From the
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 K
Termination of protein synthesis is induced by soluble release
factors (RFs).
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
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.
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
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
Under the conditions introduced by
Goldstein et al. (1970) and, in particular, at low stop codon
concentrations (10
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.
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
prfC
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 ribosome
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
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
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.
Suppression efficiency is
estimated by replica plating to glucose minimal medium (Tucker et
al., 1989) or calculated from measurements of
The
We thank Drs. Anne-Lise Haenni and Jane MacDougall for
helpful comments on the manuscript.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
for 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.
(
)
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).
[
H]UAA
ribosome 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 RF
UAA
ribosome intermediates are
actively dissociated by these nucleotides. Thus, it is possible that
RF-3 may play a role in the dissociation of RF
mRNA
ribosome
intermediates that involves an interaction with guanine nucleotides
(Tate and Caskey, 1974; Caskey, 1977).
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-tRNA
AUG
ribosome
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).
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 ().
-galactosidase were made
on cultures of unsuppressed or suppressed lacI-Z nonsense
mutants or the wild type fusion containing no in-frame stop signal.
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
RFUAA
ribosome 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.
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 codon
ribosome
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 RF
UAA
ribosome 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.
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-tRNA
AUG
ribosome 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.
-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).
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 ().
RF-1 (or RF-2) complex that are not
relevant in vivo.
-mediated suppression of a UAA mutation
affecting miaA, the first enzyme in the pathway leading to
synthesis of ms
i
A, 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.
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.
Table:
Bacterial strains and plasmids used in this work
Table:
Efficiency of
suppression of trpA nonsense mutants
-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
-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.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.