Charging levels of four tRNA species in Escherichia coli Rel+ and Rel− strains during amino acid starvation: a simple model for the effect of ppGpp on translational accuracy1

doi:10.1006/jmbi.2001.4525 available online at http://www.idealibrary.com on

J. Mol. Biol. (2001) 307, 785±798

Charging Levels of Four tRNA Species in Escherichia coli Rel‡ and Relÿ Strains during Amino Acid Starvation: A Simple Model for the Effect of ppGpp on Translational Accuracy Michael A. Sùrensen* Department of Molecular Cell Biology, University of Copenhagen, Denmark

Escherichia coli strains mutated in the relA gene lack the ability to produce ppGpp during amino acid starvation. One consequence of this de®ciency is a tenfold increase in misincorporation at starved codons compared to the wild-type. Previous work had shown that the charging levels of tRNAs were the same in Rel‡ and Rel ÿ strains and reduced, at most, two- to ®vefold in both strains during starvation. The present reinvestigaThr Leu and tRNAHis tion of the charging levels of tRNAArg 2 , tRNA1 , tRNA1 ‡ ÿ during starvation of isogenic Rel and Rel strains showed that starvation reduced charging levels tenfold to 40-fold. This reduction corresponds much better with the decreased rate of protein synthesis during starvation than that reported earlier. The determination of the charging and tRNAThr during starvation were accurate enough levels of tRNAArg 2 1 to demonstrate that charging levels were at least ®vefold lower in the Relÿ strain compared to the Rel‡ strain. Together with other data from the literature, these new data suggest a simple model in which mis-incorporation increases as the substrate availability decreases and that ppGpp has no direct effect on enhancing translational accuracy at the ribosome. # 2001 Academic Press

Keywords: ribosome; accuracy; charging level; tRNA; ppGpp

Introduction An overwhelming amount of data has accumulated over the last 40 years concerning the role of rel and ppGpp in RNA and protein synthesis, especially during amino acid starvation. Some of the main observations are summarized below. The stringent factor (Rel) encoded by the relA gene synthesizes ppGpp1 in the presence of ribosomes with an A-site occupied by a cognate, uncharged tRNA.2 The Rel‡ strain reduces its ribosomal and messenger RNA synthesis when starved for an amino acid as opposed to the Relÿ strain, which does not.3,4 The pool of ppGpp affects the regulation of RNA synthesis5,6 but whether ppGpp is the major effector of the growth-rate control of rRNA Abbreviation used: ppGpp, guanosine 5-diphosphate 3-diphosphate. E-mail address of the corresponding author: [email protected] 0022-2836/01/030785±14 $35.00/0

synthesis is a question that seems to remain to be solved.7,8 During amino acid starvation, the rate of total protein synthesis is the same in Rel‡ and Relÿ cells9-11 but translation is more accurate in the Rel ‡ strain,12,13 the error rate being approximately ten times higher in the Relÿ cells.14 Charging levels of the starved tRNA were found to be relatively high in both strains and not different enough to account for the difference in translational accuracy between Rel‡ and Relÿ strains.15,16,17 Among the models proposed to explain how ppGpp affected translational accuracy were those suggesting that ppGpp increased ribosomal proofreading by binding to translational initiation or elongation factors.18-20 Other models suggested functions of uncharged tRNA in the ribosomal A-site21,22 or different kinetic states of the ribosome controlled by the concentration of ppGpp.23 Some recent experiments indicate that there is no direct effect of ppGpp on translating ribosomes either in vitro24 or in vivo.25 This has led to the pre# 2001 Academic Press

786 sent reinvestigation of tRNA charging levels during amino acid starvation in Rel‡ and Relÿ strains. Northern blot analysis was used to measure the tRNA charging levels directly. The four species of tRNA examined here all showed much larger reduction in charging levels during starvation than reported earlier. Furthermore, for two of the tRNAs, the signal to noise ratio was high enough to see that the residual charging levels were at least ®vefold higher in the Rel ‡ than in the Rel ÿ cells. These results suggest that ppGpp has no direct effect on the translational machinery. The reduced charging levels during starvation are in good accordance with the reduction seen in protein synthesis rate and suggest that ribosomal accuracy decreases with the decrease in the residual charging level.

Results A relA‡/relA2 isogenic pair of strains derived from CP78 and CP79, respectively, was used to investigate the residual tRNA charging level during amino acid starvation. These strains have been used extensively in earlier investigations of the effects of amino acid starvation and due to the overwhelming amount of data available, describing all aspects of their growth physiology during starvation (see below for references) they seemed to be a natural ®rst choice for the present study. The strains are auxotroph for Arg, Thr, His and Leu and are, therefore, well suited for amino acid starvation experiments. In the experiments done to measure the charging levels, the cultures were grown in minimal medium and the cells were starved by ®ltration followed by two washes and resuspension in medium lacking the appropriate amino acid. Samples of the cultures were harvested for tRNA preparation before starvation and between ®ve and 45 minutes after the onset of starvation. In this time-span, the largest ¯uctuations in the ppGpp pools of both strains take place and, consequently, it was expected that the major effects on the aminoacyl-tRNA pools would be observed during this period. Transfer RNA charging levels were assayed using a Northern blot analysis of tRNA26 that measures in vivo tRNA charging levels directly. This assay has been re®ned to yield a high signal to noise ratio during previous work in our laboratory on in vivo effects of tRNA modi®cation de®ciencies.27 Starvation for arginine The charging of the major arginine isoacceptor tRNA (tRNAArg 2 ) was examined during starvation for arginine. This tRNA decodes the codons CGU, CGC, and CGA.28 CGU and CGC are the two major arginine codons; together, they are used with a frequency exceeding 99 % in highly expressed genes.29 The charging level of tRNAArg 2

tRNA Charging Levels and Translational Accuracy

must therefore represent the majority of the ¯owrate of arginine through the pool of total arginineaccepting tRNA. Figure 1 shows the charging level of tRNAArg 2 during such a starvation experiment. As can be seen, the charging dropped from high, during steady-state growth, to levels very close to that of tRNA deacylated by high-pH treatment, within ®ve minutes after the initiation of starvation. Figure 2 shows the data used to quantify the charging levels in the lanes of Figure 1. Each panel shows the amount of radioactivity found along a vertical line in each lane of Figure 1. A small, but is present at signi®cant, peak of charged tRNAArg 2 all time in the Rel‡ strain, but is absent from the Relÿ strain even as early as ®ve minutes after starvation. Table 1 summarizes three independent experiments including those shown in Figures 1 and 2. Clearly, during arginine starvation the charging was signi®cantly higher in the level of tRNAArg 2 Rel‡ strain compared to the Relÿ strain. The apparently negative charging level found after 15 minutes of starvation of the Relÿ strain most likely indicates that the chemically deacylated tRNA samples, used as backgrounds, had not been totally deacylated by the one hour incubation at pH 9.0. When the time of deacylation was varied between 0.5 and two hours, a small, and at most a 20 %, decrease in the residual ``charged tRNA'' that could be deacylated was found (data not shown). Nevertheless, in the Rel‡ strain there is a 1-2 % residual charging level after 15 to 30 minutes of arginine starvation and the charging level in the Rel ÿ strain at this time is very close to 0 %. is Clearly, the concentration of charged tRNAArg 2 different in the two strains during starvation and, relatively, the charging level may very well be at least tenfold higher in the Rel‡ strain compared to that in the Relÿ strain. Threonine starvation The charging level during starvation for threonine was examined by hybridizing a probe to the 30 major threonine isoacceptor tRNA (tRNAThr 1 ). Thr This tRNA, together with tRNA3 , decodes the two codons ACU and ACC.28,31 These two codons are used in E. coli with a frequency exceeding 90 % of all threonine codons in highly expressed genes.29

Table 1. Charging levels of tRNAArg during arginine 2 starvation (an average of three experiments) Duration of starvation (minutes) ÿ1 (Steady-state) 5 15 30 45

% Charging (SEM) relA‡

% Charging (SEM) relA2

74 (1.6) 1.6 (0.6) 0.9 (0.4) 1.2 (0.7) 1.3 (0.8)

71.6 (0.5) 0.5 (0.4) ÿ0.4 (0.4) ÿ0.8 (0.2) ÿ0.9 (0.3)

tRNA Charging Levels and Translational Accuracy

787

Figure 1. Charging levels of tRNAArg 2 . Phosphorimager scan of a Northern blot containing total tRNA from arginine-starved cultures of the Rel‡ and Relÿ strains. The blot was probed for tRNAArg 2 . Starvation began at time zero and the samples were harvested at the time indicated above each lane. The lanes marked B each contain chemically deacylated tRNA from the same sample as the one separated in the neighbouring lane on the left side. The positions marked 1 and 2 at the left side of the image indicate charged and uncharged form of the tRNA, respectively.

To improve the background correction and minimize effects of local noise on the Northern blots, an aliquot of each sample was deacylated for two hours and separated on the gel along with the untreated sample. In the ®rst measurement of the threonine tRNA charging level, the strains were grown in minimal medium. Removal of threonine resulted in a relatively small reduction in the charging level of tRNAThr 1 . Thirty minutes after removal of threonine, the charging level was reduced by about 30 % in the Rel‡ strain and 50 % in the Relÿ strain (Figure 3 and Table 2). The difference between the residual charging level in the two strains indicated clearly that the consumption of charged tRNA is higher in the Rel ÿ strain than in the Rel ‡ strain, but the absolute concentrations of charged tRNAThr 1 Table 2. Charging levels of tRNAThr during threonine 1 starvation (data are from Figure 3) Duration of starvation (minutes) ÿ1 (Steady-state) 5 30

% Charging relA‡

% Charging relA2

77 62 54

76 39 43

during starvation were much higher than expected. In both strains, the growth-rate, measured as absorbance at 436 nm, was reduced at least tenfold to 100-fold during starvation for threonine, a reduction similar to that seen during starvation for other amino acids (data not shown and e.g. see Lazzarini et al.5 and Morris & DeMoss32). However, Yegian & Stent17 showed that removal of threonine affects the charging level of isoleucine tRNA much more than of threonine tRNA itself. ``The explanation of this ®nding is probably that the allosteric nature of the bacterial threonine deaminase, the enzyme catalyzing the ®rst step in the biosynthetic conversion of threonine to isoleucine, precludes isoleucine synthesis at low levels of threonine. Thus, removal of an exogenous source of required threonine actually effects starvation for isoleucine, while the residual intracellular pool of threonine remains suf®ciently high to keep the threonine tRNA almost completely acylated''.17 In other words, removal of threonine from a threonine auxotroph causes a reduction of the internal pool of threonine, and an even further reduction in the pool of isoleucine due to the feed-back regulation on the activity of the threonine deaminase. The effect being that the cells are starving for isoleucine upon removal of threonine.

788

tRNA Charging Levels and Translational Accuracy

relA +

relA2

-1'

20

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15 20 10 5

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-1'+Base

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Counts . 10-3

20 10

20

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15'

60

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60

30'

7.5

40

5 20

2.5 0

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45'

10

30 20

5

10 0

0 0

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mm Figure 2. Quantitative analysis of the scan shown in Figure 1. Each panel shows the counts found along a vertical line in each lane of Figure 1. The local maxima around 15 mm and 35 mm represent Arg-tRNAArg and tRNAArg 2 2 , respectively. The charging levels were calculated as the percentage of counts found above a border set at a ®xed distance from the median of the maxima representing uncharged tRNA. The % charging found in the B lane for each strain was subtracted as background from each charging level found in that strain.

tRNA Charging Levels and Translational Accuracy

789

Figure 3. Charging levels of tRNAThr 1 . Phosphorimager scan of a Northern blot containing total tRNA from threonine-starved cultures of the Rel‡ and Relÿ strains. The blot was probed for tRNAThr 1 . For details, see the legend to Figure 1.

Such an effect of threonine starvation was not desired here. To avoid regulatory interactions between the threonine, isoleucine, leucine and valine biosynthetic pathways upon threonine starvation, the strains were grown in the presence of 50 mg/ml isoleucine and valine. Leucine was already present in the medium due to the auxotrophy of the strains. Now removal of threonine charging level at least tenfold reduced the tRNAThr 1 in the Rel‡ and even more in the Relÿ strain (Figure 4 and Table 3). Clearly, there is a three- to ®vefold difference in the charging level of tRNAThr 1 during threonine starvation in the two strains. Starvation for leucine In the attempt to measure the residual charging level of Leu tRNA during leucine starvation, the 30 was major leucine isoacceptor tRNA, tRNALeu 1 probed. This major Leu tRNA decodes codon CUG

Table 3. Charging levels of tRNAThr during threonine 1 starvation in the presence of 50 mg/ml Ile, Leu and Val (an average of four experiments) Duration of starvation (minutes) ÿ1 (Steady state) 5 15 30 45

% Charging (SEM) relA ‡ 71.5 5 5 6 7

(0.36) (1.9) (1.6) (3.1) (4.0)

% Charging (SEM) relA2 70 2.1 1.5 1 1.2

(1.3) (0.7) (0.8) (1.1) (0.5)

that is found with a frequency of 89 % among all six Leu codons in highly expressed genes.29 The codon CUG is also suggested to be decoded by the 28 Figure 5 shows the leucine isoacceptor tRNALeu 3 . Northern blot from such an experiment. As can be seen technical dif®culties prevented a precise determination of the residual charging level. First, the separation of the charged and uncharged form of the tRNA is rather poor even after a 1.3 times longer electrophoresis than that employed for the other tRNAs. This is probably due to the extra ten nucleotides present in the variable loop of this tRNA.33 Furthermore, there seems to be another, low-abundance and slightly slower migrating, species of Leu tRNA that is recognized by the probe. Unfortunately, the deacylated form of this comigrates more or less with the charged form of tRNALeu 1 . The in vitro deacylated tRNA and the starved, untreated tRNA are not distinguishable on the Northern blot (Figure 5). It is unlikely, therefore, that this crosshybridizing tRNA should be other than a leucine tRNA isoacceptor, because it would otherwise not be deacylated by leucine starvation. The best interpretation of this slowermigrating species of Leu tRNA is that it is a small lackhypomodi®ed subpopulation of the tRNALeu 1 ing a negatively charged modi®cation. Differences in migration due to hypomodi®cations of tRNALeu 1 have been observed before.33 Likewise, hypomodi®cations of other tRNA species have been shown to cause changes in mobility in the Northern blot assay utilized here.27 Alternatively, the probe may

790

tRNA Charging Levels and Translational Accuracy

Figure 4. Charging levels of tRNAThr in the presence of external Ile, Leu and Val. Phosphorimager scan of Northern 1 blots containing total tRNA from threonine-starved cultures of the Rel‡ and Relÿ strains. The strains were grown in the presence of 50mg/ml each of isoleucine and valine. The upper panel shows the blot from the Rel‡ strain as indicated. The blots were probed for tRNAThr 1 . Both blots were hybridized separately with the same preparation of probe. For details, see the legend to Figure 1.

recognize another of the isoaccepting species of Leu tRNAs, but no change in the relative intensity of the bands was ever observed upon increasingly stringency washings of the ®lter. Such a difference would have been expected if the probe had bound non-speci®cally mismatches to one of the other tRNALeu species. To obtain the best estimate of the charging levels, the high background was ignored and data were treated as for arginine and threonine. With this low signal to noise ratio in mind, the two independent determinations showed that the charging level decreased from 77 % in steady-state in both strains to between 1 and 3 % during starvation. Due to scatter arising from the high background, no signi®cant difference in the charging level

during starvation in the two strains could be seen. Nevertheless, it is obvious that the charging level during leucine starvation is low in both of tRNALeu 1 strains and could differ by ®ve- to tenfold without being detected. Starvation for histidine In the case of histidine, for which there is only one tRNA, it was found that the charging level in both strains fell from approximately 50 % in steady-state to virtually undetectable levels after ®ve minutes of histidine starvation (Figure 6). The concentration of the tRNAHis in E. coli is rather low and it has so far been impossible to obtain a signal to noise ratio in the Northern blots that allows a

791

tRNA Charging Levels and Translational Accuracy

Figure 5. Charging levels of tRNALeu 1 . Phosphorimager scan of Northern blots containing total tRNA from leucinestarved cultures of the Rel‡ and Relÿ strains. The blots were probed for tRNALeu 1 . The positions marked 1* and 2* at the left side of the image indicates charged and uncharged form, respectively, of the putatively hypomodi®ed tRNALeu (see the text). Otherwise, see the legend to Figure 4. 1

®rm determination of the very low charging levels of tRNAs present in such low concentrations. A further complication could be that the aminoacyl bond at this tRNA apparently is more labile than those on other tRNAs.34 In the Northern blots, an intense smear is seen between the charged and uncharged tRNAHis bands isolated from cells in steady-state growth (Figure 6, lanes 1 and 8). This smear probably represents His-tRNAHis that became deacylated during electrophoresis. A small constant loss of histidine from His-tRNAHis during electrophoresis would produce such a smear. This interpretation would also make the in vivo charging level of tRNAHis similar to the charging level of the other six tRNAs we have measured with this assay so far. They have all shown charging levels between 70 and 80 % during steady-state growth Lys 27 , tRNAGln (Figures 1, 3 and 5, tRNAGlu 2 , tRNA 1 ). The main conclusion from the ®ve histidine starvation experiments is that the charging level

during starvation is very low (less than 5 % before electrophoresis) in both the Rel‡ and the Relÿ strains.

Discussion The charging levels during amino acid starvation determined here differ in several ways from those reported in earlier work. The method employed here allowed a direct determination of charging levels of individual tRNA species in an assay that displays the quality of the tRNA without any intermediate enzymatic steps. The low charging levels found here during starvation were ®ve- to tenfold lower than those found earlier, and a signi®cant difference in charging levels between the Rel‡ and the Relÿ strain was observed. The present results are in good accordance with other observations. First, the approximately 95 % reduction in total protein synthesis rate during

792

tRNA Charging Levels and Translational Accuracy

Figure 6. Charging levels of tRNAHis. Phosphorimager scan of a Northern blot containing total tRNA from histidine-starved cultures of the Rel‡ and Relÿ strains. The blot was probed for tRNAHis. For details, see the legend to Figure 1.

amino acid starvation is re¯ected in the residual charging level of the starved tRNA. Thus, there is no gross surplus of substrate not utilized in translation. Second, the ®ve- to tenfold difference in the translational error rate observed between starved Rel‡ and Relÿ strains14 can be explained readily by tRNA charging levels, which differ by the same factor in the two strains. These observations suggest a simple explanation for the effects of ppGpp on translation, where the error rate increases as a consequence of the low residual charging level and ppGpp does not affect ribosome accuracy directly. Instead, as will be discussed below, ppGpp reduces the mRNA synthesis to an extent where mRNA becomes limiting for translation during amino acid starvation.25 As a consequence, the Rel‡ strain can avoid complete depletion of the pool of charged tRNA and it thus escapes severe mistranslation. Charging levels during starvation Of the four major tRNA species that were examined here during starvation, the highest residual charging level that was observed was about 5 % in the case of tRNAThr 1 . Earlier studies report charging levels of around 20 % following starvation. Examples of such residual charging levels are 8-20 % for Leu, 16 % for His and 21 % for Arg,17 10-25 % for Val15 and 40 % for Leu.32 Although strain-speci®c variations, due to different mutations in different amino acid biosynthetic genes, may explain some of these differences, the discrepancy is large.

Here, the charging levels of individual species of tRNA were measured directly. All the previous experiments mentioned above are based on in vitro recharging of periodate-protected acceptor capacity. Such an assay measures the combined charging level of all isoaccepting tRNAs present for an amino acid. Each isoaccepting tRNA could then contribute with different individual charging levels during starvation as suggested by O'Farrell.13 However, since the earlier work, a more detailed knowledge of E. coli tRNAs has become available. There is only one species of tRNAHis and the other three tRNAs measured here are all major tRNAs present in high concentrations and used in translation with a high frequency. As is the major isoaccepting Arg an example, tRNAArg 2 tRNA, decoding 99 % of all arginine codons in highly expressed genes. It is unlikely, therefore, that the three minor isoacceptor Arg tRNAs can contribute much to the charging level of total arginine-accepting activity, even during arginine starvation. Yegian & Stent17 reported that periodateprotected charging capacity can include peptidyltRNA and tRNAs protected by cryptic amino acid derivatives.35 Such heteroacylated tRNA may explain the observed discrepancy between charging levels found by the Northern blot assay used here and the previous periodate protected capacity measurements. Heteroacylated tRNAs could often have been detected as aminoacylated tRNAs, although they would not be true substrates for translation. It is possible that earlier extraction procedures were able to release small peptidyl-tRNAs

793

tRNA Charging Levels and Translational Accuracy

from ribosomes, which then would be added into the pool of aminoacylated tRNA. The result would be an overestimation of the residual charging level. The amount of mischarged, or heteroacylated, tRNA in the present Northern blot assay is below the limits of detection and probably negligible. The difference in migration distance of aminoacylated and free forms of tRNA depends on the charge of the amino acid aminoacylated to the tRNA.27 This can be seen here, where the separation was greater for the positively charged amino acid residues histidine and arginine than for the other two neutral ones (compare Figures 1, 3, 5 and 6). If mischarged, or peptidyl-tRNA were present during, for example, arginine starvation, it would most probably have migrated at a position other than that of arginyl-tRNAArg 2 . Such bands representing heteroacylated tRNA were not detected, even after prolonged exposures, and can, therefore, have been present in only very small amounts. Another source of differences in residual charging levels during amino acid starvation could be the method of arresting cells. In the present study, cells were harvested by pouring the culture into warmed trichloroacetic acid (5 % (w/v) ®nal concentration at 37.0  C) before further manipulations, as recommended by Lewis & Ames.34 Cooling of cultures before killing of cells can cause aberrant results due to differential rates of tRNA esteri®cation and aminoacyl tRNA utilization as the cells are chilled.36 In short, it is proposed that the low charging levels found here during starvation for arginine, histidine, threonine and leucine re¯ect the situation inside cells. During amino acid starvation, the transcription patterns of Rel‡ and Relÿ strains diverge. The most prominent difference is in the synthesis of stable RNA. This renders the selection of appropriate loading controls, to probe for on the blots, ambiguous. Instead, all lanes on a blot ideally represent equal culture volumes. Ideally because variable recovery of tRNA, e.g. during precipitation in ethanol, cannot be excluded. Furthermore, since tRNA synthesis continues in the Relÿ strain, there was some variation in the amount of tRNA recognized by the probes in the individual experiments. Nevertheless, the method used for detecting charging levels showed a high level of reliability in the range of tRNA concentrations applied here (see Materials and Methods). The continuous synthesis of RNA in the Relÿ strain during starvation leads, after prolonged starvation, to the accumulation of hypomodi®ed forms of tRNA with altered chromatographic properties.37,38 Some of these hypomodi®ed tRNA species can be expected to participate in translation and charging reactions differently from the native forms. After 45 minutes of starvation in the Relÿ strain it can be estimated that there is about 1.5fold more tRNA than before starvation, assuming an unaltered synthesis rate of RNA. An extreme case would be that none of the tRNA synthesized

during starvation could be aminoacylated due to hypomodi®cations, and further, that it comigrated with the uncharged, fully modi®ed form. In this case, the residual charging level would be underestimated by one-third at 45 minutes after the onset of starvation. This would not be enough to account for the difference found here between the residual charging levels in the two strains. Furthermore, many of the base modi®cations that occur on tRNA will change the net charge of the molecule and thereby the mobility during electrophoresis in this assay.27 On Northern blots with the four tRNA species examined here, there was never observed any change in the patterns of bands as starvation progressed. It is therefore unlikely that hypomodi®ed tRNAs played a major role for the determination of the charging levels during the short period of starvation applied here. The protein synthesis rate and the tRNA charging level Amino acid starvation in E. coli typically causes a 90 to 98 % reduction in the protein synthesis rate.13,25,39,40 It has remained a puzzle, therefore, why the protein synthesis rate was reduced by tenfold to 50-fold during amino acid starvation while the corresponding tRNA charging levels were reduced by a factor of only 4 to 5 in Rel‡ and in Relÿ cells. One explanation has been that ppGpp bound directly to parts of the translational apparatus inhibiting protein synthesis and reducing the error rate and processivity of ribosomes.18-20 This could, of course, not explain the equally low protein synthesis rate in the Relÿ strain that is not impaired by the action of ppGpp, but probably directly limited by the availability of the substrate lacking for translation. Another more plausible explanation is that an individual isoaccepting species of tRNA could become completely deacylated and inhibit protein synthesis while the residual charging level of total isoaccepting tRNA remained high.13 As discussed above, this possibility is unlikely in light of the data presented here combined with the detailed knowledge available today about individual tRNA levels and the codon usage of E. coli. Instead, a tenfold to 50-fold reduction in the protein synthesis rate after an amino acid starvation is in good accordance with the 15 to 40-fold reduction in tRNA charging levels reported here. As discussed below, the effect of ppGpp during starvation is a reduction of mRNA synthesis that results in a slight but important restriction to the protein synthesis rate. During a starvation there is no net synthesis of protein if the mutation that renders the cell auxotrophic for a particular amino acid is non-leaky. The substrate for the residual protein synthesis arises from starvation-induced protein breakdown, which proceeds with a rate comparable to that of protein synthesis rate.41,42 In strains CP78 and CP79, which are the parental strains of those used here, an arginine starvation reduces the protein

794 synthesis rate to 5 % of the prestarved rate in both strains.9 In an analogous experiment, CP78 and CP79 displayed the same reduced protein synthesis rate after a phenylalanine starvation induced by b-thienyalanine.10 Furthermore, during a nutritional downshift, Rel‡ and Relÿ strains displayed the same rate of protein synthesis.11 This means that whether the cells are Rel‡ or Relÿ, they make new protein at the rate allowed by the supply of the limiting substrate. In other words, the rate of protein breakdown and the rate of consumption of the starved amino acid is the same in both types of cells. This must mean that the ¯ux of the starved amino acid through the pool of charged tRNA is the same in both types of cells. The only, but important, difference being that in the Rel‡ strain, the mRNA synthesis rate is reduced25 to the point where a 2-5 % residual charging level of the starved tRNA can be sustained and severe mistranslation avoided. The translational error rate and the tRNA charging level Direct and precise measurements of the translational error frequency are not simple to perform. In unstarved cells, misincorporations are rare and are dif®cult to detect. The error rate, which is codonspeci®c, has been estimated to be between 5  10ÿ5 and 1  10ÿ3 per codon translated in unperturbed cells.14,43,44 The codon speci®city arises not only from the variation in availability of tRNA capable of misreading the individual starved codon. There is also a variation in error frequency at the same codon in different sequence contexts.45 In some cases, identical experiments with Rel‡ and Relÿ cells have been used to compare the error frequency during unstarved and starved conditions. From such experiments, the generalized picture is that the error frequency rises tenfold during starvation of the Rel‡ strain but 100-fold in the Relÿ strain.14 Given all the possible sources of experimental inaccuracies and the fact that there are probably variations dependent on strains and individual amino acid starvations, these numbers are in good accordance with the data obtained here. The technique used here made it possible to and measure the low charging levels of tRNAArg 2 during starvation. The precision was suf®tRNAThr 1 cient to demonstrate the different residual charging levels in the Rel‡ and the Relÿ strains. For tRNAArg 2 , it was found that there were undetectable amounts of charged tRNA left in the Relÿ strain after 15 minutes of starvation. In contrast, there was still 1-2 % charged tRNA left in the Rel‡ strain. starvation for threonine In the case of tRNAThr 1 reduced the charging level tenfold to 15-fold in the Rel‡ strain and a further three- to ®vefold in the Relÿ strain. Such differences in charged tRNA concentrations during starvation could account for the approximately tenfold difference in translational accuracy that is observed between starved Rel‡ and Relÿ strains.14 Furthermore, the tenfold to

tRNA Charging Levels and Translational Accuracy

40-fold reduction of the charging level found here during starvation of the Rel‡ strain indicates that the tenfold increase in the error frequency found during similar starvations of Rel‡ strains14 may very well depend directly on the reduced charging level. In other words, the increase in the frequency of translational misincorporation during starvation is related directly to the reduction in the charging level of the starved tRNA. The relationship in vivo between the concentration of charged tRNA and the translational error rate probably depends mostly on the residual charging level. However, the charging levels of the near-cognate tRNAs that participate in the individual mistranslation events may play some role, and the possibility cannot be excluded that these charging levels could vary between the two strains. Unfortunately, the identities of all the tRNAs that participate in mistranslation during starvation for the individual amino acids are not known.14 Therefore, future experiments are needed to elucidate the precise identity and role of all the tRNAs that determine the translational error rate during starvation. Many different models have been proposed to explain the contradicting observations that, while the charging levels for starved tRNAs are the same in Rel‡ and Relÿ cells, the error rate is kept low only in the Rel‡ strain (see Rojas & Ehrenberg24 for a review of these models). Below, a model on the function of ppGpp in translation and its effects on accuracy is presented. In many aspects, this model is similar to that outlined by O'Farrell13 more than 20 years ago, using the data then available on tRNA charging levels during starvation. A model for effects of ppGpp on translational accuracy When amino acid starvation is induced in the Rel‡ strain, the concentration of the respective ternary complex decreases and the synthesis of ppGpp increases. The increase in the ppGpp pool is a signal to the RNA polymerase and the response is a decrease in the chain growth rate of mRNA molecules.46-48 The result is a lower synthesis rate of mRNA. The pool of ppGpp reaches a maximum about ®ve minutes after the onset of starvation.1 This is because the concentration of hungry codons decreases as a result of the reduced mRNA synthesis and decay of the mRNA pools that were present initially. After this point, protein synthesis is no longer limited solely by the availability of ternary complex, but also by mRNA. In this situation, an increase in the mRNA pool increases the protein synthesis rate by about 20 %.25 Upon further starvation, a new state is reached where the ppGpp pool is still higher than before starvation, resulting in a continuously reduced and limiting mRNA pool. This allows charging of the hungry tRNA at a 2-5 % level, where protein synthesis can proceed without a drastic increase in the translational error rate. On the other hand, if the mRNA pool is kept arti®cially high in this situation, the ppGpp

795

tRNA Charging Levels and Translational Accuracy

pool increases as a response to the increased concentration of hungry codons, and translational error frequency increases.25 This shows that there is no direct link between the concentration of ppGpp and translational ®delity, which has also been shown by careful in vitro experiments.24 The scenario in the Relÿ strain resembles that in the Rel‡ strain containing an arti®cially increased mRNA pool. As the Relÿ cells are unable to synthesize ppGpp upon amino acid starvation, RNA synthesis continues and there is no mRNA limitation for the ribosomes. As a consequence, protein synthesis is limited solely by the availability of the starved substrate, and the high concentration of ribosomes calling for the starved tRNA causes the charging level of the starved tRNA to be reduced to levels very close to zero. The isogenic Rel‡ strain, during the same conditions, mistranslates moderately14,25 due to aminoacyl-tRNA limitations. The simplest explanation for the further increased error frequency in the Relÿ strain is that the lower residual charging level causes the ribosomal A-site to be empty for a longer period, enhancing the probability that near-cognate aminoacyl tRNAs are accepted. The result is more mistranslation. A ®veto tenfold difference in charging level can easily account for the ®ve- to tenfold difference in error frequency that is observed during starvation.14,43,49 With this simple model, the only effect of ppGpp produced on ribosomes when aminoacyltRNA is limiting is to signal RNA polymerase to reduce mRNA levels. This enables the cell to keep a reasonable charging level and thereby optimize the quality of the sparse protein synthesis during starvation. Consequently, there is no need to invoke any direct effect of ppGpp on the ribosome where it was synthesized.

Materials and Methods Strains The strains used are the E. coli K12 strains CP78 and CP7950 cured for l (N. Fiil). NF915 (Rel‡): thr leu his argH thi mtl supE44 relA‡spoT‡ lÿls NF916 (Relÿ): thr leu his argH thi mtl supE44 relA2 spoT‡ l ÿls Growth conditions Cultures were grown exponentially at 37.0  C in Mops minimal medium51 supplemented with 0.4 % (v/v) glycerol, 2.5 mg/ml of thiamine, 5 mg/ml of histidine, 50 mg/ml of arginine, leucine and threonine. The growth-rate was one doubling per 76 minutes. In the experiments where 50 mg/ml of valine and isoleucine were added, the growth-rate was one doubling per 70 minutes. All cultures had been growing exponentially for at least ten generations before being used in experiments. At an A436 of approximately 0.7, cells were collected by ®ltration onto a nitrocellulose ®lter (0.45 mm pore size) at 37  C, washed twice in one volume of medium

without the amino acid to be starved for and resuspended in one volume of the same medium. Washing and resuspension took less than two minutes. The zero time-point corresponds to the time where resuspension was ®nished. Analysis of aminoacylation levels of tRNA For each sample, 4 ml of culture was poured into 4 ml of 10 % (w/v) trichloroacetic acid at 37  C. From this, tRNA was puri®ed and Northern blots were processed essentially as described26 with a few modi®cations.27 Each sample of tRNA was redissolved in 20 ml of 10 mM sodium acetate (pH 4.5), 1 mM EDTA: 4 ml of such a sample were used for each lane in the gels. The tRNA in the lanes used for background subtraction was deacylated by treatment with 0.1 M Tris-HCl (pH 9.0) at 37  C for the time indicated in a total volume of 50 ml. The tRNA was neutralized, precipitated and then dissolved in 4 ml of 10 mM sodium acetate (pH 4.5) and 1 mM EDTA. Untreated or deacylated tRNA (4 ml) was mixed with 6 ml of loading buffer (0.1 M sodium acetate (pH 5.0), 8 M urea) plus 0.05 % (w/v) bromphenol blue and 0.05 % (w/v) xylene cyanol, and separated by electrophoresis through a 6.5 % (19:1 (w/w) acrylamide/ bisacrylamide) gel containing 0.1 M sodium succinate (pH 5.0), 8 M urea. The gels were 50 or 60 cm long and electrophoresis was carried out at 10 V/cm at 4  C until the bromphenol blue reached the bottom of the gel (approximately 20 hours). Then 20 cm of the gel between the two dyes was electroblotted onto a Positive2 membrane (Appligene) at 20 V for 90 minutes with 40 mM Tris-acetate (pH 8.1), 2 mM EDTA as transfer buffer. The tRNA was crosslinked to the membrane by 0.12 J of UV light in a Stratalinker 1800. The membranes were pre-hybridized at 42  C for one hour in 6 ml of hybridization solution (0.9 M NaCl, 0.05 M NaH2PO4 (pH 7.7), 5 mM EDTA, 5  Denhardt's solution (100  Denhardt's solution is 2 % (w/v) bovine gelatine, 2 % (w/v) Ficoll and 2 % (w/v) polyvinylpyrrolidone), 0.5 % (w/v) SDS and 100 mg/ml of sheared, denatured herring sperm DNA). Hybridization was at 42  C overnight in the same solution with a radioactive probe. Membranes were washed several times in 0.3 M NaCl, 30 mM sodium citrate, 0.1 % SDS at room temperature. In addition, membranes used for detection of tRNAThr were washed for 30 min1 utes at 60  C to remove unspeci®c cross-hybridization. The radioactivity present in speci®c bands was measured using a phosphorimager scanner and the charging levels were determined as described for Figure 2. To evaluate if the charging levels found were sensitive to the concentration of tRNA loaded to the gel, the following experiment was made. Two identical, 4 ml samples of culture were harvested from each of a Rel‡ and a Relÿ strain that had been starved for arginine for 45 minutes. After tRNA preparation, each pair of identical preparations was mixed and half of each was deacylated by base treatment for two hours. Samples containing 0.25, 1.0, 2.0, 4.0 and 8.0 ml of the same tRNA preparation were brought to the same volume by loading buffer and separated by electrophoresis. In the neighboring lane of each sample, an equivalent amount of deacylated tRNA was applied to be used for background subtraction. The charging levels were analyzed from a Northern blot (not shown) probed for tRNAArg 2 . The analysis showed that the amount of radioactivity bound to the ®lter was proportional to the amount of tRNA

796

tRNA Charging Levels and Translational Accuracy difference between the concentration of stable RNA in the two strains should be maximal due to the continuous synthesis of RNA in the Rel ÿ strain. The data presented in Figure 7 show that the method applied here is insensitive to such differences in tRNA concentrations as long as the signal to noise ratio is high enough. The oligonucleotides used as probes to detect tRNA had the sequences: 50 -TCCGACCGCTCGGTTCGTAGC 0 complementary to tRNAArg 2 ; 5 -CACGACAACTGGAATCACAATCC complementary to tRNAHis; 50 -GTAAGGACACTAACACCTGAAGC complementary to tRNALeu 1 and 50 -TGGGGACCTCACCCTTACCAA complementary Thr to tRNA1 . The complementarity of these probes to the individual tRNAs were veri®ed by BLAST searches (www.ncbi.nlm.nih.gov/BLAST) in the E. coli sequence database. For each blot, 33 pmol of probe was labeled with 0.1 mCi of [32P]ATP (3000 Ci/mmol) using ten units of polynucleotide kinase (New England Biolabs) in a total volume of 8-15 ml of the buffer recommended by Boehringer for their polynucleotide kinase. After one hour at 37  C, 0.1 ml was analyzed by polyethyleneimine cellulose thin-layer chromatography to check if more than half of the radioactivity was incorporated. If not, an additional ten units of polynucleotide kinase was added, and the incubation was continued for one more hour.

(a) Total count rate in lane (cpm . 10-3)

6

4

2

0

(b)

Charging level (%)

1.5

1.0

Acknowledgements 0.5

0 0

2

4 6 Sample volume (µl)

8

Figure 7. Analysis of the charging level of tRNAArg as 2 a function of tRNA concentration in the sample. tRNA was prepared from the Rel‡ (®lled symbols) and the Relÿ (open symbols) strain after 45 minutes of arginine starvation. The samples were treated as described (see the text). (a) The amount of radioactivity found in each lane on the Northern blot as a function of the tRNA concentration in the same lane. The lines represent the curves best ®tting each set of data. (b) The charging level determined for each sample as a function of tRNA concentration. The broken lines represent the mean of the charging levels found in each set of data. The mean (SEM) is 1.4 % (0.14) and 0.7 % ( 0.09) for the charging level found in the Rel‡ and the Relÿ strain, respectively.

loaded onto the gel (Figure 7(a)). Furthermore, there was no signi®cant variation in the charging levels determined at the different concentrations of tRNA (Figure 7(b)). An increase in the scatter around the average is evident at the lowest concentrations of tRNA (Figure 7(b)). This is most probably due to local variations in the background on the blot, which affect the bands with low counting rates the most. The samples used for this analysis were collected after 45 minutes of starvation. At this point the

I thank Steen Pedersen for discussions and inspiration and also for encouragement at times where methods needed to be improved. I thank Marit Warrer for her excellent technical assistance. Thanks also to Michael O'Connor and Charlotte G. Jensen for comments on the manuscript. The work was supported by grants from the Danish Natural Science Research council to Steen Pedersen.

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Edited by D. E. Draper (Received 10 October 2000; received in revised form 30 January 2001; accepted 2 February 2001)