From the Department of Biochemistry and Centre for Gene Research, University of Otago, P. O. Box 56, Dunedin, New Zealand and § Max-Planck-Institut für Molekulare Genetik, Ihnestrasse 73, D-14195 Berlin, Germany
Received for publication, October 28, 2002
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ABSTRACT |
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The function of the decoding release
factor (RF) in translation termination is to couple cognate recognition
of the stop codon in the mRNA with hydrolysis of the completed
polypeptide from its covalently linked tRNA. For this to occur, the RF
must interact with specific A-site components of the active centers
within both the small and large ribosomal subunits. In this work, we
have used directed hydroxyl radical footprinting to map the ribosomal binding site of the Escherichia coli class I release factor
RF2, during translation termination. In the presence of the cognate UGA
stop codon, residues flanking the universally conserved
250GGQ252 motif of RF2 were each shown to
footprint to the large ribosomal subunit, specifically to conserved
elements of the peptidyltransferase and GTPase-associated centers.
In contrast, residues that flank the putative "peptide anticodon"
of RF2, 205SPF207, were shown to make a
footprint in the small ribosomal subunit at positions within well
characterized 16 S rRNA motifs in the vicinity of the decoding center.
Within the recently solved crystal structure of E. coli
RF2, the GGQ and SPF motifs are separated by 23 Å only, a distance
that is incompatible with the observed cleavage sites that are up to
100 Å apart. Our data suggest that RF2 may undergo gross
conformational changes upon ribosome binding, the implications of which
are discussed in terms of the mechanism of RF-mediated termination.
Termination of protein synthesis is signaled by the translocation
of a stop rather than a sense codon into the A-site of the ribosome (1,
2). The stop codon is recognized by a protein, the polypeptide chain
release factor (RF),1 which
triggers the hydrolytic release of the nascent polypeptide chain from
the P-site-bound peptidyl-tRNA. Recognition of the stop codon by a
protein rather than a tRNA molecule led Moffat and Tate (3) to propose
the "tRNA analogue model," which describes the RF as a decoding
molecule like tRNA.
Class I RFs are the decoding factors (4). In bacteria such as
Escherichia coli, there are two decoding RFs, RF1 (decodes UAG and UAA) and RF2 (decodes UGA and UAA). In eukaryotes, a single factor, eRF1, recognizes all three stop codons (5). With the exception
of a universally conserved GGQ motif, there is no overall sequence
homology between the eubacterial class I RFs and their eukaryotic
counterparts (6). The properties of the class I RFs are remarkably
similar to those of aminoacyl-tRNAs (reviewed in Ref. 7). RFs
compete with suppressor tRNAs for binding to the A-site of the ribosome
in response to specific recognition of the A-site codon (8, 9). This
so-called anti-suppressor phenotype implies that tRNAs and RFs are
cognate species in direct competition for stop codon binding. Both
interact with the peptidyl-tRNA located at the ribosomal P-site, and
both interact on the ribosome with translational G proteins (EF-Tu for
aminoacyl-tRNA and RF3/eRF3 for the decoding RFs).
In the tRNA analogue model (3), the decoding RF was
proposed to consist of two functionally separate domains that span the
two active centers of the ribosome in a manner analogous to that of
tRNA. One domain was responsible for codon recognition and interaction
with the decoding center in the small ribosomal subunit, and a second
domain was thought to function in triggering peptidyl-tRNA hydrolysis
at the peptidyltransferase center (PTC) of the large ribosomal subunit.
This proposal was based on functional studies, which demonstrated that
it was possible to separate the decoding and peptidyl-tRNA hydrolysis
activities of the RF (3, 10-12). It is supported by more recent
site-directed mutagenesis studies of the domain structure of the
eubacterial or organellar class I RFs (13-16).
Two key motifs within the decoding RFs have been identified. A putative
peptide "anticodon" (188PAT190 in E. coli RF1 and 205SPF207 in RF2), the site
of numerous suppressor mutations, is proposed to confer codon
specificity on the class I RFs and to bind at the decoding center of
the small ribosomal subunit (16). A universally conserved GGQ motif (6)
and surrounding residues are thought to interact with the PTC of the
large subunit. Substitution of residues within the GGQ motif interferes
with the peptidyl-tRNA hydrolysis activity of the RF
(73),2 whereas a single
cleavage six residues upstream of the GGQ site completely abolishes
this activity (3). Methylation of the glutamine residue of the GGQ
motif has also been shown to be important for hydrolysis activity (17).
These studies predict that the SPF and GGQ motifs will occupy the
extremities of the two domains of the decoding RF that span the
ribosomal decoding and peptidyltransferase centers.
The first structure of a eubacterial decoding RF, that of E. coli RF2, provided unexpected surprises (18). The GGQ
peptidyl-tRNA hydrolysis and SPF anticodon motifs were only 23 Å apart
in the structure and not ~80 Å, as expected. This is unlike the
three-dimensional structure of eukaryotic eRF1, in which the GGQ and
NIKS (believed to be close to the decoding site) motifs were positioned
at the two extremities of the protein (19). Although the tertiary
structures of the eubacterial and eukaryotic factors differ
considerably, they do resemble an overall tRNA-like L shape (RF2) or Y
shape (eRF1).
To resolve the paradox between the positions of the SPF and GGQ motifs
in the crystal structure of RF2 and the functional data that would have
placed them at the two extremities of the protein, we set out to map
the ribosomal environment of RF2 when it is bound at the ribosomal
A-site. This approach would not only allow us to test the tRNA analogue
hypothesis but would also provide important information as to whether
the RF might undergo a large conformational change upon binding to the ribosome.
Hydroxyl radical probing from Fe(II) tethered directly to the
sulfhydryl groups of cysteine residues on the surface of a protein via
the linker 1-(p-bromoacetamido-benzyl)-EDTA (BABE) has been used successfully to map the ribosomal binding sites of S5 (20), S8
(21), L9 (22), and L11 (23) as well as tRNA (24) and translation
factors, IF3 (25), EF-G (26), and RF1 (27). This approach uses Fenton
chemistry to generate hydroxyl radicals that cleave the RNA backbone in
the vicinity of the tethered Fe(II). Whereas it is possible for a
released radical to miss neighboring nucleotides and to cleave rRNA at
more distant sites, the relative distances between the cleaved rRNA and
the site of radical generation can be estimated from the intensities of
the cleavage results (28). Moreover, a series of cleavages is obtained
from each site, which aids interpretation now that the ribosome
structures are available. In the current study, two adjacent tethering
sites were selected on the RF2 protein for attachment of BABE as an additional strategy to avoid misinterpretation of the significance of
specific cleavages.
Using this approach, amino acids neighboring the GGQ motif of RF2 were
shown to map to the PTC, whereas residues flanking the SPF motif mapped
near the ribosomal decoding center. These results support the tRNA
analogue model and suggest that RF2 undergoes a significant
conformational change upon binding to the ribosome.
Materials--
Construction of the RF2 expression plasmids
pETRF2* and pETRF2*(T246S) has been described previously (12). Plasmids
were purified using a Qiagen miniprep kit and were electroporated into bacteria using an Electro Cell Manipulator® 600 (BTX). MRE600 ribosomes, [3H]fMet-tRNAfMet, and codons used
in the in vitro termination assays were kindly prepared by
T. Edgar. The BABE reagent (1-(p-bromoacetamidobenzyl)-EDTA) was synthesized using the method of DeRiemer et al. (29).
ProbeQuantTM G-50 microcolumns and YM-100 spin columns used
in the purification of the BABE-conjugated release factors, and
termination complexes were from Amersham Biosciences and Millipore
Corp., respectively, and avian myeloblastosis virus reverse
transcriptase from Promega.
Growth Media and Bacterial Strains--
All cloning work was
carried out in the JM109 strain of E. coli
[endA1, recA1, gyrA96,
thi, hsdR17 (rk Construction of RF2 Cysteine Mutants--
Mutations were
introduced into prfB* (in the plasmid pETRF2*) or
prfB*(T246S) (in the plasmid pETRF2*(T246S)) using a
two-step PCR strategy. All reactions were carried out using the Expand High Fidelity PCR system (Roche Molecular Biochemicals). In the first
reaction, a 27-mer mutagenic forward primer (C128A,
5'-GACAGCGCCGACgccTACCTCGATATT; C274X,
5'-ATCGTGACCCAG(a/g/t)(c/t)(c/g)CAGAACGACCGT; L201C,
5'-GGCGTTCACCGCtgtGTGCGTAAAAGC; S209C, 5'-AGC CCGTTTGACtgcGGCGGTCGTCGC;
V243C, 5'-CTGCGCATTGACtgtTATCGCACGTCC; or T246C,
5'-GACGTTTATCGCtgcTCCGGCGCGGGC) and the pET-T7r reverse primer
(5'-GCTAGTTATTGCTCAGCGG) were used to amplify a C-terminal fragment of RF2 (350-550 bp) containing the site-directed mutation (shown in lowercase type). Approximately 180-360 ng of the purified fragments were then denatured (95 °C for 5 min) and added to a second PCR containing the pET-T7f forward primer
(5'-TAATACGACTCACTATAGGG) to amplify the full-length mutated
prfB* gene. This process was repeated for each of the
individual site-directed mutations. All amplification reactions were
performed using 1× Expand HF buffer (supplied with the enzyme)
containing 200 µM dNTPs (dATP, dCTP, dGTP, and dTTP), 1.5 mM MgCl2, 400 nM primers, 2.6 units
of Expand DNA polymerase, ~1 ng of plasmid DNA (template), and
double-distilled H2O in a total volume of 50 µl. Using a
PTC-200, Peltier thermal cycler PCR machine (MJ Research, Inc.), the
reactions were heated at 95 °C for 5 min. The following cycle was
then repeated 30 times: annealing, 50-60 °C for 1 min; extension,
72 °C for 2 min; denaturation, 95 °C for 30 s. The final
cycle ended with a 5-min extension at 72 °C, and the reactions were
cooled to 4 °C.
Release Factor Purification--
Crude preparations of RF
proteins were obtained from the frozen bacterial pellets essentially as
described previously (12, 30). The proteins were purified further on a
ResourceQTM anion exchange column using the BioLogic HR chromatography
system (Bio-Rad) according to the manufacturer's instructions.
Protein Concentration Estimations--
The BCA assay (31) was
used to estimate the concentration of purified protein samples.
In Vitro Termination Assays--
The in vitro
peptidyl-tRNA hydrolysis and codon-dependent ribosome
binding assays were performed as described previously (12).
The 5,5'-Dithiobis-(2-nitrobenzoic acid) (DTNB) Assay for
Measuring Surface Accessibility--
The DTNB assay measures the
formation of a colored chromophore at 412 nm upon reaction of Ellman's
reagent or DTNB with the sulfhydryl group of an accessible cysteine
residue. Since the RF proteins are stored in 1 mM
dithiothreitol to maintain the cysteine residues of the factor in a
reduced form they were first purified away from the dithiothreitol
using ProbeQuantTM G-50 microcolumns (Amersham Biosciences) according
to the manufacturer's instructions. DTNB (5 µl of 1 mg/ml) was added
to the RF sample, and the reaction was incubated at room temperature
for 20 min. The absorbance of a buffer blank was subtracted from the
sample values. The concentration of accessible cysteine residues was
calculated from the change in absorbance at 412 nm, using
Conjugation of BABE to RF and Termination Complex
Formation--
The BABE reagent was loaded with Fe(II) essentially as
described by Culver and Noller (28). Prior to Fe(II) derivatization, the RF proteins were washed through ProbeQuantTM G-50 microcolumns (preequilibrated in a buffer containing 50 mM Tris-HCl, pH
8, and 50 mM KCl, according to the manufacturer's
instructions) to remove excess dithiothreitol. In a typical conjugation
reaction, 50 µl of RF (~1500 pmol) was mixed with 5.4 µl of the
Fe(II)-BABE complex (~76 nmol of Fe(II)-BABE, 50-fold molar excess)
and incubated at 37 °C for 1 h. Unbound Fe(II)-BABE was then
removed by washing the reactions through the ProbeQuantTM G-50 columns,
and the efficiency of conjugation determined was using the DTNB assay
as described above.
Two different sets of conditions were used in the formation of
ribosomal termination complexes (100-µl total volume): (i) in a
buffer containing 50 mM Tris-HCl, pH 7.2, 20 mM
Mg(OAc)2, 100 mM NH4Cl, and 10%
(v/v) ethanol, by incubating 100 pmol of 70 S ribosomes with 1000 pmol
of stop signal (normally UGAU) and 400 pmol of Fe(II)-derivatized RF2
at 4 °C for 20 min; (ii) in a buffer containing 20 mM
Hepes-KOH, pH 7.8, 6 mM Mg(OAc)2, 150 mM NH4Cl, 4 mM Site-directed Hydroxyl Radical Probing and Isolation of the
Probed rRNA--
The site-directed hydroxyl radical probing was
carried out as described previously (28). To initiate hydroxyl radical
production, 2 µl of 250 mM ascorbic acid and 2 µl of
1.25% (v/v) H2O2 were added to the purified
termination complexes. After incubating the samples for 10 min on ice,
the probing reactions were quenched with 20 µl of 20 mM
thiourea. The probed rRNA was then recovered using TRIZOL® reagent
(Invitrogen), according to the manufacturer's instructions. The probed
rRNA samples were snap-frozen on dry ice and stored at Primer Extension--
The primers used to walk through the 16 and 23 S rRNA (sequences available on request) were end-labeled with
33P by incubating 10 pmol of the oligonucleotides with 3 µl of [ Construction of a Cysteineless Variant of RF2--
A
prfB* mutant devoid of endogenous cysteine residues was
required for the construction of site-specific single cysteine
derivatives of RF2. The sulfhydryl side chains of the inserted
cysteines act as targeted tether sites for the BABE reagent. The
cysteineless (CL) variant of RF2 was also used as a control for any
unintended derivatization of noncysteine side chains and was included
in the mapping experiments to eliminate sites of nonspecific RNA cleavage. The native RF2 protein of E. coli has two cysteine
residues at positions 128 and 274 (Fig.
1). A cysteine residue is not found frequently at positions equivalent to 128 of E. coli RF2
(seen in only four of the 30 representative RF sequences aligned in Fig. 1A). In contrast, a cysteine residue at position 274 is
more highly conserved among the different class I RFs (found in 24 of
the 30 sequences; Fig. 1B). The high conservation of
Cys274 may reflect its location within the putative
peptidyl-tRNA hydrolysis domain of the factor.2 The class I
RF sequence alignments in Fig. 1 were used to select appropriate amino
acids to substitute for the endogenous cysteine residues of E. coli RF2. Alanine was chosen to replace Cys128,
because this residue is tolerated at equivalent positions in 12 of the
30 RF sequences aligned in Fig. 1A. Also, the small methyl
side chain of alanine was thought to be less likely to cause
significant changes in the secondary structure of the RF compared with
the possible alternative candidates for this site, isoleucine and
valine. Methionine and serine were each chosen along with alanine as
the possible replacement amino acid for the potentially more important
Cys274, since they were the only other two amino acids
found at this position (Fig. 1B). Substitution of
Cys128 with alanine and Cys274 with
alanine, methionine, or serine did not alter the predicted secondary
structure of the RF2 proteins (data not shown).
Exogenous expression of E. coli RF2 produces a protein with
a severely diminished capacity to stimulate peptidyl-tRNA hydrolysis (see Fig. 2, top
panel). There are at least two possible reasons for this
phenomenon: (i) a threonine residue at position 246 facilitates an
incorrect conformation when the protein is expressed at high concentrations (12, 13), and (ii) Gln252 is not fully
N5-methylated (17). Peptidyl-tRNA hydrolysis
activity is restored to overexpressed RF2 by substituting
Thr246 for serine or alanine (Fig. 2, top
panel) (12, 13) or by including 10% (v/v) ethanol in the
assay system (Fig. 2, bottom panel). For this
reason, a T246S background for RF2 was used to measure the effect of
the selected substitutions at Cys128 and Cys274
on the release activities of the RF proteins in vitro in the absence of ethanol (Fig. 2, top panel) as well as
in its presence (bottom panel).
The C274A and C274M mutations impaired significantly the release
activity of the T246S variant of RF2. The deleterious effects of these
two substitutions were substantially alleviated in the presence of
ethanol (Fig. 2, bottom panel). Interestingly,
substitution of Cys128 with alanine and Cys274
with serine, both increased the specific release activity of the
T246S RF variants, independent of whether ethanol was present. Based on
these results, a CL mutant of RF2 containing the substitutions C128A
and C274S was constructed and used as a template for the creation of
novel single cysteine RF2 variants. Because of the functional enigma of
the low peptidyl-tRNA hydrolysis activity of overexpressed RF2, and
since the T246S variants exhibited increased specific release activity,
a representative of the T246S variants was also taken through the
mapping experiments for comparison (see below).
Preparation of Single Cysteine Derivatives of RF2 for the
Topographical Mapping Studies--
For probing the ribosomal
environment of the putative decoding and peptidyl-tRNA hydrolysis
domains, published data (12, 13, 16), together with our recent results
from a comprehensive site-directed mutagenesis study,2
guided the selection of tethering sites on E. coli RF2.
Three positions were selected within the proposed peptidyl-tRNA
hydrolysis domain of RF2 (Val243, Thr246, and
Cys274), whereas Leu201 and Ser209
were chosen to represent the decoding domain of RF2, because they flank
the putative tripeptide anticodon 205SPF207 of
the factor (16). All five positions were shown to be accessible to
modification by the BABE reagent using the DTNB assay (data not shown).
Val243 was selected as a representative of the
peptidyl-tRNA hydrolysis domain of RF2, because it neighbors the most
sensitive site on the protein (between Tyr244 and
Arg245) to cleavage by chymotrypsin. This cleavage
inactivates the hydrolysis function of RF2, while potentiating its
ability to bind to the ribosome (3). The identity of the residue at
position 246 of E. coli RF2 (as described above) has been
shown to have a dramatic effect on the release activity of the factor
(12, 13), signifying the importance of this residue for RF function. It
was therefore of interest to examine the rRNA neighborhood of position
246, in the hope that it might provide further information about the role of this residue in RF function. Cys274 was the only
endogenous cysteine residue of RF2 shown to be accessible for
modification by a sulfhydryl-binding reagent (data not shown). This
highly conserved residue is important for the peptidyl-tRNA hydrolysis
activity of RF22 and as indicated in Fig. 2. We also
examined the rRNA environment of Cys274 in the T246S
variant of RF2 to determine whether the effect of this secondary
substitution in increasing the release activity of overexpressed RF2
was the result of a shift in the positioning of the RF on the ribosome.
This was suggested because the T246S mutation had the same effect on
the release activity of overexpressed RF2 as 10% (v/v) ethanol (Fig.
2), which is thought to restore peptidyl-tRNA hydrolysis activity to
overexpressed RF2 by strengthening the interaction of the factor with
the ribosome. Each of the variants were expressed and purified to the
same high standard of homogeneity as analyzed on SDS-PAGE.
The ribosomal binding and peptidyl-tRNA hydrolysis activities of the
RF2 cysteine variants engineered for the mapping
experiments before and after conjugation
with Fe(II)-BABE are presented in Tables
I and II,
respectively. The ribosomal binding activities of the variant
factors were more similar to that of the control RF2 protein (Table I)
than their release activities, which varied quite considerably (Table
II). Interestingly, the increased release activity of the C128A/T246S
variant was not restricted to the cognate UGA stop codon, with residual
activity at the noncognate UAG stop codon almost twice that of the wild
type RF2 protein at UGA (Table II). This suggests that the increased
release activity of the T246S variant (Fig. 2, bottom
panel) might be at the expense of codon specificity.
Following conjugation with Fe(II)-BABE, the ribosomal binding activity
of the V243C/CL and T246C/CL variants of RF2 decreased to ~50-66%
of their respective activities before conjugation with the reagent
(Table I). However, the binding activities of the remaining variants
were only modestly reduced following conjugation of Fe(II)-BABE. Mock
derivatization of the CL variant of RF2 did not decrease the specific
activity of the factor. Collectively, these results were promising,
because in order to map the ribosomal binding site of the RF2 proteins,
it was critical that they could bind to the ribosome following the
attachment of the bulky 10-Å tether. Furthermore, the
Fe(II)-derivatized RF variants were still able to discriminate the
cognate UGA stop codon from the noncognate UAG codon. In contrast to
their ability to bind to the ribosome, the RF variants were unable to
stimulate peptidyl-tRNA hydrolysis following conjugation of Fe(II)-BABE
(Table II). Fortunately, however, for the mapping experiments, it was
essential for the derivatized factors to bind to the ribosome in a
codon-dependent manner but not for them to retain release
activity. The diminished release activity of the variants following
conjugation with Fe(II)-BABE supports the involvement of the targeted
residues in the peptidyl-tRNA hydrolysis function of the RF2 protein.
Following conjugation of Fe(II)-BABE to the RF variants, the proteins
were incubated with ribosomes in the presence of the cognate
termination signal, and hydroxyl radicals were induced according to the
method of Culver and Noller (28). The probed rRNA was then recovered
from the ribosomal complexes, and the positions of the hydroxyl
radical-induced cleavages were identified using primer extension. These
appeared as either new bands or an increased intensity of background
level bands present with the CL control. The results presented
in Figs. 3
and 4 were obtained from termination
complexes formed in the presence of the traditional in vitro
buffer (32) with an empty P site and UGAU stop signal. However, very
similar results were obtained with a more physiological buffer
containing low salt concentrations and polyamines (33). These reactions
were carried out in the presence of oligoribonucleotide mini-mRNAs
rather than UGAU and with deacylated tRNA in the P site. At most,
subtle differences in the intensity of some cleavages were seen (data
not shown).
The relative intensities of the cleaved bands were scored
as either strong, medium, or weak when compared with the intensity of
the adjacent sequencing lanes (as described in Refs. 28 and 34). These
were used as a guide to gauge the distance between the tethered Fe(II)
and the targeted rRNA site. Discrete cleavages were detected in either
the 16 or 23 S rRNA with all six cysteine variants of RF2, but
significantly, none of the Fe(II)-derivatized variants cleaved both
rRNA molecules. Interactions between RF2 and the 5 S rRNA were not
examined in this study.
Hydroxyl Radical Footprinting of the 16 and 23 S
rRNAs--
Hydroxyl radical cleavages were observed only in the 16 S
rRNA when Fe(II)-BABE was tethered to position 201 or 209 of RF2 (Figs.
3A and 4A). In the 16 S rRNA, the targeted
elements included (i) nucleotides within the 530 loop of the 5' domain;
(ii) two nucleotides in the conserved 790 loop at the platform of the
small ribosomal subunit involved in 30/50 S subunit interactions (35); (iii) an exposed rRNA element centered around nucleotide 1230 of the 3'
major domain of the 16 S rRNA; and (iv) three regions within helix 34 (nucleotides 1053-1054, 1062-1065, and 1194-1197).
Hydroxyl radicals originating from positions 243, 246, and 274 of RF2
cleaved several functionally important elements of the 23 S rRNA (Figs.
3B and 4B). Several of the cleavages were unique to one particular tethering position, whereas others were observed from
two different locations on the RF2 protein. Cleavage positions included
(i) nucleotides near the end of a long helix of domain II (nucleotides
879-900) bridging the ribosomal subunits; (ii) nucleotides
centered around positions 1943 and 1965 of domain IV (also at the
subunit interface); (iii) the bulged nucleotide A2602, which interacts with aminoacyl-tRNA and
peptidyltransferase inhibitors (36, 37); (iv) nucleotide positions
1074-1076 in the GTPase-associated center of the 23 S rRNA; (v)
nucleotides within the A-loop of domain V of the 23 S rRNA (nucleotides
2556-2557) that are protected by the 3' terminus of the A-site-bound
tRNA (36, 38, 39); (vi) nucleotide A2451, which was
hypothesized to be the catalytic base of the ribosomal PTC (reviewed in
Ref. 40); and (vii) several positions within the central ring of domain
V of the ribosomal PTC. Interactions between these sites are now
supported by the crystallographic data of the 50 S subunit in complex
with A-site ligand mimics (41-43).
Substitution of Thr246 in E. coli
RF2 with a serine residue restored functional peptidyl-tRNA hydrolysis
activity to the overexpressed factor (Fig. 2) (12, 13). It is notable
therefore that when Fe(II) was tethered to position 274 of RF2,
nucleotides 2264-2265 of the 23 S rRNA (domain V) were cleaved, but
only when residue 246 was a serine and not a threonine (compare 274 cleavages with 274*, Fig. 3B). Furthermore, the estimated
distance between the tethered Fe(II) at position 274 of RF2 and the
cleaved bases 2253-2256 increased from the strong (12-36 Å) category
to the more modest 20-44 Å in RFs carrying the T246S mutation.
Nucleotides 2256-2257 are located within the P-loop of domain V of the
23 S rRNA and are protected by the 3' terminus of the P-site-bound tRNA
(36, 38, 39). These results suggest that the increased peptidyl-tRNA hydrolysis activity of the T246S variant of RF2 might be due to a shift
in the interaction of the RF with the ribosomal PTC as predicted by
Ref. 12. Interestingly, nucleotide A2451 is specifically
cleaved when Fe(II) is attached to residue 246 of RF2 (Fig.
3B). A2451 was initially thought to be the
catalytic base in peptide bond formation; however, recent in
vitro data have cast doubt on this hypothesis (40).
The Structures of the Decoding RFs Have Features That Mimic
tRNA
The derivation of x-ray crystal structures for protein factors
involved in the different phases of protein synthesis and, more
recently, for the ribosome and its subunits has provided a structural
framework for the interpretation of decades of biochemical research. An
important discovery from these structures was the overall similarity in
shape of the protein synthesis factors to that of tRNA (44). Of
particular interest for the polypeptide chain release factors was that,
like tRNA molecules, they decode signals in the mRNA at the A-site
of the ribosome. Earlier biochemical evidence, as encapsulated in the
tRNA analogue model (3), implied that the decoding RFs were able to
extend between the two active centers of the ribosome, from the
decoding center in the small subunit to the PTC in the large ribosomal
subunit. The current study has shown that prokaryotic RF2 does indeed
footprint to these key centers in a translational termination complex.
Since most biochemical studies have focused on the bacterial RFs, there
was much interest in the long awaited structure of E. coli
RF2 (18), published while our current work was in progress. Although
RF2 and the eukaryotic equivalent, eRF1, perform the same function in
translation termination, the lack of sequence homology between these
two factors is reflected in their three-dimensional structures. RF2 has
an elongated domain of
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, mk+),
relA1, supE44,
,
(lac-proAB),
F', traD36, proA+B+, lacIqZ
M15]
(Promega), whereas expression studies from the pET3a-based vectors
utilized the E. coli strain BL21(DE3) pLysS [hsdS
gal (
cIts 857 ind1 Sam7
nin5 lacUV5-T7 gene 1) pLysS
(camR)]. The latter strain is maintained in 30 µg/ml chloramphenicol due to the presence of the pLysS plasmid. For
expression of the RF proteins, individual freshly transformed or
streaked colonies were inoculated into LB medium supplemented with 100 µg/ml ampicillin. An overnight culture was then used to inoculate
fresh selective medium (1-5% (v/v)) and grown at 37 °C with
shaking to an A600 of 0.4-0.6. Protein
expression was induced with 1 mM
isopropyl-
-D-thiogalactopyranoside, and the cells were
grown for a further 2.5-3 h. The harvested cell pellets were frozen at
80 °C in preparation for protein purification.
412 = 13,600 M
1
cm
1 for the chromophore.
-mercaptoethanol,
50 µM spermine, 2 mM spermidine, and 10%
(v/v) ethanol by incubating 100 pmol of 70 S ribosomes with 1000 pmol
of stop signal (normally GUCAUGUGAU), 200 pmol of P-site tRNA, and 400 pmol of Fe(II)-derivatized RF2 at 37 °C for 30 min. Immediately
prior to site-directed hydroxyl radical probing, the ribosomal
termination complexes were purified from free Fe(II)-RF2 using Microcon
YM-100 spin columns (Millipore Corp.) according to the manufacturer's
instructions. The columns contain a cellulose membrane with a molecular
mass cut-off of 100 kDa. The termination complexes were therefore
trapped on the membrane, whereas unbound Fe(II)-RF2 proteins washed
through. By simply inverting the column and centrifuging again, the
complexes were easily and efficiently recovered. The volumes of the
eluted ribosomes were then adjusted to 100 µl with the specific
buffer used in the formation of the termination complex.
80 °C in
preparation for primer extension.
-33P]dATP (3000 Ci/mmol) and 10 units
of T4 polynucleotide kinase in 1× T4 polynucleotide
kinase buffer (Roche Molecular Biochemicals). The volume of the
reaction was adjusted to 10 µl with RNase-free double-distilled
H2O. The reactions were incubated at 37 °C for 30 min
and then 65 °C for 10 min to inactivate the kinase enzyme. The final
concentration of the radiolabeled primers was 1 pmol/µl. Radiolabeled
primers (3 pmol) were then annealed to 1 µg of the recovered rRNA in
1× avian myeloblastosis virus primer extension buffer (Promega). The
11-µl reactions were incubated at 70 °C for 5 min to denature the
RNA and then at the Tm of the particular primer for
20 min. The samples were cooled on ice while the extension reactions
were prepared. A "master" mixture mix (enough for six reactions)
was prepared by mixing 30 µl of 2× avian myeloblastosis virus primer
extension buffer (Promega), 12 µl of 40 mM sodium
pyrophosphate, and 24 units of avian myeloblastosis virus reverse
transcriptase in a reaction volume of 54 µl. For the sequencing
reactions, individual cocktails were prepared that each contained one
of the ddNTPs at a final concentration of 0.67 mM. The
cocktails were immediately divided into 9-µl aliquots and added to
the 11-µl annealing reactions, and the 20-µl samples were incubated
at 42 °C for 30 min. The cDNA extension products were
precipitated with 2 µl of 3 M sodium acetate pH 5.5 and
40 µl of prechilled 96% (v/v) ethanol at
20 °C for 2-16 h. The
samples were then recovered by centrifugation (12,000 × g for 30 min at 4 °C). The cDNA pellets were washed
with 100 µl of 70% (v/v) ethanol, centrifuged at 12,000 × g for 5 min at 4 °C, dried at 37 °C for 5 min, and
resuspended in a buffer containing 98% (v/v) formamide, 10 mM EDTA, 0.1% (w/v) xylene cyanol, and 0.1% (w/v)
bromphenol blue. After heating at 94 °C for 5 min, 2 µl of the
cDNA products were loaded onto a 6% (w/v) acrylamide, 8 M urea gel and fractionated by electrophoresis as described
in Ref. 28.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (90K):
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Fig. 1.
Structural analysis of the regions
encompassing the two endogenous cysteine residues of E. coli
RF2. Sequence alignment of amino acids 119-138
(A) and amino acids 264-284 (B) (E. coli RF2 numbering) of a representative group of different
prokaryotic or prokaryotic-like class I RFs. The two endogenous
cysteine residues of E. coli RF2 at positions 128 and 274 are indicated with arrows in A and B,
respectively. Release factor sequences have been grouped into RF2-like
sequences, RF1-like sequences, and mitochondrial RF1 sequences
(mRF1). Highly conserved amino acids across the three groups
of RFs are blocked in black, and conserved amino
acids are blocked in gray. Prokaryotic RF
sequences not included in the alignments were identical to or very
similar to those shown.
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Fig. 2.
Peptidyl-tRNA hydrolysis activities of the
Cys128 and Cys274 variants of RF2. Release
activities of the T246S/C128A and T246S/C274X (where X
represents Ala, Met, or Ser) variants of RF2, in the absence
(top panel) or presence (bottom
panel) of 10% (v/v) ethanol, compared with the activities
of the control wild type RF2 and variant T246S proteins. Experiments
were repeated in triplicate using duplicate samples of each RF, and
release activities are expressed as the average release of
[3H]fMet (fmol), plus the S.E.
Codon-dependent specific ribosome binding activities of the
RF2 cysteine variants before and after conjugation with Fe(II)-BABE
Specific codon-dependent peptidyl-tRNA hydrolysis
activities of the RF2 cysteine variants before and after conjugation
with Fe(II)-BABE
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Fig. 3.
Identification of a RF2 footprint on the
ribosome using directed hydroxyl radical probing. Sites of
hydroxyl radical-induced cleavages in the 16 S rRNA (A) and
23 S rRNA (B) are indicated with solid
vertical bars at the right of each
autoradiogram. The numbers at the top of each
profile indicate the Fe(II)-derivatized residues of RF2. U,
G, C, and A, sequencing lanes.
*, this variant of RF2 also carries the functionally important T246S
mutation.
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Fig. 4.
Mapping the RF2 footprint on the secondary
structures of E. coli 16 and 23 S rRNA.
A, site-directed hydroxyl radical cleavages in the 16 S rRNA
when Fe(II) was tethered to position 201 or 209 of RF2. B,
site-directed hydroxyl radical cleavages in the 23 S rRNA when Fe(II)
was tethered to position 243, 246, or 274 of RF2. The phosphodiester
bond cleavages have been classified as strong, medium, or weak
(indicated by dot size) and correspond to
resolutions of ~22, ~36, and ~44 Å, respectively (see Ref.
28).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helices (domain 1), speculated by
Vestergaard et al. (18) to occupy the decoding site. On this
disposition, a bulky structural element (domains 2-4) must also fit
into the A-site of the PTC. There was growing multifaceted biochemical
and genetic evidence of proximity if not direct involvement of the GGQ
and SPF motifs with the two widely spaced ribosomal enzyme and decoding
centers. The great paradox of the structure was that these two motifs
were placed too close together to be
compatible with such placement. Our chosen sites for hydroxyl radical
generation flanking or in the vicinity of these motifs are also
illustrated on Fig. 5A. Directed hydroxyl radical
footprinting of RF2 onto the 16 and 23 S rRNA in our study provides
important and compelling information that the bacterial factor must
undergo a significant conformational unfolding to be docked into the
A-site during decoding of a stop signal. It supports the accumulated
biochemical data indicating that the GGQ motif is a proximity marker of
RFs to the peptidyltransferase, and, in the case of bacterial RF2, SPF
is a marker for proximity to the decoding site. A specific discussion
of the data that led to these conclusions is given below.
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Fig. 5.
The ribosomal binding site of E. coli RF2. A, identification of the
Fe(II)-tethering sites on the three-dimensional structure of E. coli RF2 (PDB1GQE) (18). Space fill representations of the
tethered residues have been colored as follows: Val243
(yellow), Thr246 (purple),
Cys274 (red), Leu201
(green), Ser209 (magenta).
B, mapping the footprint sites between residues 201 and 209 of E. coli RF2 and the 16 S rRNA on the three-dimensional
structure of the small ribosomal subunit from T. thermophilus (PDB1FJF) (45). Cleavages from position 209 alone are
colored green, whereas those from both 201 and 209 are
yellow. For reference, conserved bases A1492 and
A1493 are shown in red. C, mapping
the footprint sites between residues 243, 246, and 274 of E. coli RF2 and the 23 S rRNA on the three-dimensional structure of
the large ribosomal subunit from H. marismortui (PDB1FFK)
(54). Cleavages from positions 243, 246, and 274 have been
colored yellow, blue, and
red, respectively. Where there were cleavages from two
positions, the bases have been colored as follows: positions 243 and
246, green; positions 246 and 274, purple.
D, the peptidyltransferase center. The region of the 50 S
subunit containing the peptidyltransferase domain has been amplified
and presented as a space fill representation with the cleaved
nucleotides colored as in C. For reference,
peptide bond formation products in the A- and P-site are shown in
brown (CCA in P-site) and yellow-orange
(CC-puromycin-dipeptide (Phe-Tyr)), and position A2451
(A2486) is colored in light
blue. Figures were generated with the Swisspdb viewer (72)
and rendered with POVRAY (available on the World Wide Web at
www.povray.org).
Interaction of Amino Acids Flanking the SPF Motif of RF2 with the 16 S rRNA
Radicals generated from two residues flanking the SPF motif, 201 and 209, cleaved 16 S rRNA only, and they are illustrated globally on the Thermus thermophilus 30-ribosomal subunit structure (45) in Fig. 5B. Strong cleavages were detected from residue 209 in the highly conserved 530 loop, an important element of the decoding center, which cross-links 3' to the A-site codon in the mRNA (46) and is protected by the A-site tRNA (47). Cleavages were also detected in the 16 S rRNA at nucleotide 1054, universally conserved in eubacteria (48), from both positions 201 and 209 of RF2. Substitution of C1054 for A results in elevated suppression of UGA stop codons (49) and a decrease in both the effective association rate constant kcat/Km for binding of RF2 to the ribosome and the catalytic rate of peptidyl-tRNA hydrolysis (50, 51). In support of the specific interaction of RF2 with nucleotide 1054 of the 16 S rRNA, this base was not cleaved when radicals were generated from the equivalent position of RF1 (27). The C1054A mutant caused only a very small decrease in kcat/Km for RF1-dependent termination (50, 51). This site of cleavage is compelling, because C1054 makes interactions with the third base pair (wobble position) of the A-site codon:anticodon helix (52).
Identification of a small region of the 16 S rRNA that is unique for RF2-mediated termination suggests that although the decoding release factors of E. coli have overlapping binding sites on the ribosome, additional interactions are necessary to impart codon specificity. Nucleotides immediately adjacent to position 1192 were identified as potential sites of interaction with RF2 in our studies but not with RF1 (27). Changes in nucleotide C1192 of helix 34 were shown to cause defects in both RF1- and RF2-dependent binding to the ribosome, with RF2 more affected by the mutations C1192U and C1192A than RF1 (53).
Site-directed hydroxyl radical cleavages were also detected in an exposed rRNA element surrounding nucleotide 1230 (helix 30), and in the 790 loop of helix 24 of the 16 S rRNA. In the 3-Å crystal structure of the 30 S subunit from T. thermophilus, the subunits were packed such that the spur (a protruding RNA structure) of one subunit was inserted into the P-site of the neighboring subunit (45). Several regions of the 16 S rRNA including nucleotides 1229-1230 were shown to be in contact with the spur, suggesting that residues 201 and 209 of A-site-bound RF2 also are relatively close to elements of the ribosomal P site. The 790 loop is located in the platform of the small ribosomal subunit at the subunit interface. Identical cleavages were detected in this loop from RF1 (27) and the tip of domain IV of EF-G (the tRNA anticodon mimicry domain) (26).
Interaction of Amino Acids Flanking the GGQ Motif of RF2 with the 23 S rRNA
The only obvious region of conservation between the prokaryotic and eukaryotic factors is a tripeptide motif, GGQ. In eRF1, the GGQ motif resides within an exposed loop at the tip of domain 2 (19). Site-directed mutagenesis of the two glycine residues in yeast eRF1 (19) and human eRF1 (6) completely abolished the peptidyl-tRNA hydrolysis activity of the factors, without affecting their decoding function. In E. coli RF2, the proposed equivalent GGQ motif (residues 250-252) is positioned within an exposed loop of domain 2 near a protease-sensitive site, cleavage at which was shown to inactivate the peptidyl-tRNA hydrolysis activity of the factor. From these studies, the GGQ motif or its context is presumed to play a role in the peptidyl-tRNA hydrolysis activity of both decoding RFs. Cleavages from sites close to the GGQ motif (243 and 246) are shown globally on the Haloarcula marismortui 50 S ribosomal subunit structure (54) in Fig. 5C as well as those from the naturally occurring cysteine at position 274.
The Peptidyltransferase Center-- Cleavages in the PTC are illustrated in Fig. 5D, with key reference points added for clarity. Radicals generated from residues near the GGQ motif of RF2 cleaved bases in the 23 S rRNA PTC of domain V. For example, hydroxyl radicals originating from positions 246 (and 274) of RF2 specifically cleaved A2602, a universally conserved bulged nucleotide that is protected by the aminoacyl moiety of the A-site-bound tRNA (36). This base was also cleaved when radicals were generated from the 5'-CCA terminus of the deacylated tRNA (24), and it specifically cross-links to sparsomycin, an inhibitor of the ribosomal peptidyltransferase and termination (37). Mutagenesis of A2602 selectively inhibited RF-mediated termination over transpeptidation, suggesting that it may play a specific role in peptidyl-tRNA hydrolysis.3 Nucleotide G2583, substitutions at which diminish peptidyltransferase activity (55), was also specifically cleaved when Fe(II) was tethered to position 246 of RF2.
Additional sites within the central ring of domain V of the ribosomal PTC cleaved by hydroxyl radicals originating from position 243 or 246, include nucleotides 2470-2471, 2482-2483, 2555-2556, and 2568, mutations at which have been shown to dramatically reduce peptidyltransferase activity (56). Radicals from position 246 of RF2 also specifically cleaved nucleotides (i) within the A-loop of domain V of the 23 S rRNA (nucleotides 2556-2557) (55, 57), bases that are protected by the 3' terminus of the A-site-bound tRNA (36, 38, 39); (ii) nucleotides 2253-2254 within the P-loop of domain V of the 23 S rRNA, protected by the 3' terminus of the P site tRNA (as well as from residue 274) (36, 38, 39); (iii) the universally conserved nucleotide A2451, which makes the closest approach to the transition state analogue in the crystal structure of the 50 S subunit from H. marismortui (41).
The GTPase Center-- Nucleotides 1074-1076 were cleaved when Fe(II) was tethered to positions 243 and 246 of RF2, supporting a proposed interaction of the factor with this region of the 23 S rRNA. These nucleotides form part of the GTPase-associated center of the 23 S rRNA (domain II). A 23 S rRNA mutation located within this center, G1093A, facilitates in vivo UGA-specific suppression (58) and decreases the effective rate of association of RF2 with the ribosome (50, 51). Although cleavages were not detected around nucleotide 1067 from the selected positions on RF2, E. coli RF1 was shown to footprint on both nucleotides 1067 and 1093 of the GTPase domain (27). In the crystal structure of a GTPase center fragment bound to ribosomal protein L11 (59, 60), the 1093-1098 and 1065-1073 loops are situated very close to each other. This is also seen in the Deinococcus radiodurans structure (61). This suggests that both loops of the GTPase center may interact with a compact region of the decoding RFs. L11 has been shown both in vitro (62-64) and in vivo (65-67) to be essential for RF1-dependent binding and UAG termination but to dampen RF2-dependent ribosome binding and UGA termination.
The GTPase-associated center and nucleotides within domain V of the ribosomal PTC, together with the sarcin-ricin loop (centered on position 2660), form a defined pocket on the molecular surface of the three-dimensional structure of the large ribosomal subunit. Combinations of these functionally important regions of the 23 S rRNA have previously been identified as important sites of interaction with tRNA (24), EF-G (26), and RF1 (27), suggesting that the observed structural and functional mimicry between these factors, extends to similarity in their sites of interaction with the 70 S ribosome.
The Subunit Interface-- Radicals originating from position 243 or 246 of RF2 cleaved nucleotides near the end of a long helix of domain II (nucleotides 879-900), bridging the ribosomal subunits, and nucleotides centered around positions 1943 and 1965 of domain IV (also at the subunit interface), bases also targeted from the equivalent position 229 of RF1 (27). Interaction of RF2 with nucleotides at the ribosomal subunit interface supports the earlier localization of the factor to this region of the ribosome by immunoelectron microscopy (68).
The Paradox of the Structural and Biochemical Evidence to Explain How RF2 Is Positioned on the Ribosome
These findings support the original tRNA analogue proposal that
the RF spans the two active centers of the ribosome like a tRNA but are
difficult to reconcile with the solution structure of RF2. They support
the suggestion that the RF may undergo a significant change in
conformation upon binding to the ribosome. New data from cryoelectron
microscopy of the E. coli ribosome in a post-termination
complex with RF2 (69, 70) suggests strongly that the RF has a different
conformation from that seen in the solution structure. Rather, in the
reconstructions, domain 1 of RF2 contacts the L11 domain, the SPF loop
is positioned close to the mRNA, and the GGQ motif binds close to
the PTC (69, 70). These conclusions are completely consistent with our
data. Cryoelectron microscopy has also revealed that the aminoacyl-tRNA
undergoes an accommodation and forms new contacts with the active
center during codon:anticodon recognition in the A-site. This suggests an active role for the tRNA in transmitting a signal upon faithful codon recognition for subsequent events (71). RFs may play a similar
role in codon recognition and transmit a signal that extends to the
PTC. The importance of residue 246 in RF2 for an active peptidyl-tRNA
hydrolysis domain (12) and the altered footprinting pattern when this
is switched to an active domain permanently (T246S or Ala) is a strong
indicator that faithful codon recognition transmits a conformational
signal through the RF to activate the hydrolysis event. In RF2, the GGQ
motif protrudes from a domain with structural homology to ribosomal
protein S5. In S5, this changes from a loop to a straight hairpin
upon binding to the ribosome and is inserted between RNA loops. A
similar change in RF2, if mediated by transmission of a signal from the
codon recognition domain, would allow the loop containing
250GGQ252 and its surrounding residues to reach
further into the PTC and perhaps be oriented to facilitate
peptidyl-tRNA hydrolysis.
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ACKNOWLEDGEMENTS |
---|
We thank Professor Rob Smith (Chemistry Department, University of Otago) for overseeing the synthesis of BABE and Christine Hart and Tina Edgar for assistance with in vitro assays and protein purification.
![]() |
FOOTNOTES |
---|
* This work was supported by Postgraduate Scholarships from the University of Otago and the Health Research Council of New Zealand (to D.-J. G. S.), the Royal Society of New Zealand Marsden Fund (to W. P. T.), at the beginning an International Research Scholar Award from the Howard Hughes Medical Institute (to W. P. T.), and a Human Frontier Science Program Grant Number RG32/97 (to Y. Nakamura (Tokyo), L. Kisselev (Moscow), M. Philippe (Paris), and W. P. Tate (Dunedin)).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address: Malaghan Institute of Medical Research, P.O. Box
7060, Newtown, Wellington South, New Zealand.
¶ Supported by the Alexander von Humboldt Foundation.
To whom correspondence should be addressed. Tel.:
64-3-479-7864; Fax: 64-3-479-7866; E-mail:
warren.tate@stonebow.otago.ac.nz.
Published, JBC Papers in Press, November 27, 2002, DOI 10.1074/jbc.M211024200
2 D.-J. G. Scarlett, C. F. Hart, and W. P. Tate, manuscript in preparation.
3 N. Polacek and A. Mankin, personal communication.
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ABBREVIATIONS |
---|
The abbreviations used are: RF, release factor; BABE, 1-(p-bromoacetamido-benzyl)-EDTA; CL, cysteineless (C128A/C274S) variant of RF2; DTNB, 5,5'-dithiobis-(2-nitrobenzoic acid) (Ellman's reagent); PTC, peptidyltransferase center.
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