Is the rsUGA decoded by RF2 like a typical UGA stop signal?
Vectors were constructed with the rsUGA (+1 to +3 bases) within the fdhF sequence (−9 to +52) cloned between two reporter genes (Figure 1A) to test whether changing the sequence surrounding the rsUGA affected the overall translational fate. Changes were made that were known to affect the kinetics of decoding UGA termination signals by RF2. The selenocysteine incorporation readthrough event is measured by the luciferase activity derived from expression of the luc+ downstream reporter, whereas a measure of both readthrough and termination events is determined from the activity of β-galactosidase expressed from the lacZ upstream reporter.
Figure 1. An experimental system to investigate translational fate when the selenocysteine incorporation site of fdhF is altered. (A) Variations to the wild-type fdhF sequence inserted into pBM. (B) The mean readthrough efficiency (and standard errors) for each sequence expressed as a percentage. (C) The relative RF selection rate for different constructs. The SEM are shown.
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Multiple isolates of clones containing the same construct, or containing different constructs, gave very similar β-galactosidase activities [within the standard error of the mean (SEM)], indicating similar expressions independently of the construct or clone (data not shown). The construct pBMUAA, with the rsUGA changed to UAA, was used as a control as it is unable to support translational elongation since the tRNASec anticodon is not complementary to this codon (Figure 1B). Nevertheless, a small amount of luciferase activity was observed with pBMUAA. Modification of the rsUGA context (SLG23C), where there is a base change in the apical loop of the fdhF hairpin known to abolish selenocysteine incorporation in vivo (Heider et al., 1992), gave a similar level of ‘apparent readthrough’. Therefore, both measurements are likely to reflect near-cognate readthrough and/or reinitiation downstream of the rsUGA. The rsUGA was replaced by two near-cognate sense codons, UGG (Trp) and UGC (Cys), to establish luciferase activity values when termination was precluded (100% readthrough). Both constructs gave very similar values (Figure 1B).
These modifications to the rsUGA (UAA, UGG or UGC, and SLG23C) set values for upper and lower limits of potential competition for decoding at the recoding site. The UGA and its natural surrounding context allowed almost 50% readthrough, reflecting near equal competition between the termination and elongation events under the growth conditions used for these experiments (Figure 1B, UGAC). This competition was affected by changes to the sequences surrounding the rsUGA to reflect a stronger or weaker stop signal. We have established that the native rsUGA site has an upstream sequence that supports efficient termination while the downstream sequence is relatively weak (Major, 2001). We predicted that there was potential to strengthen the termination signal modestly as well as to weaken it. The vectors UGAU and ‘S’ (strong upstream and downstream contexts) (Figure 1A) contain modified fdhF and have sequence elements predicted to increase the decoding rate of a UGA stop signal, while construct ‘W’ (weak upstream and downstream contexts) should decrease the decoding rate. Simply by changing the +4 base from C to U, a consistent decrease in luciferase activity occurred (reflecting a drop from ∼9 to 8 ribosomal passages in 20) and this was also the case with the strong context,‘S’. In contrast, the number of ribosomal passages supporting selenocysteine incorporation increased from 8 to 12 in 20 with the weak termination signal ‘W’. These data indicate that selenocysteine incorporation efficiency at rsUGA responds to parameters known to be important at a more typical UGA termination site.
The relative termination efficiencies supported by rsUGA present in each of these constructs can be analysed further by calculating the rate of RF selection at each site (Pedersen and Curran, 1991). The rate of RF selection (RRF) indicates the rate of stop signal recognition by the RF and, in this case, is the rate of termination (tr) relative to the rate of readthrough (er), i.e. RRF = tr/er. RRF for each of the constructs is shown in Figure 1C. These selection rates reflect the likelihood that a termination event will occur before selenocysteine incorporation when the rsUGA is at the A site during a given ribosomal passage. For example, a decrease in termination value observed with the weak stop signal (‘W’) (Figure 1B) reflected a 2-fold decrease in the rate of productive RF selection into the A site (Figure 1C), allowing for a relative increase in the competitiveness of Sec-tRNASec and increasing the likelihood that it would be the successful decoding molecule. In contrast, the two stronger stop signals have a 1.3- to 1.4-fold increase in RRF, decreasing the competitiveness of the Sec-tRNASec. At the fdhF selenocysteine incorporation site, just as at other sites of UGA stop signals, if the efficiency with which the signal is decoded by RF2 is increased, so is its competitiveness with cognate or near-cognate events (Major, 2001).
Is the fdhF rsUGA ‘visible’ to RF2 in the decoding site?
The in vivo studies suggested the Sec-tRNASec was able to compete effectively with premature chain termination under the conditions of our experiments. How might this be mediated? Data from structural predictions (Zinoni et al., 1990) and from use of chemical and enzymatic probes (Hüttenhofer et al., 1996a) suggest that the fdhF rsUGA is in a stem–loop, and this is a critical feature that allows the Sec-tRNASec to be ultimately competitive. However, during translation, the stem–loop must be unfolded, as decoding rsUGA by either cognate decoding molecule requires the codon to be presented as a single-stranded structure. Despite the fact that the signal can be strengthened or weakened, competition could be affected by an intrinsically poor rate of RF2 selection when the rsUGA (compared with a typical UGA signal) is occupying the A site. The specific sequences of the fdhF decoding site may make the rsUGA less accessible to the factor within the ribosomal active centre. A site-directed cross-linking strategy between the rsUGA and the factor was used to determine whether the rsUGA in the fdhF sequence context was accessible to RF2. We have shown previously that RF2 is in direct contact with a UGA termination codon positioned in the ribosomal A site. A zero-length cross-link can be formed between RF2 and a radioactively labelled mRNA analogue that contains a thioU at the +1 position of the stop signal (Brown and Tate, 1994).
Figure 2A shows the site-directed cross-linking strategy with mRNA analogues containing fdhF sequences bound to the ribosome, with the rsUGA fixed in the A site by tRNAVal recognizing the previous codon in the P site. The designed mRNAs (used in the studies described in Figures 2 and 3) are shown in Figure 2B. They contain various lengths of fdhF sequence (−N to +N where +1 is the U of the rsUGA). For example, the −9 to +52 mRNA contains three upstream codons and extends downstream to include the stem–loop. Following the cross-linking reaction and before gel analysis, the products are digested with RNase T1, which leaves the factor cross-linked to a radiolabelled tetranucleotide derived from the mRNA. This complex is found at the same position as native RF2 on an SDS–polyacrylamide gel.
Figure 2. Site-directed cross-linking studies to investigate whether RF2 can interact with the rsUGA at the selenocysteine incorporation site. (A) The experimental strategy. A designed mRNA is bound to the ribosome by tRNAVal located in the P site, allowing a thioU in the +1 position of the stop codon to form cross-links with the decoding RF in the A site. (B) The mRNA sequences used for the cross-linking studies (Figures 2 and 3). (C) Polyacrylamide gel separation of cross-linked complexes after RNase T1 digestion. The complexes were transferred to a nitrocellulose membrane and then detected by autoradiography (left panel) and immunodetection of RF2 (right panel). Lanes 1, 4 and 7, and lanes 3, 6 and 9 show cross-links formed in the presence or absence of RF2 and tRNAVal, respectively. Lanes 2, 5 and 8 show the cross-links formed in the presence of RF2 only. Cross-links between mRNA and RF2 (arrow) and S1 ribosomal protein are shown.
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Figure 3. Cross-linking and ribosomal binding studies with the quarter (+12), half (+23, hSL) and full (+52, SL) stem–loops. (A) Polyacrylamide gel separation of cross-linked complexes digested with RNase T1 then transferred to a nitrocellulose membrane, followed by autoradiographic detection. RF2- (arrow) and S1-specific cross-links are shown. RF2 and tRNAVal were present or absent from the reactions as indicated. (B) Secondary structure predictions for the quarter (−9 to +12), half (−9 to +23, hSL) and full (−9 to +52, SL) stem–loops determined by the program mFold. The free energy predictions for each structure are given. (C) The results of ribosomal binding assays with the hSL and SL stem–loops when different components of the termination complex are present.
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Figure 2C (left panel) indicates that RF2 can cross-link to the +1 thioU of UGA in mRNAs (arrowed) where just the upstream valine codon, UGA, and two more nucleotides of the fdhF sequence are present (−3 to +5, lanes 1 and 2). Increasing the upstream sequence from one to three codons of fdhF sequence (−9 to +5, lanes 4 and 5), or the downstream sequence to +19 (lanes 7 and 8) still supported the cross-link but with decreased intensity. In the absence of tRNAVal, cross-links formed with RF2 (lanes 2, 5 and 8), consistent with previous experiments, demonstrating that RF2 contributes to termination complex alignment (E.S.Poole and W.P.Tate, unpublished data). No cross-links were found when both tRNAVal and RF2 were excluded from the reaction (lanes 3, 6 and 9). A strong cross-link to ribosomal protein S1 could be seen in all lanes and appeared more intense when tRNAVal was absent, consistent with an unoccupied P site allowing the −2 thioU of the GUC codon to contribute to the cross-link reaction in addition to the +1 thioU. The right panel shows an immunoanalysis of a duplicate gel after western transfer, showing RF2 at the same position as the 32P-labelled mRNA fragment. Faint bands in lanes 3, 6 and 9 reflect a small amount of RF2 remaining in the purified ribosome preparations.
Cross-linking reactions using mRNA analogues containing the three codons of upstream sequence and a quarter (−9 to +12), half (−9 to +23) or the full stem–loop (−9 to +52) are shown in Figure 3A. In these cases with a static termination complex, inclusion of fdhF sequences supported cross-links to RF2 with the control (−3 to +5, lane 1) and quarter stem–loop (lane 2) but not with the half stem–loop sequence (lanes 3–6) or when the full-length stem–loop is present (lanes 7–10). Despite this, these mRNAs formed cross-links to ribosomal protein S1 (Figure 3A, all lanes), indicating they were able to bind to the ribosome. The S1 cross-link intensity can vary with the mRNA sequence and length, as well as whether other components of the termination complex are present, as observed in Figure 3A. However, even at very long exposures (data not shown), no cross-links to RF2 were observed with the half and full stem–loop mRNAs.
Putative secondary structures formed by these mRNAs containing fdhF sequences and surrounding reporter sequences are shown in Figure 3B. The rsUGA (bold) and possible extra secondary structure contributed by the sequences are shown. Although the −9 to +23 sequence is unable to form the canonical stem–loop structure, the RNA folding program mFold (Zucker, 1989) indicates that it has the potential to form a stable secondary structure. On the other hand, the putative secondary structure of the shorter −9 to +12 mRNA is much weaker. The mRNA secondary structures in these static experimental complexes may contribute to their inability to be positioned on the ribosome in the right orientation for optimal RF2 UGA decoding.
We then used ribosomal binding assays to measure relative binding of the half stem–loop (hSL, −9 to +23) and full stem–loop (SL, −9 to +52) mRNAs to the ribosome in the presence or absence of the other components (Figure 3C). While the two mRNAs bound to the ribosome in a complete termination complex, omission of either RF2 or tRNA improved binding and there was a 2-fold increase in bound mRNA when both RF2 and tRNAVal were omitted. The half stem–loop showed significantly increased ribosomal binding when only tRNAVal was omitted, whereas the full stem–loop mRNA required the absence of both RF2 and tRNAVal before a significant change was observed. Consistent with the observations of Hüttenhofer et al. (1996b), the percentage of the mRNA bound to the ribosome decreased as the mRNA secondary structure increased (∼10% hSL, ∼7.5% SL) and decreased further when all the termination complex components were present.
A cross-link between the +1 U of the UGA and RF2 is a measure of a productive orientation of the stop codon and factor at the decoding site. For example, we have shown previously that the cross-link intensity can be affected significantly by the identity of the P site tRNA (McCaughan et al., 1998), and the results shown in Figure 3A and C indicate that the orientation of the rsUGA mRNAs with respect to RF2 is seriously disturbed in these static termination complexes. Heating the mRNAs to relax secondary structures immediately before complex formation did not restore the cross-linked product, although that with ribosomal protein S1 was somewhat enhanced (data not shown). However, rapid reformation of secondary structure is possible under the conditions used for these experiments. In vivo, the critical moment would be when the translating ribosome disrupts the secondary structure of the stem–loop to allow RF2 and Sec-tRNASec access to the rsUGA as it reaches the A site in a single-stranded conformation. This scenario is distinct from the static experiments with the rsRNA analogues used here.
Can competition for decoding the rsUGA be influenced by changes in the concentrations of the decoding molecules, RF2 or tRNASec?
Expression vectors were constructed containing prfB (pTGRF2M) encoding RF2T246S, selC (pTGSelC) encoding tRNASec, or prfA (pTGRF1) encoding RF1, and were introduced into bacteria containing the test fdhF constructs. Expression of RF2T246S, where the threonine residue at position 246 is substituted with serine, allows more reliable production of a functionally active factor than when native RF2 is expressed, and has been used in these experiments. The host vector, pTG, was included as a control to enable any effects of the overproduction of tRNASec, RF1 or RF2 to be separated from general effects resulting from the presence of the additional vector. Overexpression allowed selection rates of the decoding factors (RF1 or RF2) and Sec-tRNASec to be influenced directly through changes in their cellular concentrations. No differential effects on the activity of the downstream product, luciferase, in the control UGG- or UGC-modified test vectors were observed with any of the pTG recombinant series (Figure 4A). They also had little effect on UAA and SLG23C (altered stem–loop) controls where decoding was near 100% termination (Figure 4A). However, at the native rsUGA, overexpression of RF2 decreased readthrough, but overexpression of tRNASec increased readthrough. When the data were analysed for relative rate of RF selection at rsUGA, there was a nearly 2-fold decrease as tRNASec was overexpressed, which contrasted with a 2-fold increase as RF2 was overexpressed (Figure 4B). Overexpression of RF2 was measured by immunoanalysis and gave a 3- to 5-fold change in cellular RF2 concentration. Significantly, overexpression of the non-cognate factor RF1 (recognizing UAG and UAA) did not influence competition.
Figure 4. The effect of increased cellular concentration of RF2 and tRNASec on selenocysteine incorporation at different signals. (A) Selenocysteine incorporation efficiency on overexpression of the control vector (pTG), tRNASec (pTGSelC) and RF2 (pTGRF2M) at variant sequences of rsUGA as indicated, and the stem–loop variant SLG23C. (B) The relative RF selection rate at the natural selenocysteine incorporation site under conditions of overexpression of the control vector (pTG, shaded bar), tRNASec (pTGSelC, closed bar), RF2 (pTGRF2M, open bar) and non-cognate RF1 (pTGRF1, hatched bar). The standard errors of the mean are shown.
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Growth conditions modulate competition at the fdhF rsUGA
To determine whether the observed competition for rsUGA decoding is regulated according to the physiological state of the bacterial cell, the effect of growth rate was investigated (Figure 5). To achieve divergent growth rates, parallel cultures were grown from the same inocula, but in rich or two different minimal media. As growth rate decreased, luciferase activity increased, indicating that readthrough at the natural rsUGA site was significantly enhanced (Figure 5A). Experiments with two different readthrough constructs (pBMUGG and pBMUGC) and a termination construct (pBMUAA) were used to assess whether background readthrough or termination rates changed under different growth conditions. No significant differences were observed. At the highest growth rate, <30% of ribosomal passages through the rsUGA resulted in selenocysteine incorporation while, at the slowest growth rates, this increased to 60% of ribosomal passages. The very slow growth rate of the E.coli FJU112 strain in this latter case results in an elevation of the background level of luciferase activity with the control plasmids pBMG23C and pBMUAA. This is likely to result from increased frequency of events that are independent of the recoding event. The RRF for the different media conditions is shown in Figure 5B.
Figure 5. The effect of growth rate on selenocysteine incorporation. (A) The effect of growth rate on selenocysteine insertion at different recoding site signals. Readthrough (%) was determined under high (Rich, doubling time = 25 min, shaded bars), medium (Min C, doubling time = 90 min, closed bars) and slow (Min G, doubling time = 125 min, open bars) bacterial growth conditions with constructs for the different fdhF recoding sites as indicated. The SEM are shown. (B) RF2 selection rates at the natural rsUGA under the different growth conditions.
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