A dynamic competition between release factor 2 and the tRNASec decoding UGA at the recoding site of Escherichia coli formate dehydrogenase H
2001; Springer Nature; Volume: 20; Issue: 24 Linguagem: Inglês
10.1093/emboj/20.24.7284
ISSN1460-2075
Autores Tópico(s)Genomics and Phylogenetic Studies
ResumoArticle17 December 2001free access A dynamic competition between release factor 2 and the tRNASec decoding UGA at the recoding site of Escherichia coli formate dehydrogenase H John B. Mansell John B. Mansell Department of Biochemistry and Centre for Gene Research, University of Otago, PO Box 56, Dunedin, New Zealand Search for more papers by this author Diane Guévremont Diane Guévremont Department of Biochemistry and Centre for Gene Research, University of Otago, PO Box 56, Dunedin, New Zealand Search for more papers by this author Elizabeth S. Poole Elizabeth S. Poole Department of Biochemistry and Centre for Gene Research, University of Otago, PO Box 56, Dunedin, New Zealand Search for more papers by this author Warren P. Tate Corresponding Author Warren P. Tate Department of Biochemistry and Centre for Gene Research, University of Otago, PO Box 56, Dunedin, New Zealand Search for more papers by this author John B. Mansell John B. Mansell Department of Biochemistry and Centre for Gene Research, University of Otago, PO Box 56, Dunedin, New Zealand Search for more papers by this author Diane Guévremont Diane Guévremont Department of Biochemistry and Centre for Gene Research, University of Otago, PO Box 56, Dunedin, New Zealand Search for more papers by this author Elizabeth S. Poole Elizabeth S. Poole Department of Biochemistry and Centre for Gene Research, University of Otago, PO Box 56, Dunedin, New Zealand Search for more papers by this author Warren P. Tate Corresponding Author Warren P. Tate Department of Biochemistry and Centre for Gene Research, University of Otago, PO Box 56, Dunedin, New Zealand Search for more papers by this author Author Information John B. Mansell1, Diane Guévremont1, Elizabeth S. Poole1 and Warren P. Tate 1 1Department of Biochemistry and Centre for Gene Research, University of Otago, PO Box 56, Dunedin, New Zealand *Corresponding author. E-mail: [email protected] The EMBO Journal (2001)20:7284-7293https://doi.org/10.1093/emboj/20.24.7284 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Factors affecting competition between termination and elongation in vivo during translation of the fdhF selenocysteine recoding site (UGA) were studied with wild-type and modified fdhF sequences. Altering sequences surrounding the recoding site UGA without affecting RNA secondary structure indicated that the kinetics of stop signal decoding have a significant influence on selenocysteine incorporation efficiency. The UGA in the wild-type fdhF sequence remains 'visible' to the factor and forms a site-directed cross-link when mRNA stem–loop secondary structure is absent, but not when it is present. The timing of the secondary structure unfolding during translation may be a critical feature of competition between release factor 2 and tRNASec for decoding UGA. Increasing the cellular concentration of either of these decoding molecules for termination or selenocysteine incorporation showed that they were able to compete for UGA by a kinetic competition that is dynamic and dependent on the Escherichia coli growth rate. The tRNASec-mediated decoding can compete more effectively for the UGA recoding site at lower growth rates, consistent with anaerobic induction of fdhF expression. Introduction During synthesis of formate dehydrogenase H (FDHH) in Escherichia coli, a cis-acting mRNA element, the selenocysteine incorporation sequence (SECIS), programmes recoding of an internal UGA as selenocysteine (Heider et al., 1992). The co-translational incorporation of selenocysteine requires tRNASec, a tRNA capable of decoding the fdhF recoding site (rsUGA) and SELB, a selenocysteyl-tRNASec-specific elongation factor. While the genetic requirements for selenocysteine incorporation in E.coli are well characterized (Böck et al., 1991), the interaction of the SELB quaternary complex with the ribosome is still not well understood. A variety of approaches indicates that SELB must be complexed with the SECIS element for a productive interaction with the ribosome. This suggests that SECIS element binding induces a conformational switch in SELB that facilitates formation of an anticodon–codon interaction between the Sec-tRNASec and the UGA codon. The SECIS element appears to act as a safety switch, preventing normal UGA termination codons being decoded as selenocysteine by SELB–GTP–Sec-tRNASec complexes (Klug et al., 1997; Hüttenhofer and Böck, 1998). Incorporation of selenocysteine also necessitates that the canonical decoding of UGA as termination be subverted. It has been found previously that C following a UGA codon, as is found at the fdhF selenocysteine insertion site, induces a much lower termination efficiency in vivo than if any of the other three bases are in that position (Poole et al., 1995). There is now considerable evidence that the nucleotides surrounding the stop codon affect termination efficiency (Salser et al., 1969; Bossi and Roth, 1980; Engelberg-Kulka, 1981; Kopelowitz et al., 1992). Indeed, an emerging picture of the protein synthesis termination signal in prokaryotes suggests that the stop codon forms the core of a larger element containing both upstream and downstream sequences (Tate et al., 1996). For example, in vitro cross-linking experiments show that the release factor (RF) is in intimate contact with not only the stop codon but also the following three bases (Poole et al., 1998), and in vivo readthrough studies indicate that the two C-terminal amino acids of the nascent polypeptide (and thereby the two codons preceding the stop codon in the mRNA) also affect termination efficiency (Mottagui-Tabar et al., 1994; Björnsson et al., 1996). The kinetics of termination at the stop signal are due largely to the composition of these different elements that are presented to the RF. Furthermore, stop signals of differing efficacies appear to offer another level of control in regulating gene expression, particularly at recoding sites (Crawford et al., 1999). Following the postulate that a consequence of having one signal to support two distinct decoding events is that there is competition for the signal, this work investigates how competition at the fdhF rsUGA is mediated in vivo. We show that the relative efficiencies of competing decoding events determine the translational fate of the fdhF rsUGA. Results 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. Download figure Download PowerPoint 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. Download figure Download PowerPoint 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. Download figure Download PowerPoint 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. Download figure Download PowerPoint 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. Download figure Download PowerPoint Discussion Termination efficiency is a determinant of selenocysteine incorporation efficiency at the fdhF rsUGA Sequence context around the fdhF rsUGA could modulate selenocysteine incorporation efficiency through a direct influence on the intrinsic selection rate of either of the two competing decoding molecules, RF2 and Sec-tRNASec. Unless higher level structures in the mRNA affect binding of one or both of these trans factors into the ribosomal A site so that they are selected at markedly different rates, increasing or decreasing the selection rate of one of the decoding molecules would be predicted to lead to a converse effect on the selection rate of the other molecule. Indeed, this simple competition seems to be operating at the fdhF selenocysteine insertion site. The sequence modification to the recoding site present in pBMUGAU favoured termination according to prediction. The substitution of U for C at the position following the stop codon has been shown previously to increase termination efficiency at UGA signals in vivo (Poole et al., 1995). The increase in termination efficiency observed for pBMUGAU is not as dramatic as the effect of C→U substitution on the efficiency of termination at the RF2 frameshift rsUGA (Poole et al., 1995), but the mechanisms of the competing events and the kinetic consequences are distinct in the two cases. Studies have demonstrated that the recruitment for suppressor tRNAs is also influenced by the base following a stop codon, although this effect was shown to be mediated through the enhanced ability of purines 3′ to the codon to form stacking interactions with the suppressor tRNA (Kopelowitz et al., 1992). The C→U transition at the base 3′ to the rsUGA in pBMUGAU would not be predicted to affect the stability of the anticodon– codon interaction between tRNASec and rsUGA. The sequence elements chosen for upstream and downstream contexts of the recoding site UGA in pBM'S' and pBM'W' have been shown to increase and decrease termination efficiency, respectively, as predicted (Major, 2001). It is most likely that the changes in selenocysteine incorporation efficiency observed here are also mediated through changes to the intrinsic rate of RF selection. The increase in termination with pBM'S' was modest, but the natural sequence context upstream of the wild-type fdhF rsUGA favours relatively efficient termination. A strong upstream termination context overcoming the effect of the weaker downstream context is consistent with observations that this can occur in the natural sequence of a poorly expressed E.coli gene (Mottagui-Tabar and Isaksson, 1997). The changes in selenocysteine incorporation efficiency in response to modifications to the sequence context are unlikely to be due to perturbations of the mRNA stem–loop that programmes UGA recoding. The stem– loops encoded by pBM'S' and pBM'W' are similar in terms of their predicted secondary structure and stabilities (data not shown), yet the sequence modifications have opposing effects on selenocysteine incorporation. This suggests that neither sequestering the rsUGA codon to prevent recognition by RF2, nor an increased ribosomal pause at the site provided by the additional stability conferred on the entire stem–loop from the lower helical region are essential for efficient selenocysteine incorporation. This is consistent with results of a mutational analysis of the structural requirements of the fdhF hairpin for selenocysteine incorporation (Liu et al., 1998). In an innovative approach, Sandman and Noren (2000) have used phage display to show that the nucleotide following the internal TGA in the fdhF gene determines whether suppression with a near-cognate tRNA competes with co-translational selenocysteine incorporation: a purine nucleotide in this position supports competition whereas a pyrimidine, such as the naturally occurring C, does not. Liu et al. (1999) propose an extended selenocysteine element in the mRNA that includes the nucleotide following the rsUGA and the two preceding codons, preventing near-cognate readthrough of the UGA when selenium is limiting. We interpret this to reflect elements that are important for the termination signal and strengthen or weaken termination efficiency (Figure 1B and C; Major, 2001). Selenocysteine occupies the active site of FDHH. Therefore, the codons immediately upstream and downstream of the rsUGA encode residues that influence the local environment of selenocysteine and, consequently, the activity of the site. Furthermore, th
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