unit as described above.The protein distribution was analyzed by one-dimen-sional electrophoresis in a 10%–19.5% gradient polyacry-lamide gel containing 0.1% (w/v) SDS (Fig. 7) or by two-dimensional PAGE (Fig. 8). Two-dimensional PAGE wasperformed essentially as described.76 The gel for the firstdimension was 8% acrylamide in 0.4 M Tris/0.5 M boratebuffer, pH 8.6, containing 6 M urea and 20 mM EDTA. Therunning buffer was 0.12 M Tris/0.15 M borate, pH 8.6,containing 6 M urea and 6 mM EDTA. The seconddimension gel was 18.6% acrylamide, 6.2 M urea, 0.94 Macetic acid, 0.05 M KOH, pH 4.6. The running buffer forthe second dimension was 0.186 M glycine, 0.027 M aceticacid. For both dimensions, electrophoresis was runtowards the negative electrode at 87 V for 30 h at 4°C toseparate the positively charged proteins.

Naming conventions

The mutant proM alleles are referred to in the text by thechange in their corresponding RNA sequence; thus, G31Ais a mutant where G in position 31 of the wild type tRNAis replaced by A in the mutant, and Um32Cm is an allelethat results in a 2′-O-methylcytidine instead of 2′-O-methyluridine at position 32.

Acknowledgements

This work was supported by grants from theSwedish Science Research Council (Project BU-2930).We thank Kerstin Jacobsson and Gunilla Jäger forexcellent technical assistance with HPLC analysesand for help with construction of some of the strainsused in this study. We are grateful to TordHagervall, Stefan Nord and Anders Byström fortheir critical reading of this manuscript. The E. colimobile plasmid orf library was a generous gift fromthe National Institute of Genetics, Japan (http://www.shigen.nig.ac.jp/ecoli/strain/top/top.jsp).


The N-terminal sequence analyses by Edman degra-dation were done by the Protein Analysis Centre(PAC), at the Karolinska Institute, Stockholm, Sweden.

Supplementary Data

Supplementary data associated with this article-can be found, in the online version, at doi:10.1016/j.jmb.2008.10.069

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The Ribosomal Grip of the Peptidyl-tRNA is Critical for Reading Frame Maintenance