in Escherichia coli - NCBI

2 downloads 0 Views 696KB Size Report
Jul 11, 1988 - We are grateful to Jeffrey Miller for providing us with the F' lacIZ donor strains. This work was ... Fluck, M. M., W. Salser, and R. H. Epstein. 1977.
JOURNAL OF BACTERIOLOGY, OCt. 1988, p. 4714-4717

Vol. 170, No. 10

0021-9193/88/104714-04$02.00/0 Copyright C) 1988, American Society for Microbiology

Effects of Release Factor Context at UAA Codons in Escherichia coli ROBIN MARTIN,t MICHAEL WEINER, AND JONATHAN GALLANT* Department of Genetics, University of Washington, Seattle, Washington 98195 Received 4 January 1988/Accepted 11 July 1988

In Escherichia coli, nonsense suppression at UAA codons is governed by the competition between a tRNA and the translational release factors RF1 and RF2. We have employed plasmids carrying the genes for RF1 and RF2 to measure release factor preference at UAA codons at 13 different sites in the lacI gene. We show here that the activity of RF1 and RF2 varies according to messenger context. RF1 is favored at UAA codons which are efficiently suppressed. RF2 is preferred at poorly suppressed sites. suppressor

The effect of mRNA context on the activity of nonsense has been extensively studied (1, 4, 6, 11). However, thus far the mechanism responsible for these effects remains unclear. Theories to account for nonsense suppression context phenomena have been divided into three categories (13): release factor effects, mRNA structure effects, and tRNA-tRNA interactions. Most investigations have focused on tRNA-mediated effects. We have been interested in the role of the translational release factors RFI and RF2. In Escherichia coli, RF1 reads UAG and UAA codons, whereas RF2 recognizes UAA and UGA. Previous work has shown that the competition by RF1 and RF2 is blind with respect to the efficiency of nonsense suppression at any one codon (R. Martin, M. Hearn, P. Jenny, and J. Gallant, Mol. Gen. Genet., in press). It follows, therefore, that an increase in the level of RF1 and RF2 would not be expected to reveal any differences between contexts for UAG or UGA suppression, respectively. Competition at each site would be of equal magnitude whatever the efficiency of suppression. In contrast, at UAA codons where both RF1 and RF2 are active, introducing additional RF1 or RF2 might be expected to reveal context effects on the preferences for one release factor versus the other. We have screened a collection of F' lacI-UAA-lacZ fusions for UAA context effects of this kind. suppressors

number of P-galactosidase determinations to be performed, bacteria were grown for enzyme assay on plates rather than in liquid medium. Cells were spread from single colonies with sterile toothpicks in streaks 1 cm wide across the width of regular 9-cm-wide petri plates containing glucose minimal medium and appropriate supplements; all plates lacked proline to maintain selection for the F' lacIZ proAB+ plasmid and contained appropriate antibiotics to maintain selection for the pACYC tRNA suppressor plasmid (chloramphenicol) and the release factor plasmids (carbenicillin). After growth for about 40 h at 37°C, bacteria were harvested with toothpicks into 300 [lI of Block buffer B (Martin et al., in press), disrupted by two 15-s periods of ultrasonication on ice separated by 30 s of rest, and centrifuged for 10 min in a Microfuge to remove cell debris. The supernatants were removed to fresh tubes and assayed as previously described (Martin et al., in press) for enzyme activity and protein. We have confirmed in many comparisons that enzyme activities determined in this way do not differ significantly from those measured in conventional liquid cultures when both lacZ+ strains and suppressed lacIZ fusion strains are used. RESULTS The purpose of the present study was to compare the action of RF1 and RF2 at UAA codons in a variety of messenger contexts. Unlike the response at UAG or UGA, preference or asymmetry in the competition by RF1 and RF2 at UAA sites would afford a demonstration of an effect of messenger context on the polypeptide factors which participate in protein chain termination. For this study, we have used 13 UAA alleles distributed throughout the E. coli lacI gene. The sequence and position of each mutant are known (7). Each UAA allele has been crossed into a lacI-lacZ gene fusion which is conveniently carried on a selectable F' factor (6). Nonsense suppression in lacI is monitored by the production of ,B-galactosidase activity from the lacZ gene. Enzyme activity is unaffected by the nature of the amino acid inserted at the site of the UAA triplet by the suppressor tRNA (6). Each of the F' lacIZ UAA mutants was crossed into host strains deleted for the chromosomal lac gene and containing the pACYC279 UAA suppressor plasmid alone or in combination with pRF1 or pPJRF2. The levels of 3-galactosidase were then determined in each of these strains and expressed as a percentage of the activity from a strain containing the same F' lacIZ fusion uninterrupted by UAA (RM317). The

MATERIALS AND METHODS Bacteria and plasmids. The bacterial strains used in this study are listed in Tables 1 and 2. RM476 contains pACYC279 (sup UAA). This plasmid carries the tRNA trp gene mutated in vitro to read UAA (8). RM477 and RM478 were constructed by transforming RM476 with pRF1 and pPJRF2, respectively. pRF1 and pPJRF2 are pBR322-based plasmids which are fully compatible with pACYC279. pACYC279, pRF1, and pPJRF2 have been described previously (Martin et al., in press). F' lacIZ fusions carrying UAA mutations in the lacI portion were crossed into RM476, RM477, and RM478 by selecting for pro' and Rif. This was accomplished by cross streaking on appropriately supplemented minimal plates as previously described (Martin et al., in press). F' lacIZ donor strains were generously provided by J. Miller. Media and ,I-galactosidase assays. Because of the large * Corresponding author. t Present address: University of Sussex, Falmer, Brighton BN1 9QG, United Kingdom.

4714

RELEASE FACTOR EFFECTS AT UAA CODONS IN E. COLI

VOL. 170, 1988 TABLE 1. Bacterial strains' Genotype

Strain

RM310 RM317

...

leu

...

As

A(lac-proB)S

RM310 but RM310 but

RM476

...

As

RM477

...

As RM476 but

RM478

...

As RM476 but

a

ara

F'

thi

A(gal-bio-att) rpoB

lacIq Alac-14 proAB+

press.

results are presented in Table 3, where the antisuppression value is simply the factor by which the presence of a plasmid-borne RF1 or RF2 gene reduces the efficiency of suppression. The UAA sites are listed in order of their suppression efficiencies, for reasons which will become apparent below. It can be seen that the presence of either plasmid-borne release factor gene reduced UAA suppression by a factor of between 1.5 and 3.4, in agreement with previous results (Martin et al., in press), an effect we term antisuppression. Our purpose in these experiments was to determine whether there were sites at which antisuppression by RF1 and RF2 differed significantly. After the first three sets of experiments on all 13 sites were done, we chose sites which appeared to show asymmetry for further testing. In some cases the asymmetries were reproduced in several additional tests, and in some cases they were not. Those cases which did show reproducible asymmetries, as well as several others, were chosen for repeated replication of the experiment. This is why different sites were subjected to different numbers of replicate experiments (Table 3, last column). The method is conservative and may have missed some cases of asymmetry.

Differences between antisuppression ratios for RF1 and RF2 were analyzed by Student's t test for paired samples (in each experiment, cultures of strains harboring the RE1 plasmid, the RF2 plasmid, or neither RF plasmid were grown, processed, and assayed in parallel). Table 3 lists t values for the antisuppression ratios at each site, together with P values (equal-tails test) for those which appeared to be significant at the 5% level. Of course, since 13 independent sets of data were analyzed, a more stringent criterion of significance should be applied. The strictest we could apply would be a P value corresponding to'a chance of 5% or less that even one such case would turn up at random in 13 sets of data. This value is 4 x 1o-3; 0.05 = 1 [1 (4 x 10-3)]13. By this strict criterion, the asymmetries in antisuppression were significant at three sites: 021 (t = 4.4, P = 3 x 10-3), 028 (t = 5.2, P = 6.0 x 10-4), and 019 (t = 7.7, P = 5.7 x 10-5). We might also note that 021 showed greater antisuppression by RF1 than by RF2 in 8 of 8 replicate experiments (P = 2-7 = 7.8 X 10-3), 028 showed greater antisuppression by RF1 than by RF2 in 10 of 10 replicates (P = 2-9 = 2 x 10-3), and 019 showed greater antisuppression by RF2 than by RF1 in 9 of 9 replicates (P = 2-8 = 3.9 x 10-3). We conclude that the context of 019 favors termination by RF2 more often than by RF1 and that the context of 028, and probably that of 021, favors the converse asymmetry. The extent of this asymmetry is not large. On average, RF1 antisuppressed 62% as efficiently as RF2 at site 019 and 189% as efficiently as RF2 at site 028. Given this modest range of variation, it is possible that asymmetries exist at other sites but were obscured by experimental variance. Two considerations suggest that this is indeed the case. First, no fewer than seven cases turned up for which the -

asymmetry appeared to be significant at the 5% level. This is exceedingly unlikely to occur at random. The chance of obtaining seven or more such P values in 13 independent sets of data is, by the binomial distribution

pACYC279 (sup-tRNAtrpUAA) pRF1 pPJRF2

Reference for all strains, Martin et al., in

4715

z

(17) (0.057) (0.956) < 10-6

This consideration strongly suggests that several of these asymmetries (not just the three associated with P values of less than 4 x 10-3) are real. Second, the antisuppression ratios appear to vary systematically with the efficiency of suppression. It is striking that all of the most weakly suppressed sites show weaker antisuppression by RF1 than by RF2, whereas the most strongly suppressed sites (the three last entries in Table 3) exhibit precisely the reverse asymmetry. Figure 1 presents the ratio of RF1 antisuppression to RF2 antisuppression plotted versus the efficiency of suppression. The correlation coefficient is 0.87, which is highly significant (t = 5.7, P = 1.3 x 10-4).

DISCUSSION All the'strains used in this study were isogenic, differing only in the positions of UAA mutations in the lacI portion of the lacIZ fusion carried on the F' plasmid. Therefore, it is most unjikely that asynumetries in antisuppression could be due to variatiQn in copy number of the pBR plasmid carrying the release factor gene or to vanation in generalized, nonspecific effects of thesd- ptasmids on the copy number of suppressor tRNA plasmids. Moreover, as we have pointed out elsewhere (Martin et al., in press), studies of noncognate suppression have already provided powerful controls for such nonspecific effects. We observed that pRF1 does not compete with UGA suppression by a suppressor tRNA, as expected, but does comnpete with noncognate suppression of a UAA at the same site. Similarly, pPJRF2 competes with a UAA suppressor reading a UAA site but does not compete with the very same tRNA when it produces weak, noncognate suppression of a UAG allele at the same site. Finally, direct measurements of release factor levels by immune precipitation in strains isogenic with the present ones have indicated that introduction of the plasmids led to a five- to eightfold increase in release factor concentration, close to the magnitudes of the observed antisuppression effects (Martin et al., in press). We are therefore confident

-

TABLE 2. F' lacIZ UAA donor strainsa Strain

5' Context

Amino acid position in lacl geneb

3' Context

F'O1 F'O11

GUG UCG GUC UGU GCG GUG CAG CCA

2 78 105 108 117 137 187 189

CCA AUU GCC

AAU CGG GAC CAA AUG

209

F'015 F'016 F'017 F'019 F'021 F'022 F'024 F'026 F'Y5 F'028

F'029 a

217 220 228 231

GCG CGC GCU ACU AGU AUU GGC AGU ACC AUG

All F' lacJZ fusions were received from J. Miller in an ara thi host.

b See reference 7.

4716

MARTIN ET AL.

J. BACTERIOL.

TABLE 3. Competition by REl and RF2 at UAA sites in lac" Allele

01 017 019 015 029 YS1 024 016 026 011 021 022 028

UAA suppression (% lac' + SE)

2.4 2.7 3.1 3.8 3.9 4.9 5.4 5.8 8.2 8.4 9.8 10.0 14.9

+ 0.34 ± 1.5

+ 0.28 ± 0.43 ± 1.2 ± 0.82 ± 0.78 ± 2.2 ± 1.25 ± 1.0 ± 1.0 ± 3.6 ± 2.0

RFI + SE 1.98 1.96 2.05 2.51 1.50 2.07 2.02 1.46 1.71 2.58 3.26 2.93 2.86

± ± ± ± ± ± ± ± ±

+ ± ± ±

0.33 0.13 0.27 0.305 0.035 0.20 0.23 0.041 0.27 0.24 0.25 0.34 0.19

Antisuppression RF2 ± SE

2.73 2.55 3.32 3.13 2.08 2.10 1.84 1.80 1.74 3.40 2.44 2.56 1.51

± ± ± ± ± ± ± ± ± ± ± ± ±

0.20 0.088 0.31 0.22 0.11 0.14 0.015 0.14 0.15 0.44 0.27 0.35 0.105

P

t

RF1/RF2

0.725 0.77 0.62 0.80 0.72 0.99 1.1 0.81 0.98 0.76 1.3 1.1 1.9

8 3 9 6 3 3 3 3 6 4 8 4 10

2.2 4.95 7.7 2.7 4.7 0.21 0.77 0.20 0.13 2.1 4.4 3.7 5.2

3.85 5.7 4.4 4.2

x 10-2 x 10x 10-3 x 10-2

3.0 x 10-3 3.4 x 10-2 6.0 x 10-4

"In each experiment, maintenance of the F' was ensured by selection for proline independence, maintenance of the tRNA suppressor plasmid was ensured by selection for chloramphenicol (30 ,ug/ml) resistance, and maintenance of the release factor plasmids was ensured by selection for carbenicillin (100 ,ug/mI) resistance. Suppression is expressed as percentage of the activity in an isogenic strain carrying an F' lacIZ fusion which was grown and assayed in parallel in most experiments. The lacl+ control specific activity averaged 2.51 EU of protein per mg, with a standard deviation of 0.34 in 26 replicates (standard error of the mean = 0.067). Antisuppression is the specific activity of a given suppressed UAA allele in the absence of both release factor plasmids divided by specific activity in the presence of either pRF1 or pJRF2, with the standard error of the mean tabulated; n is the number of replicate experiments.

that antisuppression efficiency does reveal the relative frequency of termination by the release factor concerned. Generally, three possible mechanisms have been considered which might give rise to nonsense suppression context effects: release factor mechanisms, mRNA structure, and tRNA-tRNA interactions (13). Fluck et al. (5) favored a release factor mechanism to explain the uniform response of different contexts to various genetic and phenotypic suppressors. Bossi and Roth (2) and Bouadloun et al. (3) found evidence for tRNA-specific effects. The data of Miller and Albertini (6) and Bossi (1) were consistent with release factor influences in addition to tRNA-specific effects. The present study, which revealed asymmetries in the action of RF1 and RF2 at UAA, provides good evidence that release factors are indeed subject to the effect of mRNA context. A similar conclusion can be drawn from the data of Ryden and Isaksson (9). In their study, a temperature-sensitive allele of RF1, uar (= prfA, as was shown later [101), was tested for its effect on suppression at UAG and UAA codons in the lacI gene. Reduction in RF1 activity at the nonpermissive temperature, as expected, increased s.tppression at UAG much more than at UAA, where RF2 is also active. The effect of decreasing RF1 activity at the restrictive temperature differed considerably from one UAA site to another, as if the relative contributions of RF1 and RF2 to the overall probability of termination differed. Moreover, the relative effect of RF1 restriction increased with suppression efficiency, analogous to the relationship displayed in Fig. 1. Among the lacI alleles which are common to our experiments and those of Ryden and Isaksson (017, Y5, 021, 022 and 028) (9), the two studies show a striking consistency: a plot of our RF1/RF2 asymmetry ratio versus their measurement of the effect of RF1 restriction yields a correlation coefficient of 0.87. The concordance between these two approaches-which, we note, involved different suppressor tRNAs-leaves little doubt that the relative affinities for RF1 and RF2 at UAA codons are subject to context determinants. The correlation revealed in Fig. 1 implies that components of mRNA context which determine suppression efficiency overlap with those which determine the relative affinities for RFI versus RF2. One simple interpretation of this overlap is as follows. (i) Part of the context variation in suppressibility reflects varn-

ation in the affinity for both release factors; thus, contexts which dictate a weak affinity for release factors will favor suppressor tRNA binding rather than termination and will therefore be suppressed more efficiently. (ii) These context determinants affect the affinity for RF1 less than the affinity for RF2; thus, as the probability of termination declines (and the converse probability of suppression increases), the relative contribution of termination by RFI rather than RF2 increases. It follows that context rules governing nonsense suppression (1, 6, 11) may relate as much to release factor binding as to tRNA binding. Our survey does not include a sufficient number of UAA sites to warrant statistical analysis of the kind performed by Stormo et al. (11). However, it may be significant that the sequences 5' to the least efficiently suppressed, RF2-favored sites (019 GUG, 01 GUG, 017 GCG, and 015 GUC), show similarities which have not been noted previously as context determinants. In accordance with other studies, the most efficiently suppressed, RF1favored sites were followed by a 3' A. Investigation of a much larger number of UAA sites or directed mutagenesis of test UAA sites will be required to define the context rules of release factor binding in detail. 20, ul

028

20 t

._

c 021

0 a C1

*

851

01 °1

7

2

022

026

*

0915

-02-09

01 °

0*!9

UL-

)

6

9

12

15

suppression (percent of lac+ control) FIG. 1. Correlation between antisuppression ratios and allele suppressibility .

VOL. 170, 1988

RELEASE FACTOR EFFECTS AT UAA CODONS IN E. COLI

4717

donor strains. This work was supported by Public Health Service grant GM13626-22 from the National Institutes of Health and by grant NP-2791 from the American Cancer Society.

164:59-71. 7. Miller, J. H., C. Coulondre, and P. J. Farabaugh. 1978. Correlation of nonsense sites in lacd gene with specific codons in the nucleotide sequence. Nature (London) 274:770-775. 8. Raftery, L. A., J. B. Egan, S. W. Cline, and M. Yarus. 1984. Defined set of cloned termination suppressors: in vitro activity of isogenetic UAG, UAA, and UGA suppressor tRNAs. J.

LITERATURE CITED Bossi, L. 1983. Context effects: translation of UAG codon by suppression tRNA is affected by the sequence following UAG in the message. J. Mol. Biol. 164:73-87. Bossi, L., and J. R, Roth. 1980. The influence of codon context on genetic code translation. Nature (London) 286:123-127. Bouadloun, F., T. Srichaiyo, L. A. Isaksson, and G. R. Bjork. 1986. Influence of modification next to the anticodon in tRNA on codon context sensitivity of translational suppression and accuracy. J. Bacteriol. 166:1022-1027. Edelmann, P., R. Martin, and J. Gallant. 1987. Nonsense suppression context effects in Escherichia coli bacteriophage T4. Mol. Gen. Genet. 207:517-518. Fluck, M. M., W. Salser, and R. H. Epstein. 1977. The influence of reading context on the suppression of nonsense codons. Mol. Gen. Genet. 151:137-149. Miller, J. H., and A. M. Albertini. 1983. Effects of surrounding sequence on the suppression of nonsense codon. J. Mol. Biol.

9. Ryden, M., and L. A. Isaksson. 1984. A temperature sensitive mutant of Escherichia coli that shows enhanced misreading of UAG/A and enhanced efficiency for some tRNA nonsense suppressors. Mol. Gen. Genet. 193:38-45. 10. Ryden, M., J. Murphy, R. Martin, L. Isaksson, and J. Gallant. 1986. Mapping and complementation studies of the gene for release factor 1. J. Bacteriol. 168:1066-1069. 11. Stormo, G. D., T. D. Schneider, and L. Gold. 1986. Quantitative analysis of the relationship between nucleotide sequence and functional activity. Nucleic Acids Res. 14:6661-6679. 12. Weiss, R. B., J. P. Murphy, and J. A. Gallant. 1984. Genetic screen for cloned release factor genes. J. Bacteriol. 158:362364. 13. Yarus, M., and R. C. Thompson. 1983. Precision of protein biosynthesis, p. 23-64. In J. Beckwith, J. Davies, and J. Gallant (ed.), Gene function in prokaryotes. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.

ACKNOWLEDGMENTS We are grateful to Jeffrey Miller for providing us with the F' lacIZ

1.

2. 3.

4.

5.

6.

Bacteriol. 158:849-859.