ABSTRACT
The UGA codon, usually a stop codon, can also direct the incorporation into a protein of the modified amino acid selenocysteine. This UGA decoding process requires a cis-acting mRNA element called `selenocysteine insertion sequence' (SECIS) that can form a stem-loop structure. In Escherichia coli the SECIS of the selenoprotein formate dehydrogenase (FdhH) mRNA has been previously described to consist of at least 40 nucleotides following the UGA codon. Here we determined the nature of the minimal SECIS required for the in vivo UGA-directed selenocysteine incorporation in E.coli. Our study is based on extensive mutational analysis of the fdhF SECIS DNA located in a lac'Z fusion. We found that the whole stem-loop RNA structure of the E.colifdhF SECIS previously described is not required for the UGA-directed selenocysteine incorporation in vivo. Rather, only its upper stem-loop structure of 17 nucleotides is necessary on the condition that it is located in a proper distance (11 nucleotides) from the UGA codon. Based on these observations, we present a new model for the minimal E.coli SECIS.
The results of recent research in both prokaryotic and eukaryotic systems has permitted the establishment of an extended genetic code in which UGA, usually a stop codon, can also direct the incorporation of the modified amino acid selenocysteine (for reviews see 1-4). Proteins containing selenocysteine residues are called selenoproteins and these are mostly oxido-reductases (for reviews see 5,6). That selenocysteine residues have been shown to play an essential role in the activity of selenoproteins indicates the importance of the selenocysteine incorporation pathway. For UGA to be decoded (`recoded' in ref. 7) as selenocysteine, several additional elements must be present. These include several trans elements and a stem-loop structure forming a cis-acting mRNA element called a `selenocysteine insertion sequence' (SECIS) (3,8,9; and for reviews see 10,11). Although prokaryotic and eukaryotic selenocysteine incorporation systems have some analogous elements, they nevertheless seem to operate through different mechanisms.
In Escherichia coli, a number of genes have been identified in which UGA directs the incorporation of selenocysteine instead of acting as a stop codon. These include genes fdhF (12) and fdnG (13), encoding the selenocysteine-containing enzymes formate dehydrogenase H and N, respectively. Immediately downstream from the selenocysteine specifying UGA in the mRNA of each of these polypeptides is found a SECIS that has been described as consisting of at least 40 nucleotides (nt) capable of forming a stem-loop RNA structure. Insertion of a DNA segment directing this specific SECIS into a heterologous gene such as lacZ results in the incorporation of selenocysteine into the corresponding polypeptide (9,12,14-17). A number of trans elements are also required, including tRNASec (SelC), which is a specialized tRNA containing a UCA anticodon (18). tRNASec is recognized by a special elongation factor, called SelB which has been shown in vitro to bind specifically to both tRNASec and to the mRNA stem-loop structure (formed by the SECIS) adjacent to the 3' side of the UGA codon (19). Recent in vitro work by Kromayer and colleagues (20) has shown that selenocysteinyl-tRNASec can be bound by the N-terminal part of E.coli SelB (homologous to EF-Tu), and that the C-terminal subdomain of SelB specifically binds to the SECIS stem-loop RNA structure of the E.coli selenoprotein formatedehydrogenase (FdhH). They also found that the mRNA motif recognized by SelB can be reduced to a 17 nt minihelix without loss of binding capacity. This minihelix consists of the upper part of the stem-loop RNA structure of the fdhF SECIS, a loop of 6 nt and five adjacent base-paired nucleotides together with a bulged nucleotide.
Here we have determined the nature of the mRNA SECIS motif required forUGA-directed selenocysteine incorporation in E.coli in vivo. We based our study on anextensive mutational analysis of the fdhF SECIS DNA which weinserted into a lac'Z fusion. Previously it has been suggested that the presence of the whole stem-loop structure is required for UGA directed selenocysteine incorporation (9,12,21). On the basis of our results presented here, we suggest that the presence of only 17 nt consisting of the upper stem-loop structure of fdhF, are sufficient to permit this `recoding' choice, on the further condition that this sequence is located 11 nt downstream from the UGA codon.
[75Se]selenite (350 mCi/mmol) was obtained from Amersham (Amersham, UK). Bacteria were grown in YT or in M9 minimal medium (pH 7.0) (22) supplemented with a mixture of amino acids, each at a final concentration of 20 µg/ml. Ampicillin (100 µg/ml) was added to the media in which the plasmid-carrying strains were grown.
The E.coli strains used in this study were MC4100, its [Delta]selC derivative RM1 (15), and its [Delta]selB derivative WL81300 (23).
Plasmid pRM4 was constructed by us previously (15). It carries the fused genes [lambda]cI'-lac'I''Z, into which we inserted the TGA region of the SECIS DNA (from -9 to +47, where the U of the UGA codon is designated as residue number 1) of E.coli fdhF gene in the junction of [lambda]cI'and lac'I''Z. Here we used the PCR technique for the generation of a series of plasmids carrying site-directed mutations in the SECIS of fdhF by using the required primers and with plasmid pRM4 as the template. The sequence of their mutated SECIS RNA is described in Figures 1-5.
All recombinant DNA manipulations were carried out by standard procedures (24). Restriction enzymes and other enzymes used in the recombinant DNA experiments were obtained from New England Biolabs (Beverly, MA). DNA sequencing was done with the Sequenase kit of United States Biochemicals (Cleveland, OH).
Escherichia coli cells were transformed (24) by the plasmid of choice. Single colonies of freshly transformed cells were grown at 37°Con YT plates for 6-8 h and then in M9 liquid medium at 37°C in a rolling drum for 3-4 h to the OD600 = 0.4-0.6. The cells were examined for [beta]-galactosidase activity as described by Miller (22).
Freshly transformed cells were grown in M9 liquid medium with cysteine and methionine (final concentration 200 µg/ml each) at 37°C overnight in the presence of 1.0 µCi [75Se]selenite/ml. During labeling, cold selenite was added to a final concentration of 1.5 µM. Cells were lysed, and the proteins were separated on 5% SDS-polyacryamide gel by electrophoresis and detected by autoradiography as we described previously (15).
For this work we used our plasmid pRM4 (15), which we constructed previously to carry the TGA codon context of the E.coli fdhF gene fused to the lac'Z gene (lac'Z lacking the first eight codons). pRM4 carries nt -9 to +47 of fdhF gene (where the nucleotide U of the UGA codon was designated as nt 1). The mRNA of this region of the fdhF gene has been identified as a stem-loop structure forming SECIS (Fig. 1Aa), the cis acting mRNA element described previously to be required for the in vivo UGA-directed selenocysteine incorporation into the lac'Z fusion product of E.coli (12). Here we have identified the minimal sequence required for UGA-directed selenocysteine incorporation in E.coli in vivo. We carried out extensive mutational analysis on the fdhF SECIS DNA fused to the lac'Z gene on plasmid pRM4. We always studied selenocysteine incorporation into the gene product of the lac'Z fusion in two ways: (i) by measuring UGA-directed (selC-dependent) [beta]-galactosidase activity; and (ii) by measuring the direct incorporation of [75Se]selenite into the polypeptide of the fused genes. Since measuring [beta]-galactosidase activity is the more accurate of the two methods, we used this method for our quantitative determinations of selenocysteine incorporation.
For this set of experiments, we generated site-directed mutations that cause changes in the secondary structure of the fdhF SECIS mRNA downstream from the UGA codon. First, we altered five of the nucleotides (pZL7, Fig. 1Ab) and then 11 of the nucleotides (pZL14, Fig. 1Ac) in the stem region immediately after the UGA codon. We did this such that the nucleotides on the left side of the stem are identical rather than complementary to the corresponding nucleotide on the right side of the stem. As shown, the UGA-directed selenocysteine incorporation is still permitted even when the 11 nt following UGA are not base paired to their corresponding partners in the stem (Fig. 1Bc and Cc). Quantitatively, the level of the UGA-directed [beta]-galactosidase activity is reduced only by ~25% (Fig. 1Bb and Bc). We obtained similar results by changing 11 nt at the right side of the stem to be identical rather than complementary to the unchanged left side of the stem (data not shown). These results suggest that neither the primary nor the secondary structure of this region in fdhF mRNA, designated here as the `intermediary domain' (nt 4-14 and 32-42 from the UGA), are required for the incorporation of selenocysteine into the lac'Z fused polypeptide. However, the mutated construct pZL14 (Fig. 1Ac) still carries a `mini upper-stem-loop' (a stem of 5 bp, one bulged nucleotide, and a loop of 6 nt) that may be responsible for the UGA-directed selenocysteine incorporation. Indeed, when we constructed and then studied plasmid pZL18 (Fig. 1Ad), we found that selenocysteine incorporation was almost completely abolished (Fig. 1Bd and Cd) when only one additional base-pairing is also prevented, thereby reducing the `stem' part of the mini upper-stem-loop structure from 5 to only 4 bp (Fig. 1Ad). Furthermore, selenocysteine incorporation was again permitted (Fig. 1Be and Ce) when we introduced a compensatory mutation (pZL20, Fig. 1Ae). This mutation permits 5 bp in the mini upper-stem-loop due to a C-G bp (Fig. 1Ae) instead of the G-C bp in the SECIS of the pZL14 (Fig. 1Ac). Altogether, the results described so far suggest that the mini upper-stem-loop structure downstream of the UGA on fdhF SECIS mRNA, is required for the process of selenocysteine incorporation into a polypeptide in vivo, while the intermediary domain (nt 4-11 and 32-42) is not.
It was previously suggested that the presence of a C residue, after the UGA of the E.coli SECIS is crucial because it prevents the recognition of UGA by RF2 that actually recognizes 4 nt, the UGA and the first nucleotide at its 3' side; the poorest 4 base terminator is UGAC (25). The results described herein show that the C residue following UGA in fdhF mRNA can be changed to a G residue without affecting the UGA-directed selenocysteine incorporation (Fig. 1). It seems therefore, that at least in the case of fdhF SECIS, the presence of the C residue at this position is not crucial for the prevention of the recognition of UGA by RF2.
Having shown that the base pairing of the intermediary domain of the fdhF SECIS is not required for UGA-directed selenocysteine incorporation into the polypeptide product of the fdhF-lac'Z gene fusion, the following question arose. Does the length of this intermediary domain affect the efficiency of selenocysteine incorporation? Using pZL14 (Fig. 2Ab) as the basis for these studies, we either effected deletions of 3 nt [Delta]G4A5G6 (pZL24, Fig. 2Ac) or of 6 nt [Delta]G4A5G6C7U8G9 (pZL27, Fig. 2Ad), or additions of 3 nt UCA (pZL28, Fig. 2Ae) or 6 nt, UCAUCA (pZL29, Fig. 2Af). Reducing or increasing the length of the intermediary region by 3 nt decreases the level of the UGA-directed selenocysteine incorporation by ~50% (Fig. 2Bc,e and Cc,e). Moreover, either reducing or increasing the length of the intermediary region by 6 nt almost completely abolishes the UGA-directed selenocysteine incorporation (Fig. 2Bd,f and Cd,f).
The solution structure of RNA hairpins within fdhF mRNA has recently been determined by Hüttenhofer and colleagues (21) who showed that there are three bulged nucleotides in the stem of the upper-stem-loop. What is the role in vivo, if any, of the bulged nucleotides in the mini upper-stem-loop RNA structure? As shown in Figure 3Aa, in plasmid pRM4, these are nucleotides U17, U18 and A29. Does replacing these specific bulged nucleotides affect the efficiency of in vivo selenocysteine incorporation? We found that the incorporation of selenocysteine in the lac'Z fusion polypeptide is completely prevented (Fig. 3Bb,d and Cb,d) by replacing each of the following nucleotides: U17 -> A17 (pZL42; Fig. 3Ad), U18 -> A18 (pZL38; Fig. 3Ab) and A29 -> U29 (data not shown). We could, however, cause the return of selenocysteine incorporation (Fig. 3Bc and Cc) by introducing the compensatory mutation A29 -> U29 into pZL38, creating pZL43 in which A18 and U29 are base-paired (Fig. 3Ac). Thus, selenocysteine incorporation depends not on the presence of a U residue at position 18 (which is replaced by A in the mutant) but rather on its ability to base pair with the nucleotide at position 29. Note, however, that selenocysteine incorporation was completely abolished by either the single change U17 -> A17 (pZL42, Fig. 3Ad, Bd and Cd) or the double change U17 -> A17 and the compensatory mutation A29 -> U29 (pZL44; Fig. 3Ae, Be and Ce). Furthermore, selenocysteine incorporation was also completely abolished by the single changes U17 -> C17 or U17 -> G17 (data not shown). Thus, selenocysteine incorporation appears to be dependent on both (i) the specificity of the nucleotide at position 17 as a U residue, and (ii) its presence as a bulged nucleotide. The importance of the bulged U17 has been recently shown also by a SELEX approach (26).
Having determined that selenocysteine incorporation is possible even when the base pairing of the intermediary domain of the SECIS is opened, we asked how the specific structure of the mini upper-stem-loop affects selenocysteine incorporation. We particularly wished to distinguish between the role of single- and double-stranded regions in this structure. We did this by asking how the inversion of potentially base paired nucleotides might affect selenocysteine incorporation. It is assumed that 2 nt in the stem are in a base-paired state if selenocysteine incorporation is permitted even when they are inverted. Based on this assumption, we found that the following nucleotides must be paired in the mini upper-stem-loop structure: 15 with 31 (Fig. 4Ab, Bb and Cb), 16 with 30 (Fig. 4Ac, Bc and Cc), 18 with 29 (Fig. 4Ad, Bd and Cd), and 19 with 28 (Fig. 4Ae, Be and Ce). On the other hand, since the inversion of the C20-G27 to G20-C27 abolishes selenocysteine incorporation not only when the intermediary region is opened as in pZL66 (Fig. 4Af, Bf and Cf), but also when it is closed as in a pRM4 derivative (data not shown), it is possible that the pairing between nt 20 and 27 is not required and/or that the nature of the nucleotides in this position is important for selenocysteine incorporation (see Discussion). In addition, since selenocysteine incorporation is not detected when A17 is paired with U29 (Fig. 3Ae, Be and Ce), we conclude that U17 is required as a bulged nucleotide. Our experiments also reveal that, as previously shown by Heider et al. (9), for the `closed' stem, selenocysteine incorporation is also completely prevented by single mutations in the loop region (A21 -> U21, G22 -> C22) when the intermediary region is `open' (data not shown). Based on all our results, we suggest that the mini upper-stem-loop structure required for selenocysteine incorporation consists of 17 nt including (Fig. 6): (i) a region of 4 bp of non-specific nucleotides 15-31, 16-30, 18-29 and 19-28; (ii) a bulged U at position 17; and (iii) a sequence of eight specific nucleotides from C20 to G27. Among these, C20 and G27 may be weakly paired and the rest are located in a loop (see Discussion).
Based on the chemical and enzymatic probing data of experiments designed to elucidate the solution structure of mRNA hairpins within the fdhF SECIS, Hüttenhofer and colleagues (21) have suggested that upstream from the UGA codon there is an extended helix of 7 bp in which the U and G residues of the UGA codon are included, but the A residue is bulged (Fig. 5Aa). Does this extended helix upstream from the UGA codon have a role in the incorporation of selenocysteine in vivo? To answer this question, we mutated the extended helix upstream from the UGA such that the secondary structure 5' to the UGA could not be formed. In pRM4, we changed both nucleotides at the 5' side and the 3' side of the UGA; at the 5' side 5 nt were changed to A residues and nt 42-50 at the 3' side of the UGA were changed to AUUUAAAAA, respectively (pZL40; Fig. 5Ab). As shown, these changes did not significantlyaffect selenocysteine incorporation (Fig. 5Bb and Cb). Moreover, even when the same changes were made in pZL14, so that both the upstream UGA stem and the intermediary domain were `opened' (pZL36; Fig. 5Ad), selenocysteine incorporation was unaffected (Fig. 5Bd and Cd). Compare particularly the results found for plasmids pRM4 (5Ba and Ca) and pZL36 (Fig. 5Bd and Cd): pRM4 carries the whole extended helix of fdhF (that is the region upstream from UGA), the intermediary domain, and the mini upper-stem-loop structure (Fig. 5Aa). Though in pZL36 only the mini upper-stem-loop structure is unchanged, however, while at a proper distance of 11 nt from the UGA (Fig. 5Ad), it still supports the same level of selenocysteine incorporation as does pRM4. These results further support our conclusion that in vivo selenocysteine incorporation into the polypeptide product of the fdhF-lac'Z fused genes requires only the mini upper-stem-loop structure placed at the proper position.
The UGA stop codon in the mRNA of the E.coli formate dehydrogenase gene fdhF has been described to be decoded as selenocysteine when it is in the context of a SECIS of at least 40 nt following the UGA (12). This process is dependent on the ability of this SECIS to form a stem-loop RNA structure (9). These conclusions were based on the results of mutational analyses of this stem-loop structure directed by the fusion of the fdhF SECIS DNA and the lac'Z gene in an E.coli strain MC4100 (rpsL+ rpsE). It has been shown convincingly that the specificity of each of the nucleotides in the loop and the base pairing of the adjacent two nucleotides (mostly the second) located in the stem are crucial for the process of the UGA-directed selenocysteine incorporation into the fused polypeptide (12). These analyses, however, have not dealt fully with the rest of this mRNA region, and thus do not distinguish among the following possibilities: is UGA-directed selenocysteine incorporation dependent on (i) the nucleotide sequence of the SECIS; and/or (ii) the secondary structure of the SECIS; and/or (iii) the distance of the loop from the UGA codon? Also, does the extended helix upstream from the UGA codon (21) play a role in the in vivo process of selenocysteine incorporation?
By a process of progressive elimination, we arrived at a minimal SECIS which can still facilitate in vivo UGA-directed selenocysteine incorporation into the polypeptide product of a plasmid borne fdhF-lac'Z gene fusion. We determined the level of the UGA-directed selenocysteine incorporation as reflected by: (i) the level of the selC-dependent [beta]-galactosidase activity; and (ii) [75Se]selenite incorporation into the fused polypeptide. These studies were carried out in E.coli strain MC4100 (rpsL150) (15). We found that in order to permit selenocysteine incorporation in vivo the previously described fdhF SECIS (9,12,21) can still function properly even when it is reduced to only 17 nt forming a mini upper-stem-loop RNA structure (Figs 1, 4 and 5). We found, however, that two major regions of the SECIS were not crucial for this in vivo process, and these are what we call: (i) the intermediary domain, the 11 bp double-stranded RNA region, including nt 4-14 and 32-42 downstream from the UGA codon (Fig. 1); and (ii) the upstream UGA domain, the double-stranded RNA structure, including nt -5 to 1 upstream from the UGA and nt 41-46 downstream from the UGA (Fig. 5). Preventing the formation of the secondary structure of either the intermediary domain (Fig. 1) or the upstream UGA domain (Fig. 5) caused a reduction of only ~20-30% in the level of the UGA-directed selenocysteine incorporation. We have also shown that neither the primary nor the secondary structure of the intermediary domain are required for the process. Our experiments suggest that the intermediary domain assures the existence of the proper distance of 11 nt (from the UGA to the mini upper-stem-loop structure) requisite for optimal selenocysteine incorporation. Thus, these intermediary RNA nucleotides can be in either single or double-stranded form (Fig. 2). It is the distance of the mini upper-stem-loop structure from the UGA that is crucial for the process.
Having found the lower size limit for the SECIS to be 17 nt, we further analyzed the mini upper-stem-loop RNA structure necessary for selenocysteine incorporation. By examining the effects of inverting potentially base-paired nucleotides, we particularly distinguished between single- and double-stranded RNA regions in this structure required for the process (Figs 3 and 4). Our results show that the mini upper-stem-loop structure required for selenocysteine incorporation consists of 17 nt including (Fig. 6): (i) a region of 4 bp, 15-31, 16-30, 18-29 and 19-28 of non-specific nucleotides; (ii) a bulged U at position 17; and (iii) a sequence of eight specific nucleotides from C20 to G27. In our experiments we were not able to show the involvement of the base pair C20-G27; inversion of the base pair to G20-C27 does not permit selenocysteine incorporation (see Results and Fig. 4Af, Bf and Cf). Since Heider et al. (9) have previously shown that such an inversion partially affects selenocysteine incorporation (reduction to 30%), we assume that C20-G27 base pairing is questionable. Therefore, in our model on the nature of the minimal E.coli SECIS (Fig. 6), the base pair C20-G27 is represented by adot.
Based on our results, we suggest a minimal E.coli SECIS that consists of a mini upper-stem-loop structure with a proper distance from the UGA as described in Figure 6. It is interesting that this minimal SECIS in genes fdhF and fdnG is very similar (21).
The special elongation factor SelB was shown to be structurally divided into two parts: (i) an N-terminal half which shares extensive sequence homology with EF-Tu and binds to selenocysteinyl-tRNASec and GTP; and (ii) a separate C-terminal domain, required for specifically binding to the fdhF mRNA stem-loop structure 3' adjacent to the UGA codon in vitro (19). Moreover, the mRNA motif recognized by SelB can be reduced to a 17 nt minihelix without loss of binding capacity in vitro (20). This minihelix is identical to the mini upper-stem-loop of fdhF mRNA described by us here to be the minimal SECIS required for the in vivo incorporation of selenocysteine in E.coli (Fig. 6). The C-terminal domain of SelB probably exclusively binds also in vivo to this upper stem-loop RNA structure. Hüttenhofer et al. (27) have recently proposed a model where the upper part of the fdhF mRNA hairpin which binds to the C-terminus of SelB can be placed `outside' the ribosomal mRNA track during UGA decoding. The nature of the minimal E.coli SECIS described here (Fig. 6) corroborates with such a model; only small variations are suggested. The model suggested by Hüttenhoffer et al. (27) was based on in vitro toeprint experiments with 30S ribosomal subunits. They contend that when the mRNA is unfolded by the 30S ribosomal subunit there are 16 unfolded nucleotide residues from the first nucleotide at the P site to the 3' side of the UGA of fdhF mRNA. In our in vivo experiments described here, we found that selenocysteine is incorporated when there are not more than 17 unfolded nucleotides in this region; that is 11 nt from the UGA to the mini upper-stem-loop structure (Fig. 6) plus the 6 nt included in the P and A sites. Therefore, we propose that there may be a safety measure whereby the E.coli 70S elongating ribosome can even unfold 17 nt and still permit UGA-directed selenocysteine incorporation into the translated protein product. Such a model is supported by the fact that the E.coli ribosome covers 18 ± 1 nt downstream from the P-site codon (28).
In conclusion, our results show that just the mini upper-stem-loop domain of fdhF mRNA (located at the proper distance of 11 nt from the UGA) is required for selenocysteine incorporation in vivo. However, the UGA codon context of fdhF and fdnG mRNAs generates a more extensive secondary structure (9,12,21), designated here as the `intermediary domain' and the `upstream UGA domain'. We are presently investigating the possibility that under specific physiological conditions the intermediary and upstream domains may have a role in selenocysteine incorporation into polypeptides.
We thank Dr August Böck (Munich, Germany) for kindly providing us with bacterial strains. We are deeply grateful to F. R. Warshaw-Dadon (Jerusalem, Israel) for her critical reading of the manuscript. This research was supported by grants from the United States-Israel Binational Science Foundation (BSF) and the Israel Science Foundation administrated by The Israel Academy of Science and Humanities.
Nucleic Acids Research
Pages
Introduction
Materials And Methods
Materials and media
Bacterial strains and plasmid derivatives
Molecular cloning
Bacterial growth, transformations, and measurements of [beta]-galactosidase activity
[75Se]selenite labeling and identification of its incorporation in vivo into the fused gene product
Results
A mini upper-stem-loop structure of fdhF SECIS mRNA is required for selenocysteine incorporation into a polypeptide
The distance of the mini upper-stem-loop structure from the UGA of fdhF mRNA is crucial for selenocysteine incorporation into a polypeptide
U17 is a specific and bulged nucleotide of the mini upper-stem-loop structure downstream of the UGA of fdhF mRNA that is required for selenocysteine incorporation into a polypeptide
The nature of the mini upper-stem-loop structure downstream of the UGA of an fdhF mRNA that is required for selenocysteine incorporation into a polypeptide
Only the mini upper-stem-loop structure downstream of the UGA of fdhF mRNA is required for selenocysteine incorporation into a polypeptide
Discussion
Acknowledgements
References
REFERENCES
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