(Received for publication, December 4, 1996, and in revised form, January 30, 1997)
From the We have established a new in vitro
assay for translational termination. It consists of 70 S ribosomes
bound to a synthetic RNA minimessenger via interaction with P-site
binding
fMet-tRNAfMet. If the
A-site codon is a stop signal, release activity can be measured by
quantifying hydrolyzed formylmethionine. Characteristics of this assay
in terms of reaction time, ion concentration, release factor RF1 and
RF2 concentration, and competition with A-site-decoding tRNA are
discussed. The new assay shows that polypeptide chain release activity
is directly dependent on the presence of a stop codon in the ribosomal
A-site.
Termination of protein synthesis happens through stop codon
recognition by polypeptide chain release factors RF1 or RF2 (1, 2). The
factors are codon-specific, RF1 recognizing UAG and UAA and RF2
recognizing UAA and UGA (3). A third factor, RF3, has been shown to
amplify RF1 and RF2 termination (4-6) although interpretations differ
as to whether this is a general effect (7, 8) or whether it might be
specific to less stable termination complexes (9, 10). Prokaryotic and
eukaryotic release factors have been identified by using a simplified
assay. Charged fMet-tRNAfMet is bound to
the ribosomal P-site via an AUG triplet.
fMet-tRNAfMet·AUG·ribosome complexes
are then exposed to release factors RF1 or RF2 in the presence of free
stop triplets (11). Ribosomal peptidyltransferase catalyzes hydrolysis
of the fMet pseudopeptide from P-site fMet-tRNA (12). The mechanism of
termination induction by stop codon recognition is still unknown
(13).
In first termination models, release factors RF1 and RF2 were believed
to recognize stop codons in a tRNA-like manner (12). The idea was
supported by release factors and suppressor tRNAs competing in
recognition of stop codons (14). Studies by immunomicroscopy (15)
questioned the idea of release factors entering the ribosomal A-site
like a tRNA. New models proposed stop signal recognition by base
pairing with 16 S ribosomal RNA (13, 16, 17).
Cross-linking experiments between a minimessenger and RF2 (17) on the
ribosome showed that, at least for stop signal recognition by release
factors, the stop codon needs to be exposed at the ribosomal A-site.
The authors pointed out that there is no evidence for a conformational
change when the termination complex forms. Also, functional analysis of
the release reaction points to a tRNA-like action of release factors in
termination. Early biochemical studies showed that tRNA can induce
release activity of peptidyltransferase in acetone (18). Stop codon
occupation of the A-site was not necessary. In ethanol, an excess of
bulk tRNA induced fMet-ethyl ester formation. Furthermore, the study of
the functional sites of release factors (19, 20) indicates that RF1 and
RF2 might have a tRNA-like shape, spanning the codon recognition site
and peptidyltransferase (21). Two studies have led to the proposal of a
specific interaction between the terminal tRNA in the P-site and
release factor RF1 (22) or RF2 (23), as if the release factor is
localized near the P-site tRNA.
Recent publication of the structure of the ternary complex between
EF-Tu·GTP·tRNA1 (24) and its striking
similarity to the structure of elongation factor EF-G (25, 26) showed
the first example of a protein domain (EF-G domains 3, 4, and 5)
mimicking an RNA (tRNA moiety of the ternary complex). A
tRNA-like shape for release factors has repeatedly been proposed
(21, 24, 27).
However, no one has yet reported a defined in vitro release
assay that is dependent on stop codons in the ribosomal A-site. In
1983, Tate et al. (28) reported termination by release
factor in the presence of a sense codon-occupied A-site, using free
stop triplet. In 1987, Buckingham et al. (29) presented
results with an AUGUAA mini-mRNA. No release induction by release
factors could be observed, and again, free stop triplet activated the
reaction. These results brought up the question of whether a first step in stop codon recognition might occur before entry of this codon into
the ribosomal A-site (13).
Here we show release induction by AUGUAA and other chemically
synthesized minimessengers. In all cases, stop codons were localized to
the ribosomal A-site when binding the minimessage by
fMet-tRNAfMet via an AUG codon in the
P-site. Release induction of the different complexes by release factors
RF1 or RF2 shows that exposure of a stop codon in the ribosomal A-site
is necessary and sufficient for polypeptide chain termination. In
several cases we observed partial release activation by free stop
triplet, as previously reported (28, 29). We find about 10 times higher
activity of release factors RF1 and RF2 when a stop codon in the
minimessage is exposed at the P-site compared with the use of a free
stop triplet. The presence of a sense codon in the A-site inhibits release induction by free stop triplet. Release factor codon
specificity and discrimination of out-of-frame stop signals are
dependent on reaction time, competition with A-site-binding
aminoacyl-tRNA, and ion concentration.
RNA oligonucleotides were synthesized on an ABI synthesizer.
Protection groups were eliminated by gel filtration on Sephadex G-25
(Pharmacia Biotech Inc.). Purity and sequence were controlled by mass
spectroscopy (30). Release factors have been purified from wild type
strains (31) or from overexpressed strains (32). Tight couple ribosomes
were prepared (33). fMet-tRNAfMet
(Subriden) was charged and formylated as described (31). Ribosomal binding of
f-[35S]Met-tRNAfMet fixes
the AUG codon of a given RNA oligonucleotide to the P-site of the
ribosome (34). Ribosomes and tRNA were kept in an equimolar ratio (50 pmol/50 µl of complex mix). Free AUG triplets or AUG codon-containing
minimessengers were used at 2.5 nmol in 50 µl of complex mix.
0.5 pmol of
fMet-tRNAfMet·messenger·ribosome
complex were incubated together with release factors in the presence or
absence of free stop triplet (ionic conditions are stated for each
series individually). Hydrolyzed formylmethionine was extracted with ethyl acetate at pH 1. Maximal release under these conditions was
between 50 and 70% of the total f-[35S]Met present in
the reaction mix. Assays were repeated two to five times with
independently prepared complexes, and the results were averaged.
Amounts of free fMet at zero time of each reaction series (3-8%) were
subtracted.
Release Activity with Minimessengers Is Dependent on Stop Signals
Located at the Ribosomal A-site (Table I)
Different RNA oligonucleotides were fixed by charged
f-[35S]Met-tRNAfMet to 70 S ribosomes. The complexes were incubated at different concentrations
of release factors RF1 or RF2 and in the absence or presence of free
stop triplet. In complexes where the A-site was not occupied by a stop
codon, fMet release was dependent on free stop triplet addition and
high release factor concentration (lines 1 and 2). Oligonucleotides
presenting a stop codon to the A-site allowed termination in the
presence of RF1 or RF2 at relatively low concentration and independent
of free stop triplet (lines 7-9). Addition of 5 Table I.
Stop signal recognition on minimessengers
Eccles Institute of Human Genetics,
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-UUC to a given
messenger, presumably in the ribosomal E-site, did not change the
release efficiency to a significant extent (lines 8 and 9). This was
not changed in the presence of excess deacyl-tRNAPhe (data
not shown). Free stop triplet release induction is dependent on the
accessibility of the A-site and is efficiently inhibited when a sense
codon occupies the A-site (lines 3-6) although an increase of release
activity could be observed when a free stop triplet was added (lines 4, 6, 8, and 9), indicating a certain degree of breathing of the system in
the present conditions. When the message was elongated 3
from the
A-site (line 7), free stop triplet slightly inhibited termination. We
conclude that the system is not fully independent of effects due to
free stop triplet in high excess. Release factors might recognize
triplets in competition with the minimessenger before or after having
been bound to the ribosomal complex. In addition, mRNA 3
from the
A-site might sterically inhibit access of free stop triplet to the
A-site.
E P A
RF2
(5 picounits)
RF2 (50 picounits)
RF2 (5 picounits) UGA
RF2
(50 picounits) UGA
RF1 (15 picounits)
RF1 (15 picounits)
UAA
fmol
1) AUG
10
15
40
230
10
135
2) UUCAUG
26
22
45
203
3) AUGUUC
2
8
0
0
10
20
4) AUGUUCUAA
16
15
33
50
10
20
5) AUGUUCUGAGCCCC
0
0
4
8
6) UUCAUGUUUUUCUAA
0
5
0
20
7) UUCAUGUAAGCCCC
224
214
180
169
8) UUCAUGUAA
200
215
250
280
220
9) AUGUAA
179
193
217
280
Specificity of in Vitro Peptide Release Using Minimessengers
To reproduce release factor codon specificity and stop signal frame specificity, minimessages with release factor specific stop codons or bearing incremental insertions between the AUG (Met) codon and the stop codon were synthesized.
Lack of Release Specificity at 30 mM MgCl2, 15 min at 30 °C (Table II)In classical
in vitro termination conditions established for the use of
free stop triplet, our assay was only marginally specific against a +1
frame stop codon (line 3), and no specificity of RF2 against UAG
recognition could be observed, even at a relatively low concentration
of the factor (line 8). +2 or 2 out-of-frame stops were not
recognized (lines 2 and 4). A stop signal that was localized an entire
codon downstream from the A-site (Table II, line 1, and Table I, lines
4 and 5) was also not recognized.
|
Mg2+ has been shown to play a role
in coordinating secondary structures of nucleic acids (e.g.
Refs. 35 and 36) but also in coupling of ribosomal subunits and binding
of nucleic acids (mRNA, tRNA) to ribosomes (33). The optimal
Mg2+ concentration for in vitro termination (30 mM) has been established empirically (37) and is somewhat
high compared with common Mg2+ concentrations (5-10
mM) used in translation in vitro assays (33). 30 mM is optimal for release reactions using free stop triplet, where the reaction is the third order
(fMet-tRNAfMet·AUG·ribosome
intermediate, stop triplet, and release factor have to form a complex
allowing fMet-tRNAfMet hydrolysis). In
our assay, a stop codon is already part of the fMet-tRNAfMet·messenger·ribosome
intermediate. It was therefore reasonable to ask whether a lower
Mg2+ concentration could be sufficient for a reaction of
the second order, thereby allowing efficient discrimination of
non-cognate stop codons. Release factor concentration was kept below
saturation. By diminishing reaction time, we were able to show
differences in activity of release factors for different stop codons.
Fig. 1 shows that indeed stop codon specificity of RF1
and RF2 is directly dependent on incubation time and Mg2+
concentration. At 8 mM [Mg2+], a clear
difference in reaction velocity between specific and nonspecific
reactions appeared. Codon-specific reactions were mostly completed
within the first 30 s. Nevertheless, RF2 showed less codon
specificity than RF1, which showed codon specificity over the entire
range of Mg2+ concentrations tested.
Frame Specificity of Stop Signal Recognition
We then tested
the effect of incubation time on frame specificity in stop codon
recognition (Fig. 2). UUC AUG UAA GCC CC was used as a
positive control for in-frame stop codon recognition. The +1 stop (UUC
AUG UUAA) message, which had turned out to have poor discrimination
(Table II), was tested together with +2 (UUC AUG UUUAA) and 2 (UUC
AUGA) oligonucleotides. Recognition of out-of-frame stops decreased by
half when the reaction time was reduced from 4 min to 30 s.
We wanted to see whether a tRNA recognizing the cognate A-site codon
would be able to compete with a release factor. In that case, the
presence of a cognate tRNA in the A-site should diminish out-of-frame
stop codon recognition by a release factor. It has been shown
previously that it is possible to occupy a ribosomal A-site with a
cognate aminoacyl-tRNA in the absence of EF-Tu when this site is
programmed by a message (38). In Fig. 3 we show that,
under our conditions, release factor recognition of a +1 frame stop
signal on an UUC AUG UUA A is diminished 2-fold when the ribosomal
A-site has previously been occupied by a UUA that decodes
Leu-tRNALeu. This 2-fold inhibition was constant at 30 s and 5 min of incubation time. Use of uncharged tRNALeu
did not result in enhanced out-of-frame stop signal discrimination.
Activity of RF1 and RF2 with Stop Codon-containing Messengers Versus Free Triplets
Our results (Table I) suggested a higher affinity of release
factors for messages containing an A-site stop codon versus complexes dependent on free stop triplet addition. We therefore studied
the kinetics of termination reactions dependent on release factor
concentration, comparing the two systems (Fig. 4).
Conditions of kinetics were the same as for Caskey et al.
(34). As previously shown, at a low concentration of release factors,
fMet release is linear during the first 5 min, and the velocity is
dependent on release factor concentration when using free stop triplet. 30 mM Mg2+ is optimal for efficient fMet
release with free stop triplet.
At the same release factor concentration, this reaction was completed
after 1.5 min when using a stop signal on a minimessage. We
therefore studied the kinetics of our assay at intervals of <1 min
(Fig. 5). More than half of the reaction was completed within 10 s when using RF1, and within the limits of our
experiments, we could not observe a difference in velocity at two
different release factor concentrations. RF2 seems to react at least
three times slower in comparable conditions. In both cases, a plateau is reached after <2 min. We conclude that this plateau corresponds to
the actual amount of active release factor. In this case, 5 picounits
of release factor activity (34) corresponds to 50 fmol of active
protein. Continuation of the RF2 reaction after 2 min at a
significantly lower level might be due to slow release of RF2 from
previous termination complexes.
Recognition of stop codons depends on participation of termination factors, which was initially suggested by Ganoza (39). Capecchi (40) used the mRNA of bacteriophage R17 to direct in vitro protein synthesis programmed by a mutant with a stop codon at the sixth amino acid position of the coat protein. Release of the hexapeptide was shown to be dependent on the presence of protein factors (2). A defined termination assay established by Caskey et al. (11) measured release of fMet from an fMet-tRNAfMet·AUG·ribosome intermediate. The work described here presents an important improvement of the defined in vitro termination assay. We show that a minimessenger bound to the ribosome through interaction with an fMet-tRNAfMet is able to program termination by release factor recognition. A stop codon located at the A-site is necessary and sufficient for release factor-induced termination. UAG (with RF1) and UGA (with RF2) were less efficiently recognized than UAA with both factors. This is consistent with observations of Caskey et al. (34) in their system. In our initial experiments, RF2 recognized UAG as well as UGA. Reducing reaction time and Mg2+ concentration resulted in specific recognition of UAG and UAA by RF1 and UAA and UGA by RF2, although the fidelity of RF1 was higher than RF2 under tested conditions.
In a previous publication (29), termination induction by release
factors was reported when a stop codon was located one or two stop
codons 3 from the ribosomal A-site. We did not observe termination
induction by release factors with stop signals located outside of the
A-site.
2 and +2 out-of-frame stop signals were efficiently
discriminated. However, a +1 out-of-frame stop codon was fairly active.
Again, specific discrimination of this recognition was dependent on
incubation time. Furthermore, the presence of a cognate aminoacyl-tRNA
in the A-site competed with +1 out-of-frame stop signal
recognition.
Our results and conclusions are supported by kinetic comparison between free stop triplet and entire minimessenger in in vitro termination. The activity of release factors with the fMet-tRNAfMet·messenger·ribosome complex is strikingly enhanced, resulting in an increase in velocity of 2 orders of magnitude. RF2-related termination appears to occur at least three times slower than termination by RF1. We do not know if this is due to lower affinity or a lower kcat of the factor. A lower activity rate of RF2 might be a reason for the observation that there are about five times more molecules of RF2 than RF1 present in an Escherichia coli cell (32). Additionally, a lower velocity in RF2-related termination might compensate for lower specificity in stop codon recognition.
The new assay could provide insights in other aspects of translational termination. RRF, ribosomal recycling factor, has been shown to release mRNA from ribosomes after peptide release has occurred. Using an entire minimessage instead of separate triplets, our new assay might be able to determine factor requirements for ribosomal recycling after polypeptide chain termination.
We thank Edward Meenen for RNA oligonucleotide synthesis, Drs. James A. McCloskey and Andy B. Whitehill for mass spectrometry analysis, and Frances M. Adamski and Matthew A. Firpo for help with overexpression of release factors and purification of ribosomes. Plasmids for RF1 and RF2 overexpression were a kind gift from Dr. Warren P. Tate. We gratefully acknowledge Dr. Mario R. Capecchi for helpful advice. Parts of the release factors used in this work were purified in the laboratory of Dr. Richard H. Buckingham. We thank him for his collaboration. Thanks to Norma M. Wills for revising the manuscript. We are indebted to Drs. Ray Gesteland and John Atkins for supporting this work.