Mapping Functionally Important Motifs SPF and GGQ of the Decoding Release Factor RF2 to the Escherichia coli Ribosome by Hydroxyl Radical Footprinting

IMPLICATIONS FOR MACROMOLECULAR MIMICRY AND STRUCTURAL CHANGES IN RF2*

Debbie-Jane G. ScarlettDagger, Kim K. McCaughan, Daniel N. Wilson§, and Warren P. Tate||

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

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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-, mk+), relA1, supE44, lambda -, Delta (lac-proAB), F', traD36, proA+B+, lacIqZDelta M15] (Promega), whereas expression studies from the pET3a-based vectors utilized the E. coli strain BL21(DE3) pLysS [hsdS gal (lambda  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-beta -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.

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 epsilon 412 = 13,600 M-1 cm-1 for the chromophore.

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 beta -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.

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 -80 °C in preparation for primer extension.

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 [gamma -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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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).


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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.

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).


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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.

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.


                              
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Table I
Codon-dependent specific ribosome binding activities of the RF2 cysteine variants before and after conjugation with Fe(II)-BABE
Binding assays were performed at UGA and UAG using equimolar ratios of ribosome/RF. Experiments were repeated in triplicate using duplicate samples of each RF, and codon-dependent ribosome binding activities are expressed as the average RF·70S·[32P]stop codon complex formed (pmol) ± S.E. NA, not applicable.


                              
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Table II
Specific codon-dependent peptidyl-tRNA hydrolysis activities of the RF2 cysteine variants before and after conjugation with Fe(II)-BABE
Release assays were performed at UGA and UAG using 5 pmol of RF protein. Experiments were repeated in triplicate using duplicate samples of each RF, and peptidyl-tRNA hydrolysis activities are expressed as the average [3H]fMet released (fmol) ± S.E. NA, not applicable.

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).


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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).

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).

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha -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 beta  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.

    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.

Dagger 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.

    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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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