©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Function of Polypeptide Chain Release Factor RF-3 in Escherichia coli
RF-3 ACTION IN TERMINATION IS PREDOMINANTLY AT UGA-CONTAINING STOP SIGNALS (*)

Guido Grentzmann , Dominique Brechemier-Baey , Valerie Heurgué-Hamard , Richard H. Buckingham (§)

From the (1) Unité de Recherche Associée 1139 du CNRS, Institut de Biologie Physico-Chimique, 75005 Paris, France

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Two protein release factors (RFs) showing codon specificity, RF-1 (UAG, UAA) and RF-2 (UAA, UGA), are required for polypeptide chain termination in Escherichia coli. We recently reported the localization and characterization of the gene encoding RF-3 ( prfC), a third protein component previously described as stimulating termination without codon specificity. RF-3 is a GTP-binding protein that displays much sequence similarity to elongation factor EF-G. In a termination assay in vitro, RF-3 lowers the Kfor terminator trinucleotides and is thought to act in termination signal recognition. The gene prfC was identified by transposon insertion mutagenesis leading to enhanced nonsense suppression of UGA. We report here that (i) RF-3 inactivation significantly enhances the suppression of termination in vivo only at UGA-dependent stop signals; (ii) the codon-dependent contribution to the stimulation of fMet release in vitro by RF-3 is significantly greater with UGA termination triplet than UAG termination triplet; (iii) RF-3 increases dramatically the affinity of RF-2 to the UGA termination complex in vitro but not that of RF-1 to the UAG termination complex; (iv) RF-3 inactivation leads to a positive feedback on the autoregulation of RF-2 synthesis in vivo, dependent on the competition between frameshifting and termination. These findings are discussed in terms of the mechanism of involvement of RF-3 in translation termination.


INTRODUCTION

Termination of protein synthesis is induced by soluble release factors (RFs).() In Escherichia coli, two codon-specific polypeptide chain release factors, RF-1 (UAG, UAA) and RF-2 (UGA, UAA), have been shown to recognize messenger-encoded stop signals, to induce stop codon trinucleotide binding to ribosomes, and to lead to peptidyl-tRNA hydrolysis in a termination assay in vitro (Ganoza, 1966; Capecchi, 1967; Scolnick et al., 1968). The ribosomal binding sites of the two factors overlap but are not identical (Tate et al., 1990a, 1990b). The ribosomal protein L11 is implicated in RF binding and is required for the activity of RF-1 in vitro; paradoxically, however, the absence of L11 increases the activity of RF-2 (McCaughan et al., 1984).

Several reports indicate that the interaction of RF-1 and RF-2 with the ribosome are stimulated in vitro by a third soluble protein factor, RF-3 (Scolnick and Caskey, 1969; Goldstein et al., 1970; Goldstein and Caskey, 1970). At low stop codon trinucleotide concentrations, significant stimulation by RF-3 of fMet release in vitro has been demonstrated for RF-2-dependent termination in response to UAA or UGA (Goldstein et al., 1970) and for RF-1-dependent termination in the presence of UAA (Goldstein and Caskey, 1970). To our knowledge, no detailed kinetic data concerning the amplification by RF-3 of RF-1/UAG-dependent termination have been reported. RF-3 stimulates the formation of RF-1 or RF-2[H]UAAribosome intermediates (Goldstein and Caskey, 1970). This led to the suggestion that RF-3 might act in terminator codon recognition and binding of RF-1 or RF-2 to ribosomes. RF-3 contains a consensus sequence for a GTP binding site and binds GTP in vitro. The stimulation by RF-3 of termination at low stop codon concentrations is inhibited by the addition of GTP or GDP (but not by GMP), and RFUAAribosome intermediates are actively dissociated by these nucleotides. Thus, it is possible that RF-3 may play a role in the dissociation of RFmRNAribosome intermediates that involves an interaction with guanine nucleotides (Tate and Caskey, 1974; Caskey, 1977).

The properties of RF-3 in vitro suggested that inactivation of the gene encoding the factor might not be lethal but should enhance the activity of tRNA nonsense suppressors by diminishing the efficiency of polypeptide chain termination. Transposon insertion mutants of the locus have indeed been isolated and lead to enhanced suppression of UGA (Grentzmann et al., 1994; Mikuni et al., 1994). These mutants show that RF-3 is not essential to cell viability but that the factor nevertheless plays an active role in translation termination.

We describe here in more detail the effects in vivo of inactivating prfC and further characterization of RF-3 activity in vitro. These data run counter to previous conclusions concerning the lack of stop codon specificity in RF-3 action. The results with one system we have employed to study termination, that employs the autoregulatory sequence from the prfB gene, suggest that prfC inactivation should stimulate RF-2 synthesis. These findings are discussed in terms of the role of RF-3 in translation termination.


MATERIALS AND METHODS

Bacteria, Plasmids, and Episomes

Details of strains and plasmids are given in . F` lacI-Z donor strains with O12 and O17 were generously provided by Dr. M. Springer (IBPC, Paris), those with A6 and A10 by Dr. L. Bossi (CGM, Gif-sur-Yvette, France), those with U4 and U6 by Dr. Monica Rydén-Aulin (Stockholm University), and those with O1, O16, O19, O21, and O28 by Dr. Robin Martin (University of Leeds, United Kingdom). Details may be found in Bossi (1983), Miller and Albertini (1983), and Martin et al. (1988). RF-3-inactivated versions of strains are not systematically indicated in Tables I or II, but were constructed by phage P1 transduction of the Kan transposon in strain GG3. Plasmids pJC212, pJC216, and pJC27 were received from Dr. James F. Curran (Wake Forest University, Winston-Salem, NC) and pACYC279 from Dr. Robin Martin.

Assay for Termination Factor Activity

RF-3 amplification of fMet release was determined in 50-µl reaction mixes incubated for 20 min with different concentrations of RF-1 and RF-2 in 50 mM Tris acetate, pH 7.2, 75 mM ammonium acetate, 30 mM magnesium acetate, and 2.5 nmol of trinucleotide (Caskey et al., 1971b). Release reactions dependent on amplification by RF-3 at low trinucleotide concentration were performed for 8 min in 50 mM Tris acetate, pH 7.2, 50 mM potassium acetate, and 30 mM magnesium acetate (Goldstein et al., 1970). All reactions were incubated at 30 °C and contained, in 50 µl, 4-6 pmol of f[S]Met-tRNAAUGribosome complex. Background release was about 5% of the total signal. RF-1 and RF-2 were purified by high performance liquid chromatography as described by Tate and Caskey (1990). The purification of RF-3 has been described previously (Grentzmann et al., 1994); if necessary, RF-3 fractions were concentrated by ultrafiltration using Centricon 10 cones (Amicon, Inc.).

Recombinant DNA and Genetic Manipulations

General procedures for DNA recombinant techniques, plasmid extraction, etc., were performed as described by Sambrook et al. (1989), P1 lysates, transductions, conjugations of F` episomes, and -galactosidase assays as described by Miller (1992). Tryptophan synthetase assays were performed as described by Sörensen et al. (1990). For -galactosidase measurements, cells were grown in glucose minimal A medium or LB medium (Miller, 1992).


RESULTS

Suppression of Termination in Vivo by prfC Interruption

prfC has been isolated as an UGA (RF-2-dependent) antisuppressor (Grentzmann et al., 1994). In order to study the phenotype of prfC mutants with respect to UAG (RF-1-dependent) termination, a P1 lysate on the prfC-interrupted strain GG3 was used to transduce a prfCthr287::Tn 10kan insertion into suppressed trpA (UGA234) or trpA (UAG234) mutant strains, selecting for growth on kanamycin. The trpA gene encodes the -subunit of tryptophan synthetase and normally encodes a glycine residue in position 234 of the polypeptide. Only glycine and to a lesser extent alanine at this position allow -subunit activity (Murgola, 1985). Nonsense suppression leading to active tryptophan synthetase in the UGA mutant may be obtained by glycine-inserting tRNA nonsense suppressors. Termination suppression was measured in these strains in two ways: (i) by effects on cell growth after replica plating to glucose minimal medium (Tucker et al., 1989) and (ii) by the determination of tryptophan synthetase activity in cell extracts from the suppressed trpA strains (Sörensen et al., 1990). The constructions using UGA nonsense mutations in different contexts at position 234 in the trpA gene showed uniformly enhanced growth in the prfC strains, indicating increased UGA suppression (). However, no such increase was apparent in the UAG nonsense strains, in the presence of either glyU- or glyT-derived suppressors. The specific enhancement in UGA and UAG nonsense strains was confirmed by measurement of tryptophan synthetase activity in extracts from strains containing either glyT(SuUGA/G) or glyT(SuUAA/G) nonsense suppressors ().

To obtain further quantitative confirmation of the effect in vivo of RF-3 inactivation on termination at each of the three stop codons in various stop codon contexts, we made use of the set of nonsense mutants in lacI-Z fusions located on F` episomes which has been employed for detailed study of context effects on nonsense suppression by Miller and Albertini (1983), Bossi (1983), and Martin et al. (1988). The wide variety of nonsense mutants available in these fusions allowed us to construct combinations of nonsense mutations in stringent or leaky contexts with strongly or weakly suppressing tRNAs. Measurements of -galactosidase were made on cultures of unsuppressed or suppressed lacI-Z nonsense mutants or the wild type fusion containing no in-frame stop signal.

Interruption of prfC resulted in a 3- to 5-fold enhancement of the suppression of UGA stop signals in all the contexts selected in the presence of a glyT-derived UGA suppressor (I, lines 3-5). The leaky UGA mutants at positions 189 and 220 in the LacI-Z fusion protein were also examined in the absence of known tRNA nonsense suppressors or in the presence of a UAG-specific tRNA that should not translate UGA codons. The level of UGA read-through was also found to be enhanced about 4-fold in prfC strains (I, lines 1, 2, and 5). In clear contrast, prfC inactivation had no significant enhancing effect on the suppression of UAA or UAG stop signals due to any of three tRNA suppressors (I, lines 8-20) or on UAG stop codon read-through in the leaky context at position 26 of the fusion protein (I, lines 6 and 7). Similar results were obtained with cultures grown in rich medium (data not shown). These observations using lacI-Z fusions are thus in good agreement with the initial data obtained with trpA nonsense mutants, indicating a specific effect of RF-3 on UGA-containing stop signals.

In Vitro Stimulation of fMet Release at Low Trinucleotide Concentrations

The data previously reported concerning stimulation by RF-3 of UAA-mediated release in vitro do not suggest any major differences between RF-1- (Goldstein and Caskey, 1970) and RF-2-dependent release (Goldstein et al., 1970). Furthermore, RF-3 has a very similar stimulatory effect on RFUAAribosome complex formation, irrespective of whether the RF used is RF-1 or RF-2 (Scolnick and Caskey, 1969, 1970). In order to determine whether a parallel to our observations in vivo could be demonstrated in vitro, we compared the stimulatory effect of RF-3 on UGA- and UAG-mediated termination reactions in vitro.

Under the conditions introduced by Goldstein et al. (1970) and, in particular, at low stop codon concentrations (10 M), we observe that large amounts of RF-3 (6 units as defined by Grentzmann et al. (1994)) provoke uncoupling of RF-1-mediated termination from the presence of UAG trinucleotide (Fig. 1 A). Release is amplified by a factor of 2 to 2.5 by the addition of 1 unit of RF-3. Thus, addition of RF-3 did stimulate fMet release in the presence of RF-1, but the effect was largely independent of the UAG triplet.


Figure 1:A and B, RF-3 stimulation of f[S]Met release dependent on trinucleotide (UAG in A or UGA in B) and RF-3 concentrations. Each reaction was incubated at 30 °C for 8 min and contained in 50 µl: 0.2 microunit of RF-1 or RF-2 (Goldstein et al., 1970), trinucleotides as indicated, and additional components as described under ``Materials and Methods.'' f[S]Met extracted at zero time (5% of total) is subtracted from each value. The data in the absence of termination trinucleotides ( front columns) are shown as lines on the other columns to indicate the trinucleotide-independent contribution to the overall fMet release.



Under the same conditions but with UGA and RF-2, release remains much more dependent on the trinucleotide, and UGA-dependent release is about 6-fold stimulated by the addition of 1 unit of RF-3 (Fig. 1 B), in agreement with previously published results (Goldstein et al., 1970). RF-3 amplification of UAA-dependent termination with RF-1 and RF-2 has been shown to lie in the range of 6- to 10-fold (Goldstein and Caskey, 1970; Goldstein et al., 1970). As previously reported, GTP abolished release stimulation by RF-3 under these conditions (data not shown).

Stimulation of fMet Release at Low Concentration of Release Factors RF-1 or RF-2

If RF-3 plays a role in termination, it should be expected to recognize the complex of release factors RF-1 or RF-2 with the ribosome. Furthermore, if RF-3 activity depends on its binding to the RFstop codonribosome termination complex, it is not surprising that the factor stabilizes this complex. Indeed, it has been shown by measuring [H]UAA incorporation that RF-3 can stimulate the formation of a RFUAAribosome intermediate (Goldstein and Caskey, 1970). Stimulation of fMet release at low stop codon concentration (Goldstein et al., 1970) can be considered as an indirect way of observing the stabilization of the termination complex by RF-3 and is a poor model for polypeptide chain termination. Since stop codons are part of the mRNA and are positioned by the movement of the messenger during the passage through the ribosome during translation, natural termination should be a reaction of zero order with respect to the stop codon. Differences in the stimulation of termination by RF-3 for different stop triplets at low concentrations might therefore reflect characteristics which are not relevant to the natural termination process. We therefore looked at in vitro stimulatory effects of RF-3 at near-saturating concentrations of trinucleotide stop codons (Caskey et al., 1971a), varying the concentration of RF-1 or RF-2. Under these conditions, a far more striking distinction is seen between the effects of RF-3 on RF-1/UAG-dependent release as compared to RF-2/UGA-dependent release.

In the absence of RF-3, relatively little difference is seen between RF-1- or RF-2-dependent release, although release as a function of concentration is notably nonlinear at low concentrations of factor in the case of RF-1 (Fig. 2). At higher RF concentrations, RF-2/UGA release is rather less efficient than RF-1/UAG release. This result is consistent with data of Scolnick and Caskey (1969) on codon-specific RF-1 and RF-2 binding to ribosomes, which indicate that UGA binding to RF-2 is less tight than that of UAA binding to RF-2 or that of either UAA or UAG binding to RF-1.


Figure 2: f[S]Met release dependent on RF-1 and UAG or RF-2 and UGA in the absence ( open symbols) or presence ( filled symbols) of 1 of unit RF-3 (units defined by Grentzmann et al. (1994)). Each reaction is incubated at 30 °C for 20 min and contains, in 50 µl: 5 pmol of f[S]Met-tRNAAUGribosome complex, the indicated quantity of RF-1 or RF-2 in units according to Caskey et al. (1971a), and other components as indicated under ``Materials and Methods.'' The amount of f[S]Met present at zero time (5% of total) was subtracted from each value.



Addition of RF-3 led to a striking increase in UGA-dependent release, which became almost complete at release factor concentrations where UAG-specific release was barely observable. It is likely that these results reflect the difference in RF-3 termination activation that we observe in vivo. The modest amplification of around 2-fold or less, observed with UAG (Fig. 2), which corresponds well to previously published observations for RF-1 and RF-2 using UAA at near-saturating concentration (Milman et al., 1969; Goldstein and Caskey, 1970; McCaughan et al., 1984), may be explained by a general stabilizing effect of RF-3 on the termination complex. Effect of the prfCMutation on the RF-2 Translational Autoregulatory System-Apart from their function in termination, stop codons sometimes form part of recoding sites in mRNA that permit alternative responses to termination (Gesteland et al., 1992; Tate and Brown, 1992). The presence of the stop codon results in a pause in chain elongation, and the probability of a recoding event, such as frameshifting, is dependent on the duration of the pause, as well as on other elements of the recoding site. The recoding event is thus in competition with the termination process and depends on the efficiency of RF action.

The prfB gene encoding RF-2 contains an in-frame UGA codon early in its coding sequence and requires a +1 frameshift at this codon for RF-2 synthesis. This constitutes an autoregulatory system for prfB expression (Craigen et al., 1985); at adequate concentration, RF-2 can terminate its own synthesis by acting at the intragenic UGA codon. In contrast, at low concentrations of RF-2, the UGA codon may not be read as a terminator and a +1 frameshift allows translation to continue. Fusions incorporating the prfB frameshift site into the 5` region of lacZ have been used to study the competition between suppression, frameshifting, and termination (Curran and Yarus, 1988). Since this system is sensitive to factors that alter termination efficiency, we have measured the effect of interrupting prfC on -galactosidase production from fusions containing the wild type regulatory sequence from prfB, and a second fusion in which the UGA codon is replaced by UAG (Curran and Yarus, 1988).

prfC mutants and wild type cells were transformed with plasmids containing the RF-2- lacZ fusions: pJC212 (UGA codon) and pJC216 (UAG codon), comporting 23 nucleotides related to the frameshift site of RF-2, or the control plasmid pJC27 which does not require a frameshift for -galactosidase synthesis. The prfC mutation decreases the termination probability by a factor of about 3, whereas a much less significant decrease, by a factor of 1.3, was observed for the construct containing UAG ().


DISCUSSION

The hydrolytic activity required for release of the polypeptide chain from peptidyl-tRNA during translation termination is believed to be an integral part of the 50 S ribosomal subunit (for a review see Caskey (1977)). Release of ribosome-bound peptide in vitro requires RF-1 or RF-2 and a stop signal on the messenger, which can be replaced by stop codon trinucleotides. This reaction in vitro does not require RF-3, nor does peptidyl-tRNA hydrolysis in vivo, since prfC is a gene that is not essential for cell growth (Grentzmann et al., 1994; Mikuni et al., 1994). Previous work has described RF-3 as a factor stimulating termination but showing no stop codon specificity. From the experiments presented here, we conclude that the effect of RF-3 varies greatly according to the nature of the stop codon, both in vivo and under appropriate conditions in vitro, and is largely confined to termination involving UGA codons.

In the work described here, the effect of RF-3 on the efficiency of RF-2-dependent termination at stop signals containing UGA codons has been demonstrated in several ways in vivo. The suppression of nonsense mutants was measured both in the trpA gene and in lacI-Z fusions in the presence of tRNA nonsense suppressors. Read-through was measured in the case of leaky UGA nonsense mutations in lacI-Z. Finally, competition between frameshifting and termination at the RF-2 frameshift site was employed to estimate changes in termination efficiency. In each case, changes in suppression, read-through, or frameshifting are observed that correspond to a decrease of severalfold in the efficiency of RF-2-dependent termination when prfC is inactivated. These observations are consistent with the previous identification of prfC as a UGA antisuppressor, following earlier characterization of RF-3 in vitro. Furthermore, it is probable that our measurements underestimate the influence of RF-3 on RF-2 function in vivo, since no account is taken of the phenomenon of autocontrol in prfB expression. Since RF-2 synthesis is negatively controlled by the efficiency of termination at the frameshift site in prfB, the inactivation of prfC should result in partial compensation for the decreased termination efficiency by increased RF-2 synthesis.

In contrast, very little influence is seen on termination in vivo at UAG or UAA codons by any of the approaches we have used, in spite of the fact that several such codons in different messenger contexts were studied. This is an unexpected observation in view of previous conclusions, based largely on the study of termination in vitro, that RF-3 acts nonspecifically on termination at all three nonsense codons. Termination efficiency depends on stop codon context, a fact reflected by distinct context preferences in genes (Brown et al., 1990; Buckingham, 1994). UAA is decoded by both RF-1 and RF-2 and is the most frequently used stop codon in E. coli. RF-1 is thought to be the preferred factor at UAA codons efficiently suppressed by tRNA nonsense suppressors whereas RF-2 is favored at poorly suppressed signals, those preferred in highly expressed genes (Martin et al., 1988). In our experiments, UAA suppression was poorly enhanced by the inactivation of prfC irrespective of whether the context of the stop codon favored suppression.

Several tests have previously been applied in vitro to study RF-3 function. Thus, the release of fMet from initiation complexes has been studied under limiting conditions of either the stop triplet or RF; other tests have measured the incorporation of radioactive stop triplets or radioactive RF into a termination complex. Termination in vitro can be rendered largely dependent on RF-3 by reducing 10- to 20-fold the concentration of stop codon trinucleotide. Under these conditions, we observe that RF-3 enhances UGA-dependent termination more than UAG-dependent termination (about 6-fold as opposed to twice). Similar experiments with UAA show an even more striking amplification by RF-3, of about 10-fold, irrespective of whether RF-1 or RF-2 is used (Scolnick and Caskey, 1969, 1970). However, since natural termination is not limited by stop codon concentration, and since these observations diverge from what we observe in vivo, we consider that they probably reflect characteristics of the binding of different trinucleotides to the ribosomeRF-1 (or RF-2) complex that are not relevant in vivo.

A 2-fold amplification by RF-3 of termination in vitro under nearly saturating conditions of UAA trinucleotide, with varying concentrations of RF-1 or RF-2, has been previously reported (Caskey et al., 1971a). Since these conditions correspond more closely to natural termination, we applied a similar assay to UAG- and UGA-dependent termination. The amplification with UAG was approximately the same as reported for UAA. In contrast, UGA-dependent termination was amplified more than 10-fold, consistent with the difference we observe between UGA and the two other stop codons in vivo.

The conclusions we report here are not intended to imply that RF-3 does not interact in vivo with termination complexes involving UAG or UAA, but that the interaction affects peptide release rather little in comparison to release at UGA codons, at least under the growth conditions corresponding to those of our experiments. The greater effects of RF-3 in termination at UGA is probably related to a lesser stability of termination complexes involving UGA compared to others (Scolnick and Caskey, 1969). Furthermore, some observations strongly suggest that RF-3 can interact with RF-1 in vivo. Thus, Mikuni et al. (1994) have reported that some mutations affecting prfC can suppress the temperature sensitivity of a prfA mutant. This indicates that RF-3 can indeed stabilize termination complexes involving RF-1, although it may generally contribute little to termination efficiency, at least in the case of termination complexes of normal stability, involving wild type RF1. A second observation, initially difficult to reconcile with the findings we report here, concerns the prfC-mediated suppression of a UAA mutation affecting miaA, the first enzyme in the pathway leading to synthesis of msiA, a hypermodified base present in many tRNAs. This suppressor, named miaD, probably represents the first reported mutation affecting prfC (Connolly and Winkler, 1991). However, the UAA mutant (originally designated trpX) of the miaA gene that is suppressed by miaD is quite atypical of UAA codons in that it is highly leaky, and the mutant strain normally permits about 10% hypermodification of tRNA substrates. Secondly, the system employed by Connolly and Winkler (1991) to isolate miaD, making use of a miaA, trpT(SuUGA/G) lacZ(UGA) strain, is sensitive to the absence of RF-3 independently of whether UAA suppression is enhanced or not.

The effects of RF-3 in vitro at low stop codon concentration (the activation of fMet-tRNA release and stop trinucleotide binding to ribosomes) are abolished by adding GTP at a concentration of 10 M. This has led to the suggestion that RF-3 has a role in the dissociation of the termination complex following peptidyl-tRNA hydrolysis (Caskey, 1977). RF-3 possesses a GTP binding site in the N-terminal part of the protein (Grentzmann et al., 1994; Mikuni et al., 1994). Three other factors in E. coli necessary for protein synthesis are GTPases: the initiation factor IF-2 and the two elongation factors EF-Tu and EF-G. In each case, it is thought that GTP hydrolysis is associated with a conformational change in the protein that weakens its affinity for the ribosome and accelerates release.

The small effect on cell growth of inactivating prfC (Grentzmann et al., 1994; Mikuni et al., 1994) raises the question as to whether the role fulfilled by RF-3 is itself inessential and rather one of optimizing a cellular process affecting termination, or whether the role is essential but can be fulfilled by another protein in the absence of RF-3. A possible candidate for a protein able to functionally replace RF-3 is the elongation factor EF-G, in view of the extensive sequence similarities between the proteins (Grentzmann et al., 1994; Mikuni et al., 1994), although other GTPases of unknown function are present in the cell. Although we have little reason at present to doubt that the stimulation of RF-2 function could adequately explain the presence of RF-3 in the cell, it is reasonable to speculate about alternative functions for the protein that might nevertheless be reflected in termination activity. One possibility concerns the decomposition of the messenger-ribosome complex following peptidyl release from the ribosome. Another protein factor is believed to be necessary in order to complete the process of polypeptide chain termination. Ribosomal release factor (RRF) releases the 70 S ribosome from the messenger RNA after peptide release has occurred. In an in vitro translation system in the absence of RRF, ribosomes reinitiate translation immediately after the stop codon, rather than dissociating from the ribosome (Ryoji et al., 1981). In vitro reactions using puromycin, EF-G, GTP, and RRF converted polysomes to single ribosomes. The need for EF-G and GTP for this activity in vitro suggests that some step resembling elongation is necessary for ribosome dissociation from mRNA (Hirashima and Kaji, 1972). It cannot be excluded that RF-3 is used preferentially, when present, in such a termination-associated elongation step in vivo. The possible involvement of RF-3 in the dissociation of the mRNA-ribosome complex is open to experimental study.

  
Table: Bacterial strains and plasmids used in this work


  
Table: Efficiency of suppression of trpA nonsense mutants

Suppression efficiency is estimated by replica plating to glucose minimal medium (Tucker et al., 1989) or calculated from measurements of -catalytic activity and -catalytic activity tryptophan synthetase-specific activities in cell extracts, performed as described by Sorensen et al. (1990), and is equal to the ratio (-catalytic activity/-catalytic activity) for each strain, expressed as a percentage of the same ratio for an isogenic trpA strain. DBB strains are described in Table I.


  
Table: Effect of prfC inactivation on stop codon read-through and suppression

The -galactosidase activity produced by different lacI-Z fusions in the absence or presence of different suppressors was determined as described under ``Materials and Methods.'' These values are expressed as a percentage of the activity of the parental lacI-Z fusion assayed in the same genetic background. The Su strains were IB188, the trpT-derived suppressor (lines 16-20) was carried by plasmid pACYC279 (Martin et al., 1988), and other Su strains can be identified by reference to Table I.


  
Table: Effect of prfC inactivation on -galactosidase production dependent on frameshifting at a UGA or UAG stop codon

-Galactosidase activities were determined as described under ``Materials and Methods.'' The host strain was IB188. The insert in plasmid pJC212 encodes the RF-2/ lacZ fusion protein containing UGA and pJC216 the fusion containing UAG, the control plasmid pJC27 encodes a pseudowild type lacZ allele and serves as a control not requiring a frameshift for -galactosidase activity. Relative activity = activity as a percentage of that with pJC27. The ratio of the rate constants for termination and frameshifting (termination/shift) is calculated from the relative activity as follows: termination/shift = 1/(relative activity) - 1.



FOOTNOTES

*
This research was supported by Grant URA1139 from the CNRS, and grants from l'Association pour la Recherche sur le Cancer, the Fondation pour la Recherche Medicale, France, E.I. du Pont de Nemours and Co., and the Human Capital and Mobility Programme of the European Community. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed. Tel.: 33-1-4354-1972; Fax: 33-1-4046-8331.

The abbreviations used are: RF, release factor; RRF, ribosomal release factor; EF, elongation factor.


ACKNOWLEDGEMENTS

We thank Drs. Anne-Lise Haenni and Jane MacDougall for helpful comments on the manuscript.


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