(Received for publication, June 5, 1995; and in revised form, July 31, 1995)
From the
Reconstitution of protein synthesis from purified translation factors on ribosomes from Escherichia coli has revealed the requirement for a protein, W, that affects chain elongation and is essential to reconstitute the process (Ganoza, M. C., Cunningham, C., and Green, R. M.(1985) Proc. Natl. Acad. Sci. U.S.A. 82, 1648-1652).
We report that W has no effect on initiation complex formation by 30 or 70 S ribosomes or on the association of ribosomal subunits, peptide bond synthesis, or binding Ala-tRNA, which is the second amino acid of the coat protein of the MS2 RNA virion.
W has a pronounced effect on tripeptide synthesis, and is obligatory for the synthesis of the coat protein or of the hexapeptide encoded by f2am3 RNA. Extracts from a temperature-sensitive mutant of the translocase, EF-G, were purified free of the W protein and were used to score for translocation defects. W is required for binding Ser-tRNA, the third N-terminal amino acid of the MS2 or f2 RNA coat protein to ribosomes bearing fMet-Ala-tRNA, as well as for the ejection of deacyl-tRNA from ribosomes, which occurred concomitant with the binding of the Ser-tRNA.
We propose that W functions by ejecting tRNAs from ribosomes in a step that precedes the movement of mRNA during translocation.
Our understanding of the mechanisms underlying translation depends, in large part, on the biochemical reconstitution of this process(1, 2, 3, 4, 5, 6) . Specific factors that affect the initiation, elongation, and termination of translation have been isolated and have been used to examine the mechanism of each reaction on ribosomes programmed with homo- or heteropolymeric mRNAs.
In contrast, only two studies have
been published on the reconstitution of translation using native mRNA
templates. In one of these studies, the lacZ gene was
transcribed by RNA polymerase and pure translation factors and
aminoacyl-tRNA synthetases were used to translate these transcripts on Escherichia coli ribosomes (7) . The requirements for
the N-formylmethionyl-tRNA
transformylase and for the RR-F
(``ribosome recycling
factor'') (8, 9) were established by these means.
In other studies, reconstitution of translation was examined by programming synthesis with amber mutants of the coat protein gene of f2 bacteriophage. The requirement for aminoacyl-tRNA synthetases was bypassed by using the appropriate aminoacyl-tRNAs in the reactions(10, 11) . This simpler system enabled identification of a set of proteins which occur bound to 70 S ribosomes(12, 13, 14, 15) .
These
proteins have been purified to homogeneity, and the sequence of two of
their genes has been established (15) . In this
communication we examine the site of action of one of these proteins
called W, by studying stepwise synthesis of the coat protein's
N-terminal six amino acids, examining the intermediates. We propose
that W stimulates translocation by accelerating the ejection of cognate
tRNAs from ribosomes.
[S]Met (550 Ci/mmol),
[
H]Ser (38 Ci/mmol), [
H]Ala
(82.7 Ci/mmol), [
H]Thr (5.3 Ci/mmol),
[
H]Phe (18.0 Ci/mmol), and
[
H]Lys (100 Ci/mmol) were purchased from Amersham
Corp. or DuPont NEN. The E. coli K12 tRNA and the
trinucleotide codon AUG came from Sigma. Poly(U), poly(A), and MS2 RNA
were from Boehringer Mannheim.
E. coli K12 mid-log or MRE 600 cells were purchased from the Miles Chemical Corp. or from the University of Alabama Fermentation Center (University of Alabama, Birmingham, AL). MRE 600 and Q13 cells were obtained from B. Bachmann (Yale University, New Haven, CT). Mutants of fusA were obtained from Tocchini-Valentini, G.P. (International Laboratory of Genetics and Biophysics, Naples, Italy). MRE 600, Q13, and the fusA mutant cells used to prepare ribosomes were grown to mid-log phase as described previously(16) .
f[S]Met-tRNA
,
[
H]Ala-tRNA, [
H]Phe-tRNA,
[
H]Thr-tRNA, [
H]Ser-tRNA,
and [
H]Lys-tRNA were acylated as described
previously using unfractionated E. coli K12 tRNA(16) .
When appropriate, the incubations contained a 10
M solution of each unlabeled amino acid. The source of the
enzyme used to prepare these fully charged aminoacyl-tRNAs was an S100
from MRE 600 cells (containing about 10 mg/ml protein) or the S100
purified through DEAE-cellulose columns (16) . The later
partially purified synthetases enabled the labeling of each specific
aminoacyl-tRNA without apparent contamination. This was done by using
the appropriate single amino acid or amino acid mixture. The fractions
were assayed for each of 20 labeled amino acids prior to use to ensure
that each aminoacyl-tRNA was fully charged. The extent of formylation
was assessed on aliquots of the
f[
S]Met-tRNA
after
hydrolysis in 0.3 N NaOH followed by extraction of
f[
S]Met from ethyl acetate in 0.1 N HCl(16) .
Ribosomes were isolated from E. coli Q13 or MRE 600 as described(16) . Ribosomes were washed
using 10 mM HCl-Tris, pH 7.4, containing either 1 M
NHCl and 2 mM MgCl
in the first wash
or 0.5 M NH
Cl and 10 mM MgCl
in the following two washes. The cells used to prepare the
ribosomes were grown to 4
10
viable cells/ml (16) and were routinely tested for RNase revertants as
described(17) . Sucrose density gradient analysis of ribosomes
was carried out using 5-30% sucrose in 5 mM MgCl
, 10 mM Tris-HCl buffer, pH 7.6, 50
mM NH
Cl. Two-dimensional electrophoretic analysis
of ribosomal proteins was performed by the method of Kaltschmidt and
Wittmann(18) .
The f2am3 bacteriophage, encoding the N-terminal hexapeptide of the f2 RNA coat protein, was isolated, and the RNA was extracted as described (17) and used to program synthesis as in (10) and (11) .
The elongation
factors EF-Tu, EF-Ts, and EF-G were assayed with poly(U) and Phe-tRNA
as well as by exchange of [H]GDP (19) and were purified as described(20, 21) .
EF-P was assayed and purified as in (14) . The release factor,
RF-1, was assayed and purified as described in (22) . The
``rescue'' protein was assayed by complementation of
thermolabile synthesis in extracts of strain N4316, supplemented with
Asn-tRNA synthetase. The rescue protein was purified as
described(13) .
IF-1, IF-2, and IF-3 were isolated from E. coli K12 cells and assayed with AUG or MS2 RNA (23, 24) using Millipore filters to collect the
f[S]Met-tRNA
MS2 RNA (or AUG)
ribosome
complexes(25) . S1 and S1A were detected with antibodies
specific for S1.
SDS-polyacrylamide electrophoretic analysis of the proteins was performed as described(26) , and the proteins were visualized by staining with silver(27) .
Protein concentration was determined as described(28) .
The assay for W was supplemented with EF-Tu, EF-Ts, and EF-G to insure that these proteins did not limit W-dependent synthesis. The diluted S100 was used as a source of the 20 aminoacyl-tRNA synthetases, the Met-tRNA transformylase, the rescue protein EF-P, the release factors RF-1 and RF-2, and the RR-F protein. S100 fractions had to be titrated for W activity prior to use.
Protein chain elongation entails the cyclical alignment of aminoacyl-tRNAs in response to their specific codons in mRNA, peptide-bond synthesis, and movement of mRNA relative to the ribosome (translocation). These mechanisms have been examined predominantly with homopolymeric mRNAs using ionic conditions that obviate the requirements for initiation factors and other proteins that may be essential for operations on natural mRNAs (see below) (1, 2, 3, 4) .
EF-Tu complexed to
GTP, possibly as a dimer(1, 5) , accelerates the rate
of binding of the aminoacyl-tRNA to the mRNA-programmed
ribosome(6) . GTP hydrolysis is required to proofread
aminoacyl-tRNAs that are near-cognate to the mRNA
template(29, 30, 31) . After GTP hydrolysis,
EF-TuGDP leaves the ribosome and a peptide bond can be
formed(1) . EF-Ts catalyzes the exchange of GDP in
EF-Tu
GDP with free GTP enabling, once again, the formation of the
ternary complex (6) . The elongation factor, EF-G, then
stimulates ribosomes to
translocate(1, 2, 3, 4) .
Although the mechanism of chain elongation has been examined with homopolymeric mRNAs, there is considerable evidence that mRNA structure affects this process, e.g. highly structured mRNAs tend to promote premature release of pre-translocation complexes from ribosomes. It is known that a number of such ``processivity'' errors result from faulty elongation events(32, 33) .
Reconstruction of translation programmed by native templates has revealed the requirement for several factors(10, 11) . One of these, the ``rescue'' protein(12, 13) , is required for association of native ribosomal subunits; a second factor, EF-P, is essential for synthesis of the first peptide bond on 70 S ribosomes(15) ; the third of these factors, W, is involved in chain elongation and is essential to reconstitute translation(10, 11) .
W has a relative mass of 47 kDa and a sedimentation coefficient of 4.1 S. The W protein, purified to 90% apparent homogeneity, is free of all initiation, elongation, termination factors as well as other proteins, such as the RR-F (9) required to reconstitute protein synthesis(7, 10, 11) .
The gene encoding W has been cloned and mapped on the E. coli chromosome. The requirement for synthesis, chromosome location, and the partial sequence of the protein suggest that it is a new component of translation.
Here we examine the effect of the W protein on
synthesis of the full length or of the first six N-terminal amino acids
of the MS2 or f2am3 coat protein. Fig. 1A shows a bar graph of the products of synthesis analyzed by
SDS-polyacrylamide gel electrophoresis. Synthesis products were formed
in response to MS2 RNA in a purified system dependent on the W protein.
90% of the products synthesized correspond to the coat protein of the
MS2 virion. W stimulates this synthesis at least 6-fold, but has no
effect on synthesis directed by poly(A) or poly(U) (Fig. 1, A and B). Synthesis programmed with poly(U) initiated
by N-acetyl-[H]Phe-tRNA also does not
depend on W (Fig. 1C). Thus, synthesis directed by a
natural mRNA requires W, whereas synthesis directed by these artificial
templates does not.
Figure 1:
A, effect of W on MS2 RNA-, poly(U)-,
or poly(A)-programmed synthesis. The assay and purification of W,
including the methods for isolation of each initiation and elongation
factor, ribosomes and aminoacyl-tRNA synthetases devoid of factors, are
described in (10) and (11) and under ``Materials
and Methods.'' The products of synthesis were analyzed by
SDS-polyacrylamide gel electrophoresis as described by
Laemmli(26) . 90% of the products correspond to the MS2
bacteriophage coat protein. Reactions conducted as described under
``Materials and Methods'' were programmed with 10 µg of
poly(A) (PA) or MS2 RNA using 1.0 µCi of
[H]Lys; reactions were for 15 min at 37 °C. B, lack of effect of W on poly(U)-programmed synthesis.
Reactions conducted as described under ``Materials and
Methods'' were programmed with 10 µg of poly(U) and included
(1.0 µCi) [
H]Phe; reactions were for 15 min
at 37 °C. C, lack of effect of W on incorporation of N-acetyl-Phe-tRNA
. Reactions conducted as
described under ``Materials and Methods'' were programmed
with 10 µg of poly(U) and included (0.1 µCi) of N-acetyl[
H]Phe-tRNA and 10 pmol of
[
H]Phe-tRNA; reactions were for 15 min at 37
°C.
W does not dissociate, associate, or prevent
association of 70 S ribosomes and is not required for binding
fMet-tRNA to ribosomes nor for peptide bond
synthesis, as measured by f[
S]Met-puromycin or
f[
S]Met
Ala
synthesis(10, 11) .
To determine whether W is required for elongation, we examined its effect on synthesis of amino acids 2-6 of the MS2 coat protein by labeling each of the corresponding amino acids. Fig. 2shows that there is a linear correlation between the requirement for W on synthesis and the position of the amino acid in the coat protein. The synthesis of the entire coat protein is absolutely dependent on W (10, 11) , whereas dipeptide synthesis is nearly independent of W, further confirming this relationship (Fig. 2).
Figure 2:
The
requirement for W on synthesis depends on the position of the amino
acid from the N terminus of the MS2 bacteriophage coat protein. W was
assayed as described under ``Materials and Methods'' and in (10) and (11) . Reactions contained twice washed MRE
600 ribosomes, IF-1, IF-2, IF-3, EF-Tu, EF-Ts and EF-G, and
fMet-tRNA, [
H]Ala-tRNA
or [
H]Ser-tRNA, Asn-tRNA,
[
H]Phe-tRNA, or [
H]Thr-tRNA
and MS2 RNA. The incorporation of each aminoacyl-tRNA into protein was
examined with each corresponding
[
H]aminoacyl-tRNA, supplemented with the required
unlabeled aminoacyl-tRNA, and the acid-soluble products synthesized
were analyzed by monitoring binding followed by electrophoretic
analysis as described under ``Materials and
Methods.''
The requirement for W on chain elongation was further studied by reconstructing synthesis with extracts of a thermosensitive mutant in the translocase, EF-G, which were freed of W by purification. Fig. 3A shows that, in such systems, synthesis of the coat protein of the MS2 bacteriophage requires both EF-G and W.
Figure 3:
A, effect of W on pre-translocation
intermediates synthesized with a mutant of EF-G. MS2 RNA-programmed
synthesis was scored with extracts purified from a
temperature-sensitive mutant defective in EF-G(34) . The
purification of each initiation, elongation factor, W, and
aminoacyl-tRNA synthetases as well as the assay of reconstructed
synthesis are described in (10) and (11) and under
``Materials and Methods.'' Each reaction was supplemented
with 5.0 µg of W, 5.0 µg of EF-G, or a mixture of these
proteins at these concentrations. Aliquots of 25 µl of the
reactions were withdrawn after a 15-min incubation at 37 °C. B, effect of W on the N-terminal products of synthesis formed
with a mutant of EF-G. High voltage electrophoresis of the acid-soluble
products of synthesis was conducted as described in (35) using
factors purified from cells with a temperature-sensitive mutant in
EF-G(34) . Reactions were programmed with 10 µg of MS2 RNA (1) or with 10 µg f2am3 RNA (2) in the
presence (solid bars) or in the absence (clear bars)
of W. Reactions programmed with MS2 RNA were treated for 10 min at 37
°C with chymotrypsin in order to cleave the N-terminal pentapeptide
of the coat protein. Formylated di- and tripeptides as well as the coat
protein's hexapeptide were used as standards. The migration of
the penta- and hexapeptides was confirmed in double-label experiments
using [S]Met and [
H]Phe or
[
H]Thr, which occur in the first, fifth, and
sixth position of the hexapeptide, respectively. The penta- and
hexapeptides were purified as described by Capechi(35) .
Approximately 5,000 dpm were observed without addition of MS2 RNA
f2am3 RNA, and this value was
subtracted.
As expected, in the absence of a functional EF-G protein, fMet-Ala accumulates on ribosomes resulting in arrested synthesis. Under these conditions, binding of the second aminoacyl-tRNA, Ala-tRNA, does not require W (data not shown). (W stimulates fMet-Ala synthesis by 50% upon addition of EF-G but not in the absence of EF-G, data not shown.). Synthesis of the N-terminal penta- or hexapeptide (examined electrophoretically) programmed by MS2 RNA (Fig. 3B, 1) or by f2am3 RNA (Fig. 3B, 2) requires not only EF-G but also W. W stimulates hexapeptide synthesis about 2-fold. Using more highly purified ribosomes, a 10-20-fold stimulation is observed (data not shown).
As shown in Fig. 4, binding of the third
aminoacyl-tRNA, Ser-tRNA, of the MS2 bacteriophage coat
protein to the 70 S
mRNA
fMet-Ala-tRNA
EF-Tu complex
depends on addition of both EF-G and W. This experiment suggests that W
functions with EF-G in translocation.
Figure 4:
Effect of W on binding of
[H]Ser-tRNA to a pre-translocation complex and on
the ejection of [
P]tRNA from ribosomes.
Reactions (0.15 ml final volume) were programmed with MS2 RNA using
initiation and elongation factors and ribosomes purified from a mutant
of EF-G as described in Fig. 2and under ``Materials and
Methods.'' Where indicated, reactions were supplemented with 5
µg of EF-G and 7.1 µg of W (S-200 fraction) and
[
H]Ser-tRNA (104,000 dpm). Aliquots (25 µl of
each reaction) were withdrawn after 15 min at 35 °C and assayed for
[
H]Ser-tRNA bound to ribosomes (A, open bars). Reactions were also supplemented with
[
P]tRNA (approximately 80,000 dpm) and the
indicated factors (B, solid bars).
[
P]tRNA (unfractionated E. coli K12)
was labeled as described (36) and was bound to ribosomes using
initiation factors of concentration given under the assay for W (see
``Materials and Methods''). The amount of
[
P] bound to ribosomes was estimated as
described under ``Materials and
Methods.''
The synthesis of tripeptides involves the movement of mRNA relative to the ribosomal complex and requires EF-G. This event is preceded by the release of deacyl-tRNA from ribosomes after peptide-bond synthesis. Translocation is required to move the nascent peptide into a site where it can be active in peptide-bond formation and where recognition of the next codon on the mRNA can occur.
Synthesis of the initial dipeptide is followed by
ejection of the deacyl-tRNA (tRNA, in this
case) from the ribosome. We therefore examined the effect of W on
ejection of deacyl-tRNA from the pre-translocation complex
(ribosome
MS2 RNA
fMet-Ala-tRNA). The data in Fig. 4indicate that W indeed ejects
[
P]tRNA bound to ribosomes, concomitant with
binding of [
H]Ser-tRNA. EF-G does not substitute
for W in this reaction (Fig. 4).
Since W strongly stimulates
binding of the third, but not of the second, aminoacyl-tRNA to the
fMet-tRNAEF-Tu
mRNA
ribosome complex, as well as
ejection of tRNA from ribosomes, it may increase the affinity of the A
site by ejecting tRNAs from the E or P site after they have
participated in synthesis. To begin examining this possibility, the
kinetics of binding and tRNA ejection were examined using the
pre-translocation complex.
As shown in Fig. 5, W stimulates
the rate of binding of [H]Ser-tRNA to MS2
RNA
ribosome complexes that contain fMet-Ala-tRNA. In the absence
of W, very little binding of [
H]Ser-tRNA is
observed and the [
P]tRNA is stably bound to the
ribosomes. Addition of W results in the ejection of
[
P]tRNA from these complexes concomitant with
the binding of the [
H]Ser-tRNA. Since
unfractionated tRNA was used in these experiments, the tRNA released
from these complexes was identified by acylation with
[
S]Met using Met-tRNA synthetase. Thirty to 40%
of the deacyl [
P]tRNA ejected from the ribosome
is tRNA
.
Figure 5:
Effect of W on the kinetics of
[H]Ser-tRNA binding to and on the ejection of
[
P]tRNA from ribosomes. The
[
P]tRNA was labeled with
[
P]ATP and T
polynucleotide kinase
as described(36) . Reactions (155 µl) were conducted at 37
°C using the buffers, mono- and divalent ions described under
``Materials and Methods.'' Each reaction also contained
ribosomes purified from MRE 600 cells, IF-1, IF-2, IF-3, EF-Tu, EF-G,
Met-, Ala-, and Ser-tRNA synthetases [
H]Ser,
[
P]tRNA and W where indicated. Aliquots of 25
µl were withdrawn at the indicated times, and the amount of
radioactive [
H]Ser-tRNA bound in the presence of
W (
-
) or in the absence of W (
- - -
)
as well as the [
P]tRNA bound in the absence of
(
- - -
) or in the presence of W
(
-
) was
determined.
Three sites, A, P, and E, are
known to be involved in binding, respectively, aminoacyl-tRNA,
peptidyl-tRNA, or deacyl-tRNA to ribosomes(2, 3) . Two
models for the elongation reaction have been recently proposed. Both
are based on analysis of intermediates in polyphenylalanine synthesis
programmed with poly(U). In the first model it is suggested that two
sites (A and P) exist on the 30 S, and three (A, P, and E) on the 50 S
particle. Hybrid states of the ribosome are proposed to explain the
altered ``foot printing'' pattern observed on addition of the
50 S particle, EF-Tu, and EF-G(2) . According to this model,
the peptidyl-tRNA or fMet-tRNA bind to a P/P
site which is equivalent to the P site. The
EF-Tu
GTP
aminoacyl-tRNA binds to an A/T state.
EF-Tu
GDP is released after hydrolysis of GTP allowing the
aminoacyl-tRNA to interact with the 50 S A (AA) site. During peptide
bond formation, both the peptidyl and the deacyl-tRNAs move relative to
the 50 S subunit such that the deacyl-tRNA moves from the P/P to the
P/E site and the peptidyl-tRNA moves from the AA to the A/P site.
During translocation, EF-G and GTP promote movement of the
peptidyl-tRNA relative to the 30 S subunit from the A/P site to the P/P
or P site and the deacyl-tRNA from the P/E to the E site. These data
are also explained by a scheme involving two steps, where the subunits
and not the tRNAs move relative to each other after peptide-bond
formation and translocation(2) .
In the second model, a pre-translocation state is defined by the occupancy of tRNA in the P site and peptidyl-tRNA in the A site. In the post-translocation state, the tRNA occupies the E site and the peptidyl-tRNA the P site. EF-G is thought to effect the conversion of the pre- to the post-translocation states(3) . Occupation of the E site is known to induce a low affinity A site, and occupation of the A site induces a low affinity E site effecting the release of tRNA(3) .
It has been proposed
that the filling of the E site with deacyl-tRNA induces a low affinity
A site, which enables the tRNAribosome complexes to
dissociate(3) . Since W stimulates Ser-tRNA binding to
ribosomes concomitant with the ejection of deacyl-tRNA from the
particles, it could be stimulating cognate tRNA ejection by affecting
the affinity of the E site. The relative contribution of W to the exit
of cognate tRNAs from the E site of the ribosome is under study using
suitable heteropolymeric mRNAs. Other experiments are required to
establish whether W affects an essential hybrid site utilized during
chain elongation.
W has no effect on poly(U)- or poly(A)-programmed
synthesis from Phe-tRNA or Lys-tRNA
. For
these cases, a more rapid release of the cognate tRNAs from the
ribosome may occur, which may not be detected under our experimental
conditions. Alternatively, it is possible that W ejects only certain
tRNAs from ribosomes, e.g. deacyl-tRNA
. The specificity of W in the
binding and unbinding of tRNAs is also under investigation.
The combined results presented here suggest that the W protein could have pronounced effects on the efficiency of translation programmed by native mRNAs by removing tRNAs from ribosomes that are spent during synthesis. The discovery of this protein has enabled reconstitution of translation from pure components permitting the study of these and other reactions that regulate translation of mRNA transcripts.