SELB differs from EF-Tu mainly in its larger size due to a C-terminal extension. This additional domain does not exhibit
The EMBO Journal vol. 1 1 no. 1 0 pp.3759 - 3766, 1992
Coding from a distance: dissection of the mRNA determinants required for the incorporation of selenocysteine into protein Johann Heider, Christian Baron and August BockN Lehrstuhl fuir Mikrobiologie der Universitfit Munchen, Maria-Ward-Strasse la, W-8000 Munchen 19, FRG Corresponding author Communicated by A.Bock
Incorporation of selenocysteine into proteins is directed by specifically 'programmed' UGA codons. The determinants for recognition of the selenocysteine codon have been investigated by analysing the effect of mutations in fdhF, the gene for formate dehydrogenase H of Escherichia coli, on selenocysteine incorporation. It was found that selenocysteine was also encoded when the UGA codon was replaced by UAA and UAG, provided a proper codon-anticodon interaction was possible with tRNAs. This indicates that none of the three termination codons can function as efficient translational stop signals in that particular mRNA position. The discrimination of the selenocysteine 'sense' codon from a regular stop codon has previously been shown to be dependent on an RNA secondary structure immediately 3' of the UGA codon in the fdhF mRNA. It is demonstrated here that the correct folding of this structure as well as the existence of primary sequence elements located within the loop portion at an appropriate distance to the UGA codon are absolutely required. A recognition sequence can be defined which mediates specific translation of a particular codon inside an mRNA with selenocysteine and a model is proposed in which translation factor SELB interacts with this recognition sequence, thus forming a quaternary complex at the mRNA together with GTP and selenocysteyl-tRNAS'. Key words: mRNA context/RNA binding protein/RNA secondary structure/termination codon/translational control
Introduction Escherichia coli is able to synthesize three selenocysteine containing polypeptides which are constituent subunits of the formate dehydrogenase (FDH) isoenzymes, FDHH, FDHN (Cox et al., 1981) and FDHo (Sawers et al., 1991). The biosynthesis of selenocysteine and its incorporation into protein is accomplished by the products of the sel genes (Bock et al., 1991). During biosynthesis an ester-bonded seryl residue at a special tRNA (tRNAsec) is converted to a selenocysteyl residue by selenocysteine synthase, the selA gene product. This reaction proceeds through an aminoacrylyl -tRNA intermediate (Forchhammer and Bock, 1991) and involves a phosphate-activated selenium donor molecule which is synthesized by the selD gene product (Forchhammer et al., 1991; Ehrenreich et al., 1992; Veres et al., 1992). (C Oxford University Press
The resulting selenocysteyl -tRNASeC is specifically recognized by an alternate elongation factor, SELB, which has been shown to replace EF-Tu in binding this particular tRNA and delivering it to the ribosome (Forchammer et al., 1989; Forster et al., 1990). Specific incorporation of selenocysteine into distinct proteins is directed by in-frame UGA codons in the mRNAs derived from all known selenoprotein genes (Bock et al., 1991). Since UGA codons are normally used as translation termination codons, the existence of a specific mechanism to discriminate them from UGA codons directing selenocysteine insertion is obvious (Bock et al., 1991). This discrimination does not require a UGA codon per se, because selenocysteine incorporation into the E. coli fdhF gene product still occurred when the UGA was mutated. After mutation at the third ('wobble') position (to a cysteine codon, UGC or UGU), significant selenocysteine incorporation could be observed in wild-type strains; this is reduced, but not abolished by restrictive rpsL alleles (Zinoni et al., 1987, 1990). After mutation at the second position to a serine (UCA) codon, selenocysteine incorporation was abolished but could be rescued when a compensatory exchange was introduced into the anticodon of tRNASec (Baron et al., 1990). In these cases selenocysteine insertion into polypeptides by tRNAs' efficiently competed with the insertion of cysteine or serine via the 'normal' tRNAs (Baron et al., 1990). These data suggest that additional mRNA elements outside of the UGA codon are responsible for the recognition of selenocysteine codons. Indeed, it was demonstrated recently for the E. coli fdhF gene (encoding the 80 kDa subunit of FDHH) and the fdnG gene (Berg et al., 1991b) that the presence of a 3'-flanking sequence is required in addition to the UGA codon to obtain efficient incorporation. This mRNA region correlates with the existence of a putative mRNA secondary structure (Zinoni et al., 1990; Berg et al., 1991b). Independent support for the relevance of this structure came from the generation of a new selenoprotein by mutating the gene for the a-subunit of Methanobacteriumfonnicicum FDH, a cysteine-containing homologue of the selenopolypeptide of FDHH (Zinoni et al., 1987). This was achieved firstly by changing the cysteine codon into UGA, and secondly, by introducing an mRNA secondary structure downstream of the UGA which closely resembled that identified for fdhF mRNA (Heider and Bock, 1992). The aim of this study was to determine the detailed structural requirements within the fdhF niRNA that are responsible for the specific incorporation of selenocysteine. The data indicate the existence of a recognition sequence located 3' of the UGA codon which (i) precludes recognition of the UGA as a termination signal, and (ii) directs selenocysteine insertion, most probably by the existence of a SELB/selenocysteyl-tRNA/GTP ternary complex preformed on the mRNA. 37 59
J.Heider, C.Baron and A.Bock
Results Requirements for codon anticodon interaction Stop codon recognition in E. coli involves the action of the two release factors RF-1 and RF-2. While recognition of UAG is an exclusive feature of RF- 1, UGA stop codons are recognized solely by RF-2 and UAA codons by both RF- 1 and RF-2 (reviewed by Tate et al., 1990). Since only UGA codons are so far known to be used for selenocysteine incorporation, RF-2 could possibly exert a specific function in this process. It was examined, therefore, whether it is possible to direct selenocysteine incorporation by the other stop codons, UAG and UAA. Plasmid pFM323 contains 59 nucleotides around the selenocysteine codon (UGA140) offdhF cloned in-frame into the lacZ gene (Zinoni et al., 1990); the UGA140 codon was changed into UAG and UAA by site-directed mutagenesis (Figure 1A). Complementary changes were introduced into the anticodon of tRNAsec, namely from UCA to CUA and UUA (Figure 1B). Single copies of mutated selC genes were then introduced at the X-att site of the chromosome of a AselC strain, as described by Baron et al. (1990). The resulting strains contain the same selC fragment as strain WL81460/selC2013 which harbours an integrated wild-type selC gene (Baron et al., 1990). These strains were transformed with plasmids carrying the constructed gene fusions and selenocysteine incorporation into fusion proteins was determined. Table I shows the effects on readthrough over the different 'stop' codons measured by f-galactosidase activity. the 'wildtype' UGA codon can only be decoded by wild-type tRNAsec; whereas the UAG and UAA codons are solely -
GU c
A
GAC-G U
recognized by tRNASeC variants containing mutated anticodons. The highest readthrough was obtained with the wild-type UGA/UCA pair, followed by the UAA/UUA and UAG/CUA pairs. Interestingly, a third position 'wobble' interaction seems to allow the decoding of UAA and UAG codons by each of the mutated anticodons, with the UAG/UUA pair at a reduced rate and to a marginal extent with the UAA/CUA pair (Table I). A [75Se]selenite labelling experiment proved that the readthrough values measured via f-galactosidase activity corresponded to the incorporation of selenocysteine into the fusion proteins. As shown in Figure 2, the amount of 75Selabelled polypeptides paralleled the readthrough values shown in Table I. In addition, the incorporation of [75Se] into the 10 kDa selenopolypeptide of FDHo (Sawers et al., 1991) was undetectable when the anticodon of tRNAS' was mutated to UUA or CUA (Figure 2). Requirements for specific recognition of selenocysteine codons Since the UGA codon directing selenocysteine insertion could be replaced by a number of other codons without loss of coding capacity, there must be additional mRNA elements that determine its specificity. Indeed, it has been shown Table I. Readthrough analysis with fdhF-lacZ gene fusions containing either of the three stop codons at the site of the selenocysteine codon of fdhF. Values are given in units according to Miller (1972)
Anticodon UCA UUA CUA
Codon UGA
UAA
UAG
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