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Sequestration of specific tRNA species cognate to the last sense codon of an overproduced gratuitous protein

Sequestration of specific tRNA species cognate to the last sense codon of an overproduced... © 2000 Oxford University Press Nucleic Acids Research, 2000, Vol. 28, No. 23 4725–4732 Sequestration of specific tRNA species cognate to the last sense codon of an overproduced gratuitous protein Jeanne Menez, Valérie Heurgué-Hamard and Richard H. Buckingham* UPR9073 du CNRS, Institut de Biologie Physico-Chimique, 13 rue Pierre et Marie Curie, F-75005 Paris, France Received August 8, 2000; Revised October 6, 2000; Accepted October 19, 2000 ABSTRACT Escherichia coli (4,5). In cells deficient in Pth, protein synthesis is arrested and the cells finally die due to starvation High-level expression of non-functional model for tRNA, which becomes sequestered as peptidyl-tRNA and proteins, derived from elongation factor EF-Tu by the unavailable for protein synthesis. When deprived of Pth by the deletion of an essential domain, greatly inhibits the transfer to a non-permissive temperature of cells containing a growth of Escherichia coli partly deficient in Lys thermosensitive enzyme, tRNA becomes limiting considerably peptidyl-tRNA hydrolase. High-level expression in before other tRNA species (6). The reasons for this are not yet wild-type cells has little effect on growth. The inhibitory fully understood, and may be related to a higher rate of drop- Lys effect is therefore presumably due to the sequestration off of peptidyl-tRNA species involving tRNA . One possible Lys of essential tRNA species, partly in the form of free explanation is that peptidyl-tRNA is a poor substrate for Pth compared with peptidyl-tRNAs that accumulate slowly. peptidyl-tRNA. The growth inhibitory effect can be Recent measurements, however, show that the differences are modulated by changing the last sense codon in the not large enough to explain the large variation in accumulation genes encoding the model proteins. Thus, replacement rates (V.Heurgué-Hamard, unpublished results). of Ser by Lys or His at this position increases growth Several aspects of peptidyl-tRNA drop-off remain poorly inhibition. The effects of 11 changes studied are understood. It is unclear what fraction of drop-off events related to the rates of accumulation previously occurs at codons cognate to the tRNA in question, and what observed of the corresponding families of peptidyl- fraction occurs at near-cognate codons, as a result of errors of tRNA. Two non-exclusive hypotheses are proposed selection of tRNA that escape the proofreading mechanism (7). to account for these observations: first, the last Studies of the suppression of a thermosensitive Pth mutant sense codon of mRNA is a prefered site of peptidyl-tRNA showed that drop-off was not merely a passive event, but drop-off in cells, due to the slow rate of translation involved translation factors RRF and RF3 in vivo (8,9). termination compared with sense codon translation; However, attempts to demonstrate that mutants affecting RRF secondly, the relatively long pause of the ribosome at and RF3 increased translational processivity using the lacZ the stop codon (of the order of 1 s), results in signifi- monomer-dimer approach of Jørgensen and Kurland (2) were unsuccessful (V.Heurgué-Hamard and R.H.Buckingham, cant temporary sequestration on the ribosome of the unpublished work). One explanation for this unexpected result, tRNA cognate to the last sense codon. apart from technical reasons related to the difficulties of this experimental approach, is that drop-off events involving RRF INTRODUCTION and RF3 occur predominantly at the beginning or end of mRNAs and would therefore not be detected. A significant proportion of ribosomes that initiate translation The drop-off of short peptidyl-tRNAs has been the subject of of an mRNA fail to terminate by the normal mechanism, which extensive recent study (6,9–14). The expression of very short involves the recognition of messenger-encoded stop signals, open reading frames, encoded by ‘mini-genes’, can lead to the hydrolysis by the ribosome of peptidyl-tRNA and release of rapid accumulation of peptidyl-tRNA and the arrest of protein the completed polypeptide. Truncated proteins arise at a –4 synthesis even in cells with normal amounts of Pth. In such frequency of ∼3 × 10 per codon, which implies that about cases drop-off does indeed appear to occur at a codon read by one-third of the ribosomes that initiate β-galactosidase the cognate tRNA, namely, the last sense codon encoded by the synthesis fail to reach the stop signal (1–3). Measurements of mini-gene, as the inhibitory effect can be relieved (at least in the synthesis of β-galactosidase dimers indicate that the the limited number of cases that have been studied) by over- frequency of such processivity failures is affected by mutations production of the tRNA species cognate to the last sense in genes encoding ribosomal proteins S12, S4 and S5 (3), codon. The presence of a stop codon very early in an mRNA known to control the accuracy of translation. It is believed that the majority of processivity failures result in the dissociation of favours drop-off and peptidyl-tRNA accumulation for several peptidyl-tRNA from the ribosome (2), or ‘drop-off’. The recycling reasons. These include the catalysis of drop-off by translation of these molecules requires peptidyl-tRNA hydrolase (Pth), a factor and the phenomenon of 30S ribosome recycling on short ubiquitous enzyme known to be essential for viability in mRNAs (9,14). Another important factor probably arises *To whom correspondence should be addressed. Tel: +33 1 4354 1972; Fax: +33 1 4046 8331; Email: rhb@ibpc.fr The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors 4726 Nucleic Acids Research, 2000, Vol. 28, No. 23 Table 1. Bacterial strains Strain Genotype Reference/construction FTP4993 metB, glyV55, trpA(AGC233, UGA234) F.T.Pagel IB188 ara, ∆ (lac–proB), nalA, metB Derivative of XA102 (40) GY5552 ∆ (lac, pro), thi, strA, endA, sbcB15, hsdR4F′traD36, proAB, lacIqZ∆ M15, recA938::Cm Marsh and Walker (41) VH7 supE44, thi1, thr1, leuB6, lacY1, tonA21, pth(ts), fadR13::Tn10 Heurgué-Hamard et al. (6) VH287 ara, ∆ (lac–proB), nalA, metB, pth(ts), fadR13::Tn10, recA938::Cm This work, IB188 and P1 lysates on VH7 and GY5552 VH733 metB, glyV55, pth(ts) Heurgué-Hamard et al. (8) VH804 metB, glyV55, trpA(AGC233, UGA234), recA::Cm This work, FTP4993 and P1 lysate on GY5552 VH805 metB, glyV55, pth(ts), recA::Cm This work, VH733 and P1 lysate on GY5552 directly from the slow kinetics of the termination reaction are derivatives of pHD67 (3), which contains the partially compared with the translation of most sense codons. The deleted tufB gene from plasmid pTuB12.1 (21) cloned in the limiting factor in the termination reaction is probably the low expression vector pTrc99c (22). Plasmid pJMM1 contained a –1 value of k , of the order of 1 s (15). The pausing of ribo- further truncation in the tufB gene (encoding ∆∆ EF-Tu), and cat somes for substantial periods at stop signals has been observed wasobtainedbydigestion of pHD67with EagI and religation both in vitro (16,17) and in vivo (18). of the plasmid. Variants of both plasmids pHD67 and pJMM1 Since drop-off is an important phenomenon when the were constructed by a method employing two PCR steps. To ribosome is paused at the termination signal of very short introduce different codons before the stop codon, a first PCR coding sequences, it is of interest to know whether the drop-off was performed using a mutagenic oligonucleotide covering the of peptidyl-tRNAs with complete polypeptide chains represents a stop signal and a second oligonucleotide upstream of the EagI significant part of total drop-off. The reason for the high rate of (951) site (see Fig. 1) site in pHD67 or pJMM1. The product of Lys peptidyl-tRNA accumulation in cells deprived of Pth (19) is this PCR step was used in a second PCR step in conjunction not well understood, but may simply be a reflection of a generally with a further oligonucleotide downstream of an SgfI site increased frequency of adenosine (and consequently of Lys located 181 nucleotides beyond the tufB stop codon. The codons) close to initiation and termination codons (20). Little product was restricted with SgfIand PshAI or EagIand used to is known of the distribution of polypeptide chain lengths in replace the corresponding fragment in pHD67 or pJMM1 peptidyl-tRNA molecules arising by premature peptidyl-tRNA respectively. All variants of these plasmids were verified by dissociation from the ribosome and we cannot yet say whether sequencing (23). They are listed in Table 3 together with the the beginnings or ends of coding regions are preferred regions corresponding final sense codon. Plasmids pVH1 and for drop-off to occur. pJMM19, carrying the pth and lysV genes, respectively, are To search for effects on peptidyl-tRNA accumulation related derivatives of low-copy-number plasmid pWSK129 (24). to the coding sequence just upstream of the stop signal, we Plasmid pJMM19 was made by recloning the ApaI–BamHI have examined the overexpression of a series of model fragment carrying lysV from pVH119 (6) between the same sites proteins that differ with respect to the last sense codon. The in pWSK129; like pVH119 it results in an increase of ∼2-fold in experiments are performed under conditions of limitation for Lys the quantity of tRNA in the cell. Pth, so that cell growth becomes sensitive to the degree of sequestration of different tRNA species. The results show that Growth conditions the identity of the last sense codon does indeed affect the Luria–Bertani (LB) broth was employed as rich medium. Anti- degree of toxicity due to high-level expression of the model biotics were added as necessary at the following final concen- protein and that the effect is related to the rate of accumulation trations: kanamycin 50 µ g/ml, ampicillin 200 µ g/ml. In of the corresponding peptidyl-tRNA species described by experiments with thermosensitive Pth strains, precultures were Menninger (19). Two non-exclusive mechanisms can account grown at 30°C in LB broth containing appropriate antibiotics. for the observations: first, drop-off occurring at a significant After dilution to an OD of 0.05, growth was continued at level at the last sense codon, and secondly, the relatively slow 37°C (or as otherwise indicated in figure legends) to an OD process of termination and ribosome recycling that sequesters of 0.5. Synthesis of ∆ EF-Tu or ∆∆ EF-Tu was induced by the tRNA corresponding to the last sense codon for a sufficient addition of isopropyl-β-D-thiogalactopyranoside (IPTG) to a period that the pool of free, charged tRNA is significantly final concentration of 1 mM after a further dilution to an OD depleted. of 0.05. For labelling of proteins with [ S]Cys, cells were grown in M9-glucose medium (25) supplemented with all MATERIALSAND METHODS amino acids except Cys. An overnight culture was diluted to ∼2 × 10 cells/ml and labelled for two generations by addition Bacteria and plasmids of [ S]Cys (1 Ci/µ mol) to a concentration of 15 µ Ci/ml. IPTG Escherichia coli K12 strains are listed in Table 1. All plasmid was added to a concentration of 1 mM and labelling continued constructions expressing ∆ EF-Tu and variants of this protein for 3 h. Nucleic Acids Research, 2000, Vol. 28, No. 23 4727 Figure 1. Deleted regions of EF-Tu in model proteins ∆ EF-Tu and EF-Tu. The deleted regions are shown in relation to the 3D structure of EF-Tu (A) and to the tufB gene coding sequence (B). The region in red is absent from the ∆ EF-Tuprotein andboththisand theregioninbluefrom ∆∆ EF-Tu. The model is based on the crystallographic structure of EF-Tu (42) and the sequence is from GenBank, accession no. M30610. Recombinant DNA techniques and genetic manipulations protein, called ∆ EF-Tu, lacks part of the GTP-binding motif G3 and motif G4 as a result of the deletion of a SmaI–SmaI General procedures for recombinant DNA techniques, plasmid fragment in tufB covering nucleotides 248–491 (29). This extraction, agarose gel electrophoresis, etc., were performed as removes a region of the native sequence corresponding to a describedbySambrook et al. (26). DNA fragments from well-defined domain in the three-dimensional structure of the agarose gels were extracted using Jetsorb gel (Bioprobe) protein (30). A further truncation was also made by deleting an according to the manufacturer’s instructions. Phage P1 lysates, EagI–EagI fragment (nucleotides 951–1140), yielding a transductions and transformations were performed as second protein (∆∆ EF-Tu), lacking about two-thirds of domain described by Miller (27). 3 (Fig. 1) as well as the GTP-binding motifs. Quantification of proteins In plasmidpHD67encoding ∆ EF-Tu, transcription depends on the trc promoter and is induced by IPTG (3). Continuous Induced cultures labelled with [ S]Cys as described above 35 35 labelling experiments with [ S]Cys or [ S]Met indicate that were centrifuged and the cells lysed for 3 min at 100°Cinlysis 3 h after induction of pth wild-type cells transformed with buffer (50 mM Tris–HCl, pH 6.8, 100 mM dithiothreitol, 2% pHD67, ∼25% of total cell protein is ∆ EF-Tu, confirming the SDS, 0.1% bromophenol blue). The proteins were separated by observations of Dong et al. (3). Under the conditions of our electrophoresis on 10% polyacrylamide gel as described by experiments the expression of ∆ EF-Tu has little effect on the Laemmli (28). Gels were dried and the radioactivity in growth of pth wild-type cells. In contrast, in cells partly individual bands and complete tracks was determined using a deficient in Pth, growth is partially or completely inhibited. All PhosphorImager (Molecular Dynamics). experiments in Pth-deficient cells have made use of the thermo- sensitive Pth mutant isolated by Atherly and Menninger (4), RESULTS ANDDISCUSSION transduced into various genetic backgrounds by selection for a nearby transposon. In liquid culture in rich media, the pth(ts) Overproduction of an inactive protein inhibits growth of strain VH805 grows at temperatures up to 42°C, and grows at Pth-deficient cells 37°C asfast asthe pth parental strain VH804. When trans- The study of tRNA sequestration and its dependence of mRNA formed with plasmid pHD67, strain VH805 grows normally at sequence required model proteins that could be expressed at a 37°Cinthe absence of ∆ EF-Tu expression, but ceases to grow shortly after induction of the gene carried in pHD67 (triangles, high level in E.coli without deleterious effects on the growth of Fig. 2). Cotransformation of cells with a plasmid pVH1 carrying cells with normal levels of Pth activity. In order to avoid tRNA the wild-type pth gene suppresses this growth inhibition (not usage that might affect tRNA pool sizes we chose a gene with codon usage typical of highly expressed genes in E.coli,a shown). A similar behaviour is seen on plates (Fig. 3A, truncated version of tufB, encoding protein synthesis elongation compare segments pHD67 and pTrc99c). At 30°C, where factor EF-Tu (3,29). As shown in Figure 1, the truncated intracellular Pth activity is less affected by the thermolability ∆∆ 4728 Nucleic Acids Research, 2000, Vol. 28, No. 23 Table 2. Growth inhibition at different temperatures of Pth-deficient cells due to plasmid-directed synthesis of ∆ EF-Tu and ∆∆ EF-Tu Strain/plasmid pth(ts)/control pth(ts)/∆∆ EF-Tu pth(ts)/∆ EF-Tu pth /∆ EF-Tu VH805/pTrc99 VH805/pJMM1 VH805/pHD67 VH804/pHD67 IPTG: – +– + – +– + 30°C ++ ++ ++++++ ++ ++++ 34°C ++ ++ ++++++ – ++ ++ 37°C ++ ++ +++ ++ – ++++ 39°C ++ ++ ++– ++ – ++++ 43°C – – –––– ++ + Transformed cells were streaked on to LB-ampicillin plates with or without 1 mM IPTG and incubated at the temperature shown in the first column. The table shows inhibited growth (+) or no growth (–) in comparison with the normal growth (++) of pth cells transformed with control plasmid pTrc99C. Figure 2. Inhibition of cell growth in partially Pth-deficient strains following induction of ∆ EF-Tu synthesis. Tranformants of the Pth thermosensitive strain VH805 carrying plasmid pHD67 encoding ∆ EF-Tu were grown at 37°Cin LB-ampicillin medium to ∼4 × 10 cells/ml. Growth was monitored by measurement of OD as a function of time after induction with 1 mM IPTG; induced culture (open triangles) and non-induced culture (filled circles). of the enzyme, induction of ∆ EF-Tu expression does not affect growth. At temperatures of 34°C and above (in the case of strain VH805), pth(ts) cells transformedwithpHD67nolonger grow in the presence of IPTG, whereas non-induced cells or Figure 3. Growth inhibition on solid medium due to ∆ EF-Tu or EF-Tu synthesis cells transformed with the control plasmid pTrc99C grow and the effect of mutations in the gene encoding ∆∆ EF-Tu. Tranformants of normally at temperatures up to 39°C (Table 2). The threshold for Pth thermosensitive strains with pHD67 or pJMM1 (SerAGC) and variants of thermosensitivity is thus lowered by the remarkable magnitude of the latter plasmid altered in the last sense codon were streaked on LB–agar plates 9°Cwhen ∆ EF-Tu is expressed. In other strain backgrounds with or without 1 mM IPTG, and incubated at the temperature indicated. Growth the pth(ts) mutation leads to more or less thermosensitive on plates is shown for (A) transformants of VH805 with plasmids pHD67 and pJMM1, encoding ∆ EF-Tu and ∆∆ EF-Tu, respectively, and control plasmid growth, and the onset of thermosensitivity of the transformed, pTrc99c; (B) transformants of VH287 with plasmid pJMM1 and variants altered induced cells is correspondingly shifted to lower or higher in the last sense codon of tufB from SerAGC to LysAAA/G or HisCAC/U. temperatures, respectively. Two broadly different explanations can account for the observation that overproducing one gratuitous protein can uniform but to vary according to the mRNA (31). The second lower the threshold of thermosensitivity in the Pth mutant class of explanation would depend on particular properties of strain. The overproduction does not lead to a marked increase the mRNA for ∆ EF-Tu. Thus, drop-off of peptidyl-tRNA in the total amount of protein synthesis occurring in the cell. during synthesis of ∆ EF-Tu may be higher than the average Rather, it diverts part of the cell’s capacity for protein frequency of drop-off, and increase the total amount of drop- synthesis to one protein at the expense of others. Thus, the off in the cell. Alternatively, even if the overall amount is not proportion of any particular protein (including Pth) being synthesised may fall in relation to total protein synthesis as a increased, the pattern of tRNAs accumulating as peptidyl-tRNA may change due to particular drop-off points characteristic of the result of the extra imposed ‘protein burden’. In fact, the degree to which the synthesis of different cell proteins is affected by mRNA, and hasten the onset of starvation for an essential the induction of a plasmid-carried gene is known to be not tRNA species. Further insight into these and other explanations ∆∆ ∆∆ Nucleic Acids Research, 2000, Vol. 28, No. 23 4729 Figure 4. Suppression of growth inhibition due to EF-Tu synthesis by over- Lys production of tRNA from a low-copy-number plasmid. Strain VH805 was Figure 5. Increased growth inhibition due to EF-Tu synthesis on substitution of cotransformed with plasmid pJMM1 expressing ∆∆ EF-Tu and either plasmid Lys codons at the last sense position of the ∆∆ EF-Tu mRNA. Tranformants of pJMM19 carrying lysV or the parent plasmid pWSK129 with no insert. the Pth thermosensitive strain VH805 with plasmid pJMM1 or variants of the Transformants were grown at 38.5°C in LB-ampicillin-kanamycin medium to plasmid altered in the last sense codon of ∆ tufB from SerAGC to LysAAA 7 7 ∼3 × 10 cells/ml, and EF-Tu synthesis was then induced with 1 mM IPTG. were grown at 38°C in LB-ampicillin medium to ∼3 × 10 cells/ml, and EF-Tu Growth was monitored by measurement of OD as a function of time after synthesis was induced with 1 mM IPTG. Growth was monitored by measurement induction for induced cultures (open circles, pJMM1 and pJMM19; open of OD as a function of time after induction for induced cultures (open circles, triangles, pJMM1 and pWSK129) and non-induced cultures (filled circles, pJMM1; open squares, pJM-LysAAA) and non-induced cultures (filled circles, pJMM1 and pJMM19; filled triangles, pJMM1 and pWSK129). pJMM1; filled squares, pJM-LysAAA). was obtained by experiments in which specific modifications Lys Growth inhibition is suppressed by tRNA were made to the ∆ EF-Tu mRNA. overproduction Deletion of a C-terminal fragment of ∆ EF-Tu reduces the The fact that the growth inhibitory effect of overproducing growth inhibition ∆ EF-Tu was seen only in mutant cells partially deficient in Pth strongly suggests that the mechanism of growth inhibition is It has been reported that a region near the C-terminus of EF-Tu, likely to be the same as in severely Pth-limited cells. Previous including residue G375, is important for an autoregulatory work has shown that under semi-permissive conditions for mechanism of tufB expression, and that this regulatory effect is pth(ts) strains (at ∼42°C in the case of strain VH805), growth conserved in ∆ EF-Tu (32,33). This suggested that the toxicity is inhibited due to starvation for tRNA and in particular for associated with ∆ EF-Tu expression might in part be related to Lys tRNA (6). If tRNA starvation due to sequestration as regulatory effects on tufB expression. The ∆ tufB in plasmid peptidyl-tRNA results from the induction of ∆ EF-Tu synthesis, pHD67 was therefore modified by deletion of a region of the then the effects should be at least partly suppressed by the over- gene encoding 63 amino acids close to the C-terminus of ∆ EF-Tu, Lys production of tRNA similarly to the original observation (6). In including residue G375 of TufB and surrounding residues, the experiment presented in Figure 4, the lysV gene is carried resulting in plasmid pJMM1. The deleted fragment corresponds to on a low-copy-number plasmid pJMM19 compatible with an EagI–EagI fragment in the ∆ tufB gene (Fig. 1). The expression pJMM1 and results in an ∼2-fold increase in the level of of this doubly deleted protein ‘∆∆ EF-Tu’ remains inhibitory to Lys tRNA (6). The growth inhibition resulting from the induction of growth in pth(ts) cells, but the threshold temperature for ∆∆ EF-Tu synthesis in the strain cotransformed with pJMM1 inhibition is shifted upwards by ∼5°C. The behaviour of pth(ts) and the parent plasmid pWSK129 is seen to be almost cells transformed with plasmid pJMM1 is intermediate completely eliminated when the plasmid carries a lysV insert. between pHD67-transformed cells and cells transformed with the control plasmid pTrc99C (Table 2, columns 6 and 7). Thus, Changing the last codon of the ∆∆ EF-Tu gene modulates at 34–38°C IPTG induction of the pth(ts) strain VH805 trans- the growth inhibition formed with pJMM1 still grows, whereas the same strain Recent data suggest that the termination step in protein transformed with pHD67 ceases growth completely (Table 2; see also Figs 4 and 5). In contrast, at 39°C EF-Tu expression synthesis catalysed by release factors RF1 and RF2 is slow in completely inhibits cell growth although VH805 transformed comparison with an elongation step (15,18,34). This suggests with pTrc99C grows as well as the pth parental strain VH804 that the last sense codon might be more prone to peptidyl- tRNA drop-off than the average sense codon of an mRNA. To at this temperature. Other experiments (not shown) show that ∆∆ EF-Tu is synthesised with similar translational efficiency to look for evidence of such an effect, we have introduced mutations ∆ EF-Tu. The difference between the two model proteins could that change the last amino acid of ∆∆ EF-TuinpJMM1 (or be accounted for by the ‘protein burden’ type of explanation, ∆ EF-TuinpHD67) andlooked for modulation ofthe growth as a significant part of the polypeptide chain is removed in this inhibitory effects in Pth-deficient cells. Previous work has Lys second deletion. It is also possible that the second deletion identified tRNA as the tRNA isoacceptor most likely to removes drop-off sites encoded within the EagI–EagI fragment of become fully sequestered as peptidyl-tRNA in Pth-limited tufB. cells (6). The data of Menninger (19) provide further valuable ∆∆ ∆∆ ∆∆ ∆∆ ∆∆ 4730 Nucleic Acids Research, 2000, Vol. 28, No. 23 information concerning the probable relative rates of accumu- significant effect on the degree of toxicity on induction. The lation of different peptidyl-tRNA species. Thus, peptidyl- comparison of the inhibitory effects of the entire family of Thr His Ile tRNA , peptidyl-tRNA and peptidyl-tRNA as well as plasmids with altered last sense codons is summarised in Lys peptidyl-tRNA accumulate rapidly under these conditions, Table 3, showing that an increase in inhibition was observed Gly Cys whereas peptidyl-tRNA and peptidyl-tRNA accumulate only in the case of ∆ EF-Tu variants in which the last amino acid Lys only very slowly. The rapid accumulation of peptidyl-tRNA corresponded to one of the three most rapidly accumulating does not appear to arise either from an abnormally high rate of peptidyl-tRNA species identified by Menninger (19). Lys peptidyl-tRNA drop-off at Lys codons or from an unusually Lys poor substrate activity of peptidyl-tRNA for Pth Table 3. Summary of toxicity of pJMM1-derived plasmids with changed last (V.Heurgué-Hamard, unpublished results). As the data of sense codon Menninger (19) can be difficult to interpret in cases of multiple isoacceptors for an amino acid, we have concentrated our Plasmid Amino acid Codon Growth Drop-off changes on introducing new codons belonging to codon families read by a single tRNA isoacceptor. pJM/pHD67-LysA Lys AAA – 0 Ten new codons have been introduced by site-directed pJM/pHD67-LysC Lys AAG – 0 mutagenesis into the site preceding the stop codon in the gene pJM-HisC His CAC – 2 for ∆∆ EF-Tu in plasmid pJMM1, in place of the ACG Ser pJM-HisU His CAU – 2 codon found in tufB. The presence of a single EagI site upstream of the stop codon (as opposed to two sites in pHD67) pHD67-Ile Ile AUA – 2 facilitated these changes. The relative growth of cells trans- pJM-PheU Phe UUU 0 4 formed by the variant plasmids was studied both in liquid pJM-PheC Phe UUC 0 4 medium and on plates. In the first method, cells in liquid pJM-Arg Arg CGG 0 6 culture were induced with IPTG at a density of ∼4 × 10 cells/ml and growth was monitored until the end of exponential growth pJMM1/pHD67 Ser AGC 0 12 or until growth ceased due to the toxic effect of ∆∆ EF-Tu pJM/pHD67-Gly Gly GGC 0 18 induction. Growth experiments showed that four of these pJM-CysU Cys UGU 0 20 modified plasmids led to considerably increased growth inhibition in the pth(ts) strain VH805 following induction at 37°C, pJM-CysC Cys UGC 0 20 compared with the parent plasmid pJMM1. Both the Lys codons AAA/G and the His codons CAU/C were associated Growth under conditions of induction (1 mM IPTG) of strain VH805 transformed with the plasmids shown in first column is shown as similar to (0) with increased growth inhibition. Figure 5 shows the growth or less than (–) relative to transformants with the appropriate control plasmid inhibition following induction of the LysAAA variant of [line 9: pJMM1 (SerAGC) or pHD67 (SerAGC)] under various experimental ∆∆ EF-Tu under these conditions. conditions (see text). These results were confirmed by comparisons on agar plates. Drop-off observed by Menninger (19) for amino acid-accepting families of This approach had two experimental advantages. First, it was peptidyl-tRNA: time required (min) for the accumulation of 25% of acceptance capacity as peptidyl-tRNA. The lines are ordered with respect to this parameter. easy to distinguish faster-growing revertants when they Refers to variant of ∆ EF-Tu. occurred. Secondly, multiple parallel experiments enabled different conditions of growth, notably temperature, to be easily compared. Thus, it was possible to identify temperatures The most striking result of these experiments is that small at which cells expressing the normal SerAGC ∆∆ EF-Tu grew changes to the EF-Tu gene can modulate the growth inhibitory well on plates whereas neither of the LysAAA/G variants grew effect to a considerable extent, without changing the level of visibly at all (Fig. 3B). The Pth mutant strain used in this expression of the gene. This implies that protein burden effects experiment, VH287, is more thermosensitive than VH805, cannot easily provide the full explanation for the effects of hence the lower temperature employed. The rare Ile codon overproducing our gratuitous model proteins, but that seques- AUA was associated with a significant increase in toxicity in tration of essential tRNA species is an important part of the ∆ EF-Tu but a much smaller effect, if any, in ∆∆ EF-Tu. With phenomenon. Secondly, the effect of different codons at the this exception similar effects were observed with mutant position of the last sense codon is clearly related to the rate at plasmids derived from pJMM1 and from pHD67, encoding which the corresponding tRNA species was found to accumulate EF-Tu and ∆ EF-Tu, respectively, in the cases where variants as peptidyl-tRNA by Menninger (19). were constructed of both parent plasmids, namely, SerAGC, The most evident hypothesis to explain these observations is LysAAA, LysAAG and GlyCAU. As expected (see above), the that drop-off of peptidyl-tRNA does indeed occur when the temperature thresholds for thermosensitive growth were ribosome is paused at the last sense codon. However, another always lower in the case of pHD67 and its variants. Lys mechanism may also be contributing to the tRNA starvation, Experiments in which tRNA was overproduced in cotrans- due directly to the slow kinetics of termination. In both cases, formants of VH805 by pHD67-LysA/G and pJMM19 showed that the particular hierachy observed should be expected, since the suppression of growth inhibition due to the LysV-containing even before induction of ∆ EF-Tu or ∆∆ EF-Tu synthesis, the plasmid was much less marked than in the case of the parent cells are partially starved for Pth and therefore for certain plasmid pHD67 (results not shown). Similar experiments with Lys His derivatives of pJMM1 where the C-terminal Ser codon was tRNAs (notably, tRNA and tRNA ). The hierachy is hence replaced by either the Phe codon UUU or UUC or the Gly pre-established by the shortage of Pth. This would explain why codon GGC, showed that none of these changes had any only the codons corresponding to tRNAs accumulating rapidly ∆∆ ∆∆ Nucleic Acids Research, 2000, Vol. 28, No. 23 4731 as peptidyl-tRNA (AAA/G, CAC/U and AUA; see Table 3) introduces a translational pause (38,39). In conclusion, the result in growth inhibition when introduced at the end of ∆ EF-Tu introduction of Lys at the two last positions modifies the or ∆∆ EF-Tu. As might be expected, measurements of the phenomenon of growth inhibition whereas addition or removal degree to which different tRNA species are sequestered as of Lys at various upstream positions does not. While not peptidyl-tRNA under semi-permissive conditions for growth conclusive, these observations tend to argue in favour of the rank in the same way as the rates at which they accumulate second explanation for tRNA sequestration, i.e. temporary under non-permissive conditions (results not shown), sequestration on the ribosome. They further suggest that the confirming the observations of Menninger (19). Four acceptor binding of RF to the A-site in response to a stop signal does not activities were tested: Lys and His (high accumulation), Gly result in the rapid ejection of tRNA from the E-site. (low accumulation) and Phe (intermediate accumulation). The second mechanism that may contribute arises directly from the Table 4. Toxicity of pJMM1-derived plasmids with changed last EF-Tu relatively long pause of the ribosome at the stop codon (16,17), of tetrapeptide the order of 1 s (15). This could result in temporary sequestration on the ribosome of the tRNA cognate to the last sense codon. Plasmid Terminal tetrapeptide Relative growth A rough calculation shows that ∼5% of the ribosomes synthe- pJMM1 Lys-Val-Leu-Ser (wild type) +++ sising the model protein could be paused at the stop codon. This might immobilise ∼5% of the total of a tRNA isoacceptor pJM-CVLS Cys-Val-Leu-Ser +++ Lys such as tRNA at any moment, a fraction that is small but pJM-CKLS Cys-Lys-Leu-Ser +++ could be significant in the case of a tRNA already in short pJM-KVLK Lys-Val-Leu-Lys ++ supply as a result of sequestration as peptidyl-tRNA due to pJM-KKLS Lys-Lys-Leu-Ser + drop-off. pJM-KVKS Lys-Val-Lys-Ser + A variety of attempts has been made to demonstrate contri- butions to tRNA starvation by sequestration as peptidyl-tRNA pJM-KVKK Lys-Val-Lys-Lys 0 on or off the ribosome, using mutations in translation factors pJM-KVLH Lys-Val-Leu-His ++ that affect the kinetics of termination and drop-off (8,9). In pJM-KVHH Lys-Val-His-His + general, the effects are difficult to interpret in an unambiguous pJM-KVHK Lys-Val-His-Lys + manner. Further light has, however, been thrown on the problem by studying the effect of changing codons just upstream of the last sense codon in the mRNAs for ∆ EF-Tu Relative rates of growth under conditions of induction (1 mM IPTG) of strain VH805 transformed with the plasmids shown in first column. Temperatures of and ∆∆ EF-Tu. incubation were chosen between 35.5 and 37°C soastoallow some growth of the slowest-growing strain being plated. The last two sense codons modulate tRNA sequestration Translocation factor EF-G is responsible for moving peptidyl- Conclusion tRNA from the A-site to the P-site on the ribosome, with the concurrent movement of deacylated tRNA from the P-site to Previous work using Pth-limited cells showed that the translation the E-site and translocation of mRNA by one codon (35). Two factors RRF and RF3 were involved in a significant proportion of tRNAs are considered to be bound to the ribosome at any time drop-off events in E.coli (6,8,9). However, failure to demonstrate (36,37), due to anti-cooperativity between the A- and E-sites of that mutants affecting RRF and RF3 increased translational the binding of tRNA. Whether binding of RF to the A-site processivity focused attention on the possibility the the proces- induces tRNA release from the E-site in the way that is thought sivity failures involving these factors occurred predominantly to occur when tRNA binds in a stable manner to the A-site is at the beginning or the end of polypeptide chain synthesis. The unknown. We have introduced Lys and in some cases His involvement of the two factors in processivity failures early in codons at positions upstream of the last sense codon to see the translation of an mRNA has been amply borne out by whether this leads to an effect comparable to their presence as recent in vitro experiments (8,9,14). Here, we have attempted the last sense codon (the Nth codon). As shown in Table 4, to demonstrate drop-off at the last sense codon of an mRNA by when codon (N–1), normally a CTG Leu codon, is replaced by an looking for effects of changing that codon in a system where AAA Lys codon (pJM-KVKS) the inhibitory effect is similar to altered levels of drop-off are reflected in cellular growth rate. replacing the Nth codon by the Lys codon. When both the N and Such effects have been clearly demonstrated. However, the (N–1) codons are changed to Lys codons (pJM-KVKK), the fact that the penultimate sense codon also affects growth under effect is cumulative. In contrast, replacement of the Lys conditions of Pth limitation suggests that drop-off at the last residue normally present at position N–4 by Cys (yielding sense codon may not be the only (or indeed the principal) pJM-CVLS) has no effect. The introduction of Lys at N–3 into mechanism for the observed phenomenon. We suggest that the latter plasmid (producing pJM-CKLS) is also without temporary sequestration of tRNA while the ribosome is paused at effect. Similarly, replacement by Gly of either or both of the a stop signal, either as peptidyl-tRNA at the P-site or deacylated two Lys residues in ∆ EF-Tu encoded within the EagI–EagI tRNA at the E-site, may contribute to tRNA starvation in a codon- fragment (and therefore absent in ∆∆ EF-Tu) has no effect. It specific manner. This may occur when Pth limitation has should be noted that the presence of Lys residues at both the N–3 already established partial starvation for tRNA species that and N–4 positions (pJM-KKLS) does increase growth inhibition. accumulate preferentially under these conditions, such as However, this is a known effect due to the presence of two Lys His similar adjacent codons read by a tRNA in short supply, which tRNA or tRNA . ∆∆ 4732 Nucleic Acids Research, 2000, Vol. 28, No. 23 18. Björnsson,A. and Isaksson,L.A. (1996) Nucleic Acids Res., 24, 1753–1757. ACKNOWLEDGEMENTS 19. Menninger,J.R. (1978) J. Biol. Chem., 253, 6808–6813. We thank Måns Ehrenberg and Miklos de Zamaroczy for 20. Watanabe,H., Gojobori,T., Terabe,M., Wakiyama,M. and Miura,K. helpful comments on the manuscript. This work was supported (1997) Nucleic Acids Symp. Ser., 37, 297–298. 21. Van Delft,J.H., Schmidt,D.S. and Bosch,L. (1987) J. Mol. Biol., 197, 647–657. by the Centre National pour la Recherche Scientifique 22. Amann,E., Ochs,B. and Abel,K.J. (1988) Gene, 69, 301–315. (UPR9073), l’Association pour la Recherche sur le Cancer, the 23. Sanger,F., Nicklen,S. and Coulson,A.R. (1977) Proc. Natl Acad. Sci. Fondation pour la Recherche Medicale, INTAS and the Human USA, 74, 5463–5467. Capital and Mobility Programme of the European Community. 24. Wang,F.W. and Kushner,S.R. (1991) Gene, 100, 195–199. 25. Miller,J.H. (1972) Experiments in Molecular Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. REFERENCES 26. Sambrook,J., Fritsch,E.F. and Maniatis,T. (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, 1. Manley,J.L. (1978) J. Mol. Biol., 125, 407–432. Cold Spring Harbor, NY. 2. Jørgensen,F. and Kurland,C.G. (1990) J. Mol. Biol., 215, 511–521. 27. Miller,J.H. (1992) A Short Course in Bacterial Genetics. 3. Dong,H., Nilsson,L. and Kurland,C.G. (1995) J. Bacteriol., 177, 1497–1504. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 4. Atherly,A.G. and Menninger,J.R. (1972) Nature (Lond.), 240, 245–246. 5. Schmitt,E., Mechulam,Y., Fromant,M., Plateau,P. and Blanquet,S. (1997) 28. Laemmli,U.K. (1970) Nature (Lond.), 227, 680–685. EMBO J., 16, 4760–4769. 29. Van Delft,J.H.M., Verbeek,H.M., DeJong,P.J., Schmidt,D.S., Talens,A. 6. Heurgué-Hamard,V., Mora,L., Guarneros,G. and Buckingham,R.H. and Bosch,L. (1988) Eur. J. Biochem., 175, 355–362. (1996) EMBO J., 15, 2826–2833. 30. Kjeldgaard,M. and Nyborg,J. (1992) J. Mol. Biol., 223, 721–742. 7. Menninger,J.R. (1977) Mech. Ageing Dev., 6, 131–142. 31. Vind,J., Sorensen,M.A., Rasmussen,M.D. and Pedersen,S. (1993) 8. Heurgué-Hamard,V., Karimi,R., Mora,L., MacDougall,J., Leboeuf,C., J. Mol. Biol., 231, 678–688. Grentzmann,G., Ehrenberg,M. and Buckingham,R.H. (1998) EMBO J., 32. Van Delft,J.H. and Bosch,L. (1988) Eur. J. Biochem., 175, 375–378. 17, 808–816. 33. Van der Meide,P., Vijgenboom,E., Dicke,M. and Bosch,L. (1982) 9. Dinçbas,V., Heurgué-Hamard,V., Buckingham,R.H., Karimi,R. and FEBS Lett., 139, 325–330. Ehrenberg,M. (1999) J. Mol. Biol., 291, 745–759. 34. Freistroffer,D.V., Pavlov,M.Y., MacDougall,J., Buckingham,R.H. and 10. Ontiveros,C., Valadez,G., Hernandez,J. and Guarneros,G. (1997) Ehrenberg,M. (1997) EMBO J., 16, 4126–4133. J. Mol. Biol., 269, 167–175. 35. Noller,H.F. (1991) Annu. Rev. Biochem., 60, 191–227. 11. Hernández,J., Ontiveros,C., Valadez,C., Buckingham,R.H. and 36. Nierhaus,K.H., Junemann,R. and Spahn,C.M.T. (1997) Proc. Natl Acad. Guarneros,G. (1997) Biochimie, 79, 527–531. Sci. USA, 94, 10499–10500. 12. Hernández-Sánchez,J., Valadez,J.G., Herrera,J.V., Ontiveros,C. and 37. Nierhaus,K.H. (1990) Biochemistry, 29, 4997–5008. Guarneros,G. (1998) EMBO J., 17, 3758–3765. 38. Robinson,M., Lilley,R., Little,S., Emtage,J.S., Yarranton,G., Stephens,P., 13. Tenson,T., Herrera,J.V., Kloss,P., Guarneros,G. and Mankin,A.S. (1999) Millican,A., Eaton,M. and Humphreys,G. (1984) Nucleic Acids Res., 12, J. Bacteriol., 181, 1617–1622. 6663–6671. 14. Heurgué-Hamard,V., Dinçbas,V., Buckingham,R.H. and Ehrenberg,M. 39. Roche,E.D. and Sauer,R.T. (1999) EMBO J., 18, 4579–4589. (2000) EMBO J., 19, 2701–2709. 40. Coulondre,C. and Miller,J.H. (1977) J. Mol. Biol., 117, 525–575. 15. Freistroffer,D.V., Kwiatkowski,M., Buckingham,R.H. and Ehrenberg,M. 41. Marsh,L. and Walker,G.C. (1987) J. Bacteriol., 169, 1818–1823. (2000) Proc. Natl Acad. Sci. USA, 97, 2046–2051. 16. Wolin,S.L. and Walter,P. (1988) EMBO J., 7, 3559–3569. 42. Polekhina,G., Thirup,S., Kjeldgaard,M., Nissen,P., Lippmann,C. and 17. Doohan,J.P. and Samuel,C.E. (1992) Virology, 186, 409–425. Nyborg,J. (1996) Structure, 4, 1141–1151. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Nucleic Acids Research Oxford University Press

Sequestration of specific tRNA species cognate to the last sense codon of an overproduced gratuitous protein

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Oxford University Press
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0305-1048
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1362-4962
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10.1093/nar/28.23.4725
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Abstract

© 2000 Oxford University Press Nucleic Acids Research, 2000, Vol. 28, No. 23 4725–4732 Sequestration of specific tRNA species cognate to the last sense codon of an overproduced gratuitous protein Jeanne Menez, Valérie Heurgué-Hamard and Richard H. Buckingham* UPR9073 du CNRS, Institut de Biologie Physico-Chimique, 13 rue Pierre et Marie Curie, F-75005 Paris, France Received August 8, 2000; Revised October 6, 2000; Accepted October 19, 2000 ABSTRACT Escherichia coli (4,5). In cells deficient in Pth, protein synthesis is arrested and the cells finally die due to starvation High-level expression of non-functional model for tRNA, which becomes sequestered as peptidyl-tRNA and proteins, derived from elongation factor EF-Tu by the unavailable for protein synthesis. When deprived of Pth by the deletion of an essential domain, greatly inhibits the transfer to a non-permissive temperature of cells containing a growth of Escherichia coli partly deficient in Lys thermosensitive enzyme, tRNA becomes limiting considerably peptidyl-tRNA hydrolase. High-level expression in before other tRNA species (6). The reasons for this are not yet wild-type cells has little effect on growth. The inhibitory fully understood, and may be related to a higher rate of drop- Lys effect is therefore presumably due to the sequestration off of peptidyl-tRNA species involving tRNA . One possible Lys of essential tRNA species, partly in the form of free explanation is that peptidyl-tRNA is a poor substrate for Pth compared with peptidyl-tRNAs that accumulate slowly. peptidyl-tRNA. The growth inhibitory effect can be Recent measurements, however, show that the differences are modulated by changing the last sense codon in the not large enough to explain the large variation in accumulation genes encoding the model proteins. Thus, replacement rates (V.Heurgué-Hamard, unpublished results). of Ser by Lys or His at this position increases growth Several aspects of peptidyl-tRNA drop-off remain poorly inhibition. The effects of 11 changes studied are understood. It is unclear what fraction of drop-off events related to the rates of accumulation previously occurs at codons cognate to the tRNA in question, and what observed of the corresponding families of peptidyl- fraction occurs at near-cognate codons, as a result of errors of tRNA. Two non-exclusive hypotheses are proposed selection of tRNA that escape the proofreading mechanism (7). to account for these observations: first, the last Studies of the suppression of a thermosensitive Pth mutant sense codon of mRNA is a prefered site of peptidyl-tRNA showed that drop-off was not merely a passive event, but drop-off in cells, due to the slow rate of translation involved translation factors RRF and RF3 in vivo (8,9). termination compared with sense codon translation; However, attempts to demonstrate that mutants affecting RRF secondly, the relatively long pause of the ribosome at and RF3 increased translational processivity using the lacZ the stop codon (of the order of 1 s), results in signifi- monomer-dimer approach of Jørgensen and Kurland (2) were unsuccessful (V.Heurgué-Hamard and R.H.Buckingham, cant temporary sequestration on the ribosome of the unpublished work). One explanation for this unexpected result, tRNA cognate to the last sense codon. apart from technical reasons related to the difficulties of this experimental approach, is that drop-off events involving RRF INTRODUCTION and RF3 occur predominantly at the beginning or end of mRNAs and would therefore not be detected. A significant proportion of ribosomes that initiate translation The drop-off of short peptidyl-tRNAs has been the subject of of an mRNA fail to terminate by the normal mechanism, which extensive recent study (6,9–14). The expression of very short involves the recognition of messenger-encoded stop signals, open reading frames, encoded by ‘mini-genes’, can lead to the hydrolysis by the ribosome of peptidyl-tRNA and release of rapid accumulation of peptidyl-tRNA and the arrest of protein the completed polypeptide. Truncated proteins arise at a –4 synthesis even in cells with normal amounts of Pth. In such frequency of ∼3 × 10 per codon, which implies that about cases drop-off does indeed appear to occur at a codon read by one-third of the ribosomes that initiate β-galactosidase the cognate tRNA, namely, the last sense codon encoded by the synthesis fail to reach the stop signal (1–3). Measurements of mini-gene, as the inhibitory effect can be relieved (at least in the synthesis of β-galactosidase dimers indicate that the the limited number of cases that have been studied) by over- frequency of such processivity failures is affected by mutations production of the tRNA species cognate to the last sense in genes encoding ribosomal proteins S12, S4 and S5 (3), codon. The presence of a stop codon very early in an mRNA known to control the accuracy of translation. It is believed that the majority of processivity failures result in the dissociation of favours drop-off and peptidyl-tRNA accumulation for several peptidyl-tRNA from the ribosome (2), or ‘drop-off’. The recycling reasons. These include the catalysis of drop-off by translation of these molecules requires peptidyl-tRNA hydrolase (Pth), a factor and the phenomenon of 30S ribosome recycling on short ubiquitous enzyme known to be essential for viability in mRNAs (9,14). Another important factor probably arises *To whom correspondence should be addressed. Tel: +33 1 4354 1972; Fax: +33 1 4046 8331; Email: rhb@ibpc.fr The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors 4726 Nucleic Acids Research, 2000, Vol. 28, No. 23 Table 1. Bacterial strains Strain Genotype Reference/construction FTP4993 metB, glyV55, trpA(AGC233, UGA234) F.T.Pagel IB188 ara, ∆ (lac–proB), nalA, metB Derivative of XA102 (40) GY5552 ∆ (lac, pro), thi, strA, endA, sbcB15, hsdR4F′traD36, proAB, lacIqZ∆ M15, recA938::Cm Marsh and Walker (41) VH7 supE44, thi1, thr1, leuB6, lacY1, tonA21, pth(ts), fadR13::Tn10 Heurgué-Hamard et al. (6) VH287 ara, ∆ (lac–proB), nalA, metB, pth(ts), fadR13::Tn10, recA938::Cm This work, IB188 and P1 lysates on VH7 and GY5552 VH733 metB, glyV55, pth(ts) Heurgué-Hamard et al. (8) VH804 metB, glyV55, trpA(AGC233, UGA234), recA::Cm This work, FTP4993 and P1 lysate on GY5552 VH805 metB, glyV55, pth(ts), recA::Cm This work, VH733 and P1 lysate on GY5552 directly from the slow kinetics of the termination reaction are derivatives of pHD67 (3), which contains the partially compared with the translation of most sense codons. The deleted tufB gene from plasmid pTuB12.1 (21) cloned in the limiting factor in the termination reaction is probably the low expression vector pTrc99c (22). Plasmid pJMM1 contained a –1 value of k , of the order of 1 s (15). The pausing of ribo- further truncation in the tufB gene (encoding ∆∆ EF-Tu), and cat somes for substantial periods at stop signals has been observed wasobtainedbydigestion of pHD67with EagI and religation both in vitro (16,17) and in vivo (18). of the plasmid. Variants of both plasmids pHD67 and pJMM1 Since drop-off is an important phenomenon when the were constructed by a method employing two PCR steps. To ribosome is paused at the termination signal of very short introduce different codons before the stop codon, a first PCR coding sequences, it is of interest to know whether the drop-off was performed using a mutagenic oligonucleotide covering the of peptidyl-tRNAs with complete polypeptide chains represents a stop signal and a second oligonucleotide upstream of the EagI significant part of total drop-off. The reason for the high rate of (951) site (see Fig. 1) site in pHD67 or pJMM1. The product of Lys peptidyl-tRNA accumulation in cells deprived of Pth (19) is this PCR step was used in a second PCR step in conjunction not well understood, but may simply be a reflection of a generally with a further oligonucleotide downstream of an SgfI site increased frequency of adenosine (and consequently of Lys located 181 nucleotides beyond the tufB stop codon. The codons) close to initiation and termination codons (20). Little product was restricted with SgfIand PshAI or EagIand used to is known of the distribution of polypeptide chain lengths in replace the corresponding fragment in pHD67 or pJMM1 peptidyl-tRNA molecules arising by premature peptidyl-tRNA respectively. All variants of these plasmids were verified by dissociation from the ribosome and we cannot yet say whether sequencing (23). They are listed in Table 3 together with the the beginnings or ends of coding regions are preferred regions corresponding final sense codon. Plasmids pVH1 and for drop-off to occur. pJMM19, carrying the pth and lysV genes, respectively, are To search for effects on peptidyl-tRNA accumulation related derivatives of low-copy-number plasmid pWSK129 (24). to the coding sequence just upstream of the stop signal, we Plasmid pJMM19 was made by recloning the ApaI–BamHI have examined the overexpression of a series of model fragment carrying lysV from pVH119 (6) between the same sites proteins that differ with respect to the last sense codon. The in pWSK129; like pVH119 it results in an increase of ∼2-fold in experiments are performed under conditions of limitation for Lys the quantity of tRNA in the cell. Pth, so that cell growth becomes sensitive to the degree of sequestration of different tRNA species. The results show that Growth conditions the identity of the last sense codon does indeed affect the Luria–Bertani (LB) broth was employed as rich medium. Anti- degree of toxicity due to high-level expression of the model biotics were added as necessary at the following final concen- protein and that the effect is related to the rate of accumulation trations: kanamycin 50 µ g/ml, ampicillin 200 µ g/ml. In of the corresponding peptidyl-tRNA species described by experiments with thermosensitive Pth strains, precultures were Menninger (19). Two non-exclusive mechanisms can account grown at 30°C in LB broth containing appropriate antibiotics. for the observations: first, drop-off occurring at a significant After dilution to an OD of 0.05, growth was continued at level at the last sense codon, and secondly, the relatively slow 37°C (or as otherwise indicated in figure legends) to an OD process of termination and ribosome recycling that sequesters of 0.5. Synthesis of ∆ EF-Tu or ∆∆ EF-Tu was induced by the tRNA corresponding to the last sense codon for a sufficient addition of isopropyl-β-D-thiogalactopyranoside (IPTG) to a period that the pool of free, charged tRNA is significantly final concentration of 1 mM after a further dilution to an OD depleted. of 0.05. For labelling of proteins with [ S]Cys, cells were grown in M9-glucose medium (25) supplemented with all MATERIALSAND METHODS amino acids except Cys. An overnight culture was diluted to ∼2 × 10 cells/ml and labelled for two generations by addition Bacteria and plasmids of [ S]Cys (1 Ci/µ mol) to a concentration of 15 µ Ci/ml. IPTG Escherichia coli K12 strains are listed in Table 1. All plasmid was added to a concentration of 1 mM and labelling continued constructions expressing ∆ EF-Tu and variants of this protein for 3 h. Nucleic Acids Research, 2000, Vol. 28, No. 23 4727 Figure 1. Deleted regions of EF-Tu in model proteins ∆ EF-Tu and EF-Tu. The deleted regions are shown in relation to the 3D structure of EF-Tu (A) and to the tufB gene coding sequence (B). The region in red is absent from the ∆ EF-Tuprotein andboththisand theregioninbluefrom ∆∆ EF-Tu. The model is based on the crystallographic structure of EF-Tu (42) and the sequence is from GenBank, accession no. M30610. Recombinant DNA techniques and genetic manipulations protein, called ∆ EF-Tu, lacks part of the GTP-binding motif G3 and motif G4 as a result of the deletion of a SmaI–SmaI General procedures for recombinant DNA techniques, plasmid fragment in tufB covering nucleotides 248–491 (29). This extraction, agarose gel electrophoresis, etc., were performed as removes a region of the native sequence corresponding to a describedbySambrook et al. (26). DNA fragments from well-defined domain in the three-dimensional structure of the agarose gels were extracted using Jetsorb gel (Bioprobe) protein (30). A further truncation was also made by deleting an according to the manufacturer’s instructions. Phage P1 lysates, EagI–EagI fragment (nucleotides 951–1140), yielding a transductions and transformations were performed as second protein (∆∆ EF-Tu), lacking about two-thirds of domain described by Miller (27). 3 (Fig. 1) as well as the GTP-binding motifs. Quantification of proteins In plasmidpHD67encoding ∆ EF-Tu, transcription depends on the trc promoter and is induced by IPTG (3). Continuous Induced cultures labelled with [ S]Cys as described above 35 35 labelling experiments with [ S]Cys or [ S]Met indicate that were centrifuged and the cells lysed for 3 min at 100°Cinlysis 3 h after induction of pth wild-type cells transformed with buffer (50 mM Tris–HCl, pH 6.8, 100 mM dithiothreitol, 2% pHD67, ∼25% of total cell protein is ∆ EF-Tu, confirming the SDS, 0.1% bromophenol blue). The proteins were separated by observations of Dong et al. (3). Under the conditions of our electrophoresis on 10% polyacrylamide gel as described by experiments the expression of ∆ EF-Tu has little effect on the Laemmli (28). Gels were dried and the radioactivity in growth of pth wild-type cells. In contrast, in cells partly individual bands and complete tracks was determined using a deficient in Pth, growth is partially or completely inhibited. All PhosphorImager (Molecular Dynamics). experiments in Pth-deficient cells have made use of the thermo- sensitive Pth mutant isolated by Atherly and Menninger (4), RESULTS ANDDISCUSSION transduced into various genetic backgrounds by selection for a nearby transposon. In liquid culture in rich media, the pth(ts) Overproduction of an inactive protein inhibits growth of strain VH805 grows at temperatures up to 42°C, and grows at Pth-deficient cells 37°C asfast asthe pth parental strain VH804. When trans- The study of tRNA sequestration and its dependence of mRNA formed with plasmid pHD67, strain VH805 grows normally at sequence required model proteins that could be expressed at a 37°Cinthe absence of ∆ EF-Tu expression, but ceases to grow shortly after induction of the gene carried in pHD67 (triangles, high level in E.coli without deleterious effects on the growth of Fig. 2). Cotransformation of cells with a plasmid pVH1 carrying cells with normal levels of Pth activity. In order to avoid tRNA the wild-type pth gene suppresses this growth inhibition (not usage that might affect tRNA pool sizes we chose a gene with codon usage typical of highly expressed genes in E.coli,a shown). A similar behaviour is seen on plates (Fig. 3A, truncated version of tufB, encoding protein synthesis elongation compare segments pHD67 and pTrc99c). At 30°C, where factor EF-Tu (3,29). As shown in Figure 1, the truncated intracellular Pth activity is less affected by the thermolability ∆∆ 4728 Nucleic Acids Research, 2000, Vol. 28, No. 23 Table 2. Growth inhibition at different temperatures of Pth-deficient cells due to plasmid-directed synthesis of ∆ EF-Tu and ∆∆ EF-Tu Strain/plasmid pth(ts)/control pth(ts)/∆∆ EF-Tu pth(ts)/∆ EF-Tu pth /∆ EF-Tu VH805/pTrc99 VH805/pJMM1 VH805/pHD67 VH804/pHD67 IPTG: – +– + – +– + 30°C ++ ++ ++++++ ++ ++++ 34°C ++ ++ ++++++ – ++ ++ 37°C ++ ++ +++ ++ – ++++ 39°C ++ ++ ++– ++ – ++++ 43°C – – –––– ++ + Transformed cells were streaked on to LB-ampicillin plates with or without 1 mM IPTG and incubated at the temperature shown in the first column. The table shows inhibited growth (+) or no growth (–) in comparison with the normal growth (++) of pth cells transformed with control plasmid pTrc99C. Figure 2. Inhibition of cell growth in partially Pth-deficient strains following induction of ∆ EF-Tu synthesis. Tranformants of the Pth thermosensitive strain VH805 carrying plasmid pHD67 encoding ∆ EF-Tu were grown at 37°Cin LB-ampicillin medium to ∼4 × 10 cells/ml. Growth was monitored by measurement of OD as a function of time after induction with 1 mM IPTG; induced culture (open triangles) and non-induced culture (filled circles). of the enzyme, induction of ∆ EF-Tu expression does not affect growth. At temperatures of 34°C and above (in the case of strain VH805), pth(ts) cells transformedwithpHD67nolonger grow in the presence of IPTG, whereas non-induced cells or Figure 3. Growth inhibition on solid medium due to ∆ EF-Tu or EF-Tu synthesis cells transformed with the control plasmid pTrc99C grow and the effect of mutations in the gene encoding ∆∆ EF-Tu. Tranformants of normally at temperatures up to 39°C (Table 2). The threshold for Pth thermosensitive strains with pHD67 or pJMM1 (SerAGC) and variants of thermosensitivity is thus lowered by the remarkable magnitude of the latter plasmid altered in the last sense codon were streaked on LB–agar plates 9°Cwhen ∆ EF-Tu is expressed. In other strain backgrounds with or without 1 mM IPTG, and incubated at the temperature indicated. Growth the pth(ts) mutation leads to more or less thermosensitive on plates is shown for (A) transformants of VH805 with plasmids pHD67 and pJMM1, encoding ∆ EF-Tu and ∆∆ EF-Tu, respectively, and control plasmid growth, and the onset of thermosensitivity of the transformed, pTrc99c; (B) transformants of VH287 with plasmid pJMM1 and variants altered induced cells is correspondingly shifted to lower or higher in the last sense codon of tufB from SerAGC to LysAAA/G or HisCAC/U. temperatures, respectively. Two broadly different explanations can account for the observation that overproducing one gratuitous protein can uniform but to vary according to the mRNA (31). The second lower the threshold of thermosensitivity in the Pth mutant class of explanation would depend on particular properties of strain. The overproduction does not lead to a marked increase the mRNA for ∆ EF-Tu. Thus, drop-off of peptidyl-tRNA in the total amount of protein synthesis occurring in the cell. during synthesis of ∆ EF-Tu may be higher than the average Rather, it diverts part of the cell’s capacity for protein frequency of drop-off, and increase the total amount of drop- synthesis to one protein at the expense of others. Thus, the off in the cell. Alternatively, even if the overall amount is not proportion of any particular protein (including Pth) being synthesised may fall in relation to total protein synthesis as a increased, the pattern of tRNAs accumulating as peptidyl-tRNA may change due to particular drop-off points characteristic of the result of the extra imposed ‘protein burden’. In fact, the degree to which the synthesis of different cell proteins is affected by mRNA, and hasten the onset of starvation for an essential the induction of a plasmid-carried gene is known to be not tRNA species. Further insight into these and other explanations ∆∆ ∆∆ Nucleic Acids Research, 2000, Vol. 28, No. 23 4729 Figure 4. Suppression of growth inhibition due to EF-Tu synthesis by over- Lys production of tRNA from a low-copy-number plasmid. Strain VH805 was Figure 5. Increased growth inhibition due to EF-Tu synthesis on substitution of cotransformed with plasmid pJMM1 expressing ∆∆ EF-Tu and either plasmid Lys codons at the last sense position of the ∆∆ EF-Tu mRNA. Tranformants of pJMM19 carrying lysV or the parent plasmid pWSK129 with no insert. the Pth thermosensitive strain VH805 with plasmid pJMM1 or variants of the Transformants were grown at 38.5°C in LB-ampicillin-kanamycin medium to plasmid altered in the last sense codon of ∆ tufB from SerAGC to LysAAA 7 7 ∼3 × 10 cells/ml, and EF-Tu synthesis was then induced with 1 mM IPTG. were grown at 38°C in LB-ampicillin medium to ∼3 × 10 cells/ml, and EF-Tu Growth was monitored by measurement of OD as a function of time after synthesis was induced with 1 mM IPTG. Growth was monitored by measurement induction for induced cultures (open circles, pJMM1 and pJMM19; open of OD as a function of time after induction for induced cultures (open circles, triangles, pJMM1 and pWSK129) and non-induced cultures (filled circles, pJMM1; open squares, pJM-LysAAA) and non-induced cultures (filled circles, pJMM1 and pJMM19; filled triangles, pJMM1 and pWSK129). pJMM1; filled squares, pJM-LysAAA). was obtained by experiments in which specific modifications Lys Growth inhibition is suppressed by tRNA were made to the ∆ EF-Tu mRNA. overproduction Deletion of a C-terminal fragment of ∆ EF-Tu reduces the The fact that the growth inhibitory effect of overproducing growth inhibition ∆ EF-Tu was seen only in mutant cells partially deficient in Pth strongly suggests that the mechanism of growth inhibition is It has been reported that a region near the C-terminus of EF-Tu, likely to be the same as in severely Pth-limited cells. Previous including residue G375, is important for an autoregulatory work has shown that under semi-permissive conditions for mechanism of tufB expression, and that this regulatory effect is pth(ts) strains (at ∼42°C in the case of strain VH805), growth conserved in ∆ EF-Tu (32,33). This suggested that the toxicity is inhibited due to starvation for tRNA and in particular for associated with ∆ EF-Tu expression might in part be related to Lys tRNA (6). If tRNA starvation due to sequestration as regulatory effects on tufB expression. The ∆ tufB in plasmid peptidyl-tRNA results from the induction of ∆ EF-Tu synthesis, pHD67 was therefore modified by deletion of a region of the then the effects should be at least partly suppressed by the over- gene encoding 63 amino acids close to the C-terminus of ∆ EF-Tu, Lys production of tRNA similarly to the original observation (6). In including residue G375 of TufB and surrounding residues, the experiment presented in Figure 4, the lysV gene is carried resulting in plasmid pJMM1. The deleted fragment corresponds to on a low-copy-number plasmid pJMM19 compatible with an EagI–EagI fragment in the ∆ tufB gene (Fig. 1). The expression pJMM1 and results in an ∼2-fold increase in the level of of this doubly deleted protein ‘∆∆ EF-Tu’ remains inhibitory to Lys tRNA (6). The growth inhibition resulting from the induction of growth in pth(ts) cells, but the threshold temperature for ∆∆ EF-Tu synthesis in the strain cotransformed with pJMM1 inhibition is shifted upwards by ∼5°C. The behaviour of pth(ts) and the parent plasmid pWSK129 is seen to be almost cells transformed with plasmid pJMM1 is intermediate completely eliminated when the plasmid carries a lysV insert. between pHD67-transformed cells and cells transformed with the control plasmid pTrc99C (Table 2, columns 6 and 7). Thus, Changing the last codon of the ∆∆ EF-Tu gene modulates at 34–38°C IPTG induction of the pth(ts) strain VH805 trans- the growth inhibition formed with pJMM1 still grows, whereas the same strain Recent data suggest that the termination step in protein transformed with pHD67 ceases growth completely (Table 2; see also Figs 4 and 5). In contrast, at 39°C EF-Tu expression synthesis catalysed by release factors RF1 and RF2 is slow in completely inhibits cell growth although VH805 transformed comparison with an elongation step (15,18,34). This suggests with pTrc99C grows as well as the pth parental strain VH804 that the last sense codon might be more prone to peptidyl- tRNA drop-off than the average sense codon of an mRNA. To at this temperature. Other experiments (not shown) show that ∆∆ EF-Tu is synthesised with similar translational efficiency to look for evidence of such an effect, we have introduced mutations ∆ EF-Tu. The difference between the two model proteins could that change the last amino acid of ∆∆ EF-TuinpJMM1 (or be accounted for by the ‘protein burden’ type of explanation, ∆ EF-TuinpHD67) andlooked for modulation ofthe growth as a significant part of the polypeptide chain is removed in this inhibitory effects in Pth-deficient cells. Previous work has Lys second deletion. It is also possible that the second deletion identified tRNA as the tRNA isoacceptor most likely to removes drop-off sites encoded within the EagI–EagI fragment of become fully sequestered as peptidyl-tRNA in Pth-limited tufB. cells (6). The data of Menninger (19) provide further valuable ∆∆ ∆∆ ∆∆ ∆∆ ∆∆ 4730 Nucleic Acids Research, 2000, Vol. 28, No. 23 information concerning the probable relative rates of accumu- significant effect on the degree of toxicity on induction. The lation of different peptidyl-tRNA species. Thus, peptidyl- comparison of the inhibitory effects of the entire family of Thr His Ile tRNA , peptidyl-tRNA and peptidyl-tRNA as well as plasmids with altered last sense codons is summarised in Lys peptidyl-tRNA accumulate rapidly under these conditions, Table 3, showing that an increase in inhibition was observed Gly Cys whereas peptidyl-tRNA and peptidyl-tRNA accumulate only in the case of ∆ EF-Tu variants in which the last amino acid Lys only very slowly. The rapid accumulation of peptidyl-tRNA corresponded to one of the three most rapidly accumulating does not appear to arise either from an abnormally high rate of peptidyl-tRNA species identified by Menninger (19). Lys peptidyl-tRNA drop-off at Lys codons or from an unusually Lys poor substrate activity of peptidyl-tRNA for Pth Table 3. Summary of toxicity of pJMM1-derived plasmids with changed last (V.Heurgué-Hamard, unpublished results). As the data of sense codon Menninger (19) can be difficult to interpret in cases of multiple isoacceptors for an amino acid, we have concentrated our Plasmid Amino acid Codon Growth Drop-off changes on introducing new codons belonging to codon families read by a single tRNA isoacceptor. pJM/pHD67-LysA Lys AAA – 0 Ten new codons have been introduced by site-directed pJM/pHD67-LysC Lys AAG – 0 mutagenesis into the site preceding the stop codon in the gene pJM-HisC His CAC – 2 for ∆∆ EF-Tu in plasmid pJMM1, in place of the ACG Ser pJM-HisU His CAU – 2 codon found in tufB. The presence of a single EagI site upstream of the stop codon (as opposed to two sites in pHD67) pHD67-Ile Ile AUA – 2 facilitated these changes. The relative growth of cells trans- pJM-PheU Phe UUU 0 4 formed by the variant plasmids was studied both in liquid pJM-PheC Phe UUC 0 4 medium and on plates. In the first method, cells in liquid pJM-Arg Arg CGG 0 6 culture were induced with IPTG at a density of ∼4 × 10 cells/ml and growth was monitored until the end of exponential growth pJMM1/pHD67 Ser AGC 0 12 or until growth ceased due to the toxic effect of ∆∆ EF-Tu pJM/pHD67-Gly Gly GGC 0 18 induction. Growth experiments showed that four of these pJM-CysU Cys UGU 0 20 modified plasmids led to considerably increased growth inhibition in the pth(ts) strain VH805 following induction at 37°C, pJM-CysC Cys UGC 0 20 compared with the parent plasmid pJMM1. Both the Lys codons AAA/G and the His codons CAU/C were associated Growth under conditions of induction (1 mM IPTG) of strain VH805 transformed with the plasmids shown in first column is shown as similar to (0) with increased growth inhibition. Figure 5 shows the growth or less than (–) relative to transformants with the appropriate control plasmid inhibition following induction of the LysAAA variant of [line 9: pJMM1 (SerAGC) or pHD67 (SerAGC)] under various experimental ∆∆ EF-Tu under these conditions. conditions (see text). These results were confirmed by comparisons on agar plates. Drop-off observed by Menninger (19) for amino acid-accepting families of This approach had two experimental advantages. First, it was peptidyl-tRNA: time required (min) for the accumulation of 25% of acceptance capacity as peptidyl-tRNA. The lines are ordered with respect to this parameter. easy to distinguish faster-growing revertants when they Refers to variant of ∆ EF-Tu. occurred. Secondly, multiple parallel experiments enabled different conditions of growth, notably temperature, to be easily compared. Thus, it was possible to identify temperatures The most striking result of these experiments is that small at which cells expressing the normal SerAGC ∆∆ EF-Tu grew changes to the EF-Tu gene can modulate the growth inhibitory well on plates whereas neither of the LysAAA/G variants grew effect to a considerable extent, without changing the level of visibly at all (Fig. 3B). The Pth mutant strain used in this expression of the gene. This implies that protein burden effects experiment, VH287, is more thermosensitive than VH805, cannot easily provide the full explanation for the effects of hence the lower temperature employed. The rare Ile codon overproducing our gratuitous model proteins, but that seques- AUA was associated with a significant increase in toxicity in tration of essential tRNA species is an important part of the ∆ EF-Tu but a much smaller effect, if any, in ∆∆ EF-Tu. With phenomenon. Secondly, the effect of different codons at the this exception similar effects were observed with mutant position of the last sense codon is clearly related to the rate at plasmids derived from pJMM1 and from pHD67, encoding which the corresponding tRNA species was found to accumulate EF-Tu and ∆ EF-Tu, respectively, in the cases where variants as peptidyl-tRNA by Menninger (19). were constructed of both parent plasmids, namely, SerAGC, The most evident hypothesis to explain these observations is LysAAA, LysAAG and GlyCAU. As expected (see above), the that drop-off of peptidyl-tRNA does indeed occur when the temperature thresholds for thermosensitive growth were ribosome is paused at the last sense codon. However, another always lower in the case of pHD67 and its variants. Lys mechanism may also be contributing to the tRNA starvation, Experiments in which tRNA was overproduced in cotrans- due directly to the slow kinetics of termination. In both cases, formants of VH805 by pHD67-LysA/G and pJMM19 showed that the particular hierachy observed should be expected, since the suppression of growth inhibition due to the LysV-containing even before induction of ∆ EF-Tu or ∆∆ EF-Tu synthesis, the plasmid was much less marked than in the case of the parent cells are partially starved for Pth and therefore for certain plasmid pHD67 (results not shown). Similar experiments with Lys His derivatives of pJMM1 where the C-terminal Ser codon was tRNAs (notably, tRNA and tRNA ). The hierachy is hence replaced by either the Phe codon UUU or UUC or the Gly pre-established by the shortage of Pth. This would explain why codon GGC, showed that none of these changes had any only the codons corresponding to tRNAs accumulating rapidly ∆∆ ∆∆ Nucleic Acids Research, 2000, Vol. 28, No. 23 4731 as peptidyl-tRNA (AAA/G, CAC/U and AUA; see Table 3) introduces a translational pause (38,39). In conclusion, the result in growth inhibition when introduced at the end of ∆ EF-Tu introduction of Lys at the two last positions modifies the or ∆∆ EF-Tu. As might be expected, measurements of the phenomenon of growth inhibition whereas addition or removal degree to which different tRNA species are sequestered as of Lys at various upstream positions does not. While not peptidyl-tRNA under semi-permissive conditions for growth conclusive, these observations tend to argue in favour of the rank in the same way as the rates at which they accumulate second explanation for tRNA sequestration, i.e. temporary under non-permissive conditions (results not shown), sequestration on the ribosome. They further suggest that the confirming the observations of Menninger (19). Four acceptor binding of RF to the A-site in response to a stop signal does not activities were tested: Lys and His (high accumulation), Gly result in the rapid ejection of tRNA from the E-site. (low accumulation) and Phe (intermediate accumulation). The second mechanism that may contribute arises directly from the Table 4. Toxicity of pJMM1-derived plasmids with changed last EF-Tu relatively long pause of the ribosome at the stop codon (16,17), of tetrapeptide the order of 1 s (15). This could result in temporary sequestration on the ribosome of the tRNA cognate to the last sense codon. Plasmid Terminal tetrapeptide Relative growth A rough calculation shows that ∼5% of the ribosomes synthe- pJMM1 Lys-Val-Leu-Ser (wild type) +++ sising the model protein could be paused at the stop codon. This might immobilise ∼5% of the total of a tRNA isoacceptor pJM-CVLS Cys-Val-Leu-Ser +++ Lys such as tRNA at any moment, a fraction that is small but pJM-CKLS Cys-Lys-Leu-Ser +++ could be significant in the case of a tRNA already in short pJM-KVLK Lys-Val-Leu-Lys ++ supply as a result of sequestration as peptidyl-tRNA due to pJM-KKLS Lys-Lys-Leu-Ser + drop-off. pJM-KVKS Lys-Val-Lys-Ser + A variety of attempts has been made to demonstrate contri- butions to tRNA starvation by sequestration as peptidyl-tRNA pJM-KVKK Lys-Val-Lys-Lys 0 on or off the ribosome, using mutations in translation factors pJM-KVLH Lys-Val-Leu-His ++ that affect the kinetics of termination and drop-off (8,9). In pJM-KVHH Lys-Val-His-His + general, the effects are difficult to interpret in an unambiguous pJM-KVHK Lys-Val-His-Lys + manner. Further light has, however, been thrown on the problem by studying the effect of changing codons just upstream of the last sense codon in the mRNAs for ∆ EF-Tu Relative rates of growth under conditions of induction (1 mM IPTG) of strain VH805 transformed with the plasmids shown in first column. Temperatures of and ∆∆ EF-Tu. incubation were chosen between 35.5 and 37°C soastoallow some growth of the slowest-growing strain being plated. The last two sense codons modulate tRNA sequestration Translocation factor EF-G is responsible for moving peptidyl- Conclusion tRNA from the A-site to the P-site on the ribosome, with the concurrent movement of deacylated tRNA from the P-site to Previous work using Pth-limited cells showed that the translation the E-site and translocation of mRNA by one codon (35). Two factors RRF and RF3 were involved in a significant proportion of tRNAs are considered to be bound to the ribosome at any time drop-off events in E.coli (6,8,9). However, failure to demonstrate (36,37), due to anti-cooperativity between the A- and E-sites of that mutants affecting RRF and RF3 increased translational the binding of tRNA. Whether binding of RF to the A-site processivity focused attention on the possibility the the proces- induces tRNA release from the E-site in the way that is thought sivity failures involving these factors occurred predominantly to occur when tRNA binds in a stable manner to the A-site is at the beginning or the end of polypeptide chain synthesis. The unknown. We have introduced Lys and in some cases His involvement of the two factors in processivity failures early in codons at positions upstream of the last sense codon to see the translation of an mRNA has been amply borne out by whether this leads to an effect comparable to their presence as recent in vitro experiments (8,9,14). Here, we have attempted the last sense codon (the Nth codon). As shown in Table 4, to demonstrate drop-off at the last sense codon of an mRNA by when codon (N–1), normally a CTG Leu codon, is replaced by an looking for effects of changing that codon in a system where AAA Lys codon (pJM-KVKS) the inhibitory effect is similar to altered levels of drop-off are reflected in cellular growth rate. replacing the Nth codon by the Lys codon. When both the N and Such effects have been clearly demonstrated. However, the (N–1) codons are changed to Lys codons (pJM-KVKK), the fact that the penultimate sense codon also affects growth under effect is cumulative. In contrast, replacement of the Lys conditions of Pth limitation suggests that drop-off at the last residue normally present at position N–4 by Cys (yielding sense codon may not be the only (or indeed the principal) pJM-CVLS) has no effect. The introduction of Lys at N–3 into mechanism for the observed phenomenon. We suggest that the latter plasmid (producing pJM-CKLS) is also without temporary sequestration of tRNA while the ribosome is paused at effect. Similarly, replacement by Gly of either or both of the a stop signal, either as peptidyl-tRNA at the P-site or deacylated two Lys residues in ∆ EF-Tu encoded within the EagI–EagI tRNA at the E-site, may contribute to tRNA starvation in a codon- fragment (and therefore absent in ∆∆ EF-Tu) has no effect. It specific manner. This may occur when Pth limitation has should be noted that the presence of Lys residues at both the N–3 already established partial starvation for tRNA species that and N–4 positions (pJM-KKLS) does increase growth inhibition. accumulate preferentially under these conditions, such as However, this is a known effect due to the presence of two Lys His similar adjacent codons read by a tRNA in short supply, which tRNA or tRNA . ∆∆ 4732 Nucleic Acids Research, 2000, Vol. 28, No. 23 18. Björnsson,A. and Isaksson,L.A. 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Published: Dec 1, 2000

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