Coding from a distance: dissection of the mRNA determinants required for the incorporation of selenocysteine into protein.

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 tRNA(Sec). 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-tRNA(Sec).     

Solution structure of mRNA hairpins promoting selenocysteine incorporation in Escherichia coli and their base-specific interaction with special elongation factor SELB.

On the basis of chemical probing data, the solution structures of RNA hairpins within fdhF and fdnG mRNAs in Escherichia coli, which both promote selenocysteine incorporation at UGA codons, were derived with the help of computer modeling. We find that these mRNA hairpins contain two separate structural domains that possibly also exert two different functions. The first domain is comprised of the UGA codon, which is included within a complex and distorted double-stranded region. Thereby, release factor 2 might be prevented from binding to the UGA codon to terminate protein synthesis. The second domain is located within the apical loop of the mRNA hairpin structures. This loop region exhibits a defined tertiary structure in which no base is involved in Watson-Crick interactions. The structure of the loop is such that, following a sharp turn after G22 (A22 in fdnG mRNA), bases G23 and U24 are exposed to the solvent on the deep groove side of the supporting helix. Residues C25 and U26 close the loop with a possible single H-bonding interaction between the first and last residues of the loop, 04(U26) and N6(A21). The bulge residues U17 and U18 (in fdhF mRNA), or Ul7 only in fdnG mRNA, point their Watson-Crick positions in the same direction as loop residues G23 and U24 do, and at the same time open up the deep groove at the top of the hairpin helix. Chemical probing data demonstrate that bases G23 and U24 in both mRNA hairpins, as well as residues U17 and Ul7/U18 (for fdhF mRNA) located in a bulge 5' to the loop, are involved directly in binding to special elongation factor SELB in both mRNAs. Therefore, SELB recognizes identical bases within both mRNA hairpins despite differences in their primary sequence, consistent with the derived 3D models for these mRNAs, which exhibit similar tertiary structures. Binding of SELB to the fdhF mRNA hairpin was estimated to proceed with an apparent Kd of 30 nM.     

A dynamic competition between release factor 2 and the tRNA(Sec) decoding UGA at the recoding site of Escherichia coli formate dehydrogenase H.

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 tRNA(Sec) 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 tRNA(Sec)-mediated decoding can compete more effectively for the UGA recoding site at lower growth rates, consistent with anaerobic induction of fdhF expression.     

Structural basis for dynamic interdomain movement and RNA recognition of the selenocysteine-specific elongation factor SelB

Selenocysteine (Sec) is the "21st" amino acid and is genetically encoded by an unusual incorporation system. The stop codon UGA becomes a Sec codon when the selenocysteine insertion sequence (SECIS) exists downstream of UGA. Sec incorporation requires a specific elongation factor, SelB, which recognizes tRNA(Sec) via use of an EF-Tu-like domain and the SECIS mRNA hairpin via use of a C-terminal domain (SelB-C). SelB functions in multiple translational steps: binding to SECIS mRNA and tRNA(Sec), delivery of tRNA(Sec) onto an A site, GTP hydrolysis, and release from tRNA and mRNA. However, this dynamic mechanism remains to be revealed. Here, we report a large domain rearrangement in the structure of SelB-C complexed with RNA. Surprisingly, the interdomain region forms new interactions with the phosphate backbone of a neighboring RNA, distinct from SECIS RNA binding. This SelB-RNA interaction is sequence independent, possibly reflecting SelB-tRNA/-rRNA recognitions. Based on these data, the dynamic SelB-ribosome-mRNA-tRNA interactions will be discussed.     

In vitro selection of RNA aptamers that bind special elongation factor SelB, a protein with multiple RNA-binding sites, reveals one major interaction domain at the carboxyl terminus.

The SelB protein of Escherichia coli is a special elongation factor required for the cotranslational incorporation of the uncommon amino acid selenocysteine into proteins such as formiate dehydrogenases. To do this, SelB binds simultaneously to selenocysteyl-tRNA(Sec) and to an RNA hairpin structure in the mRNA of formiate dehydrogenases located directly 3' of the selenocysteine opal (UGA) codon. The protein is also thought to contain binding sites allowing its interaction with ribosomal proteins and/or rRNA. SelB thus includes specific binding sites for a variety of different RNA molecules. We used an in vitro selection approach with a pool completely randomized at 40 nt to isolate new high-affinity SelB-binding RNA motifs. Our main objective was to investigate which of the various RNA-binding domains in SelB would turn out to be prime targets for aptamer interaction. The resulting sequences were compared with those from a previous SELEX experiment using a degenerate pool of the wild-type formiate dehydrogenase H (fdhF) hairpin sequence (Klug SJ et al., 1997, Proc. Natl. Acad. Sci. USA 94:6676-6681). In four selection cycles an enriched pool of tight SelB-binding aptamers was obtained; sequencing revealed that all aptamers were different in their primary sequence and most bore no recognizable consensus to known RNA motifs. Domain mapping for SelB-binding aptamers showed that despite the different RNA-binding sites in the protein, the vast majority of aptamers bound to the ultimate C-terminus of SelB, the domain responsible for mRNA hairpin binding.     

Mutagenesis of selC, the gene for the selenocysteine-inserting tRNA-species in E. coli: effects on in vivo function.

The selenocysteine-inserting tRNA (tRNA(Sec)) of E. coli differs in a number of structural features from all other elongator tRNA species. To analyse the functional implications of the deviations from the consensus, these positions have been reverted to the canonical configuration. The following results were obtained: (i) inversion of the purine/pyrimidine pair at position 11/24 and change of the purine at position 8 into the universally conserved U had no functional consequence whereas replacements of U9 by G9 and of U14 by A14 decreased the efficiency of selenocysteine insertion as measured by translation of the fdhF message; (ii) deleting one basepair in the aminoacyl acceptor stem, thus creating the canonical 7 bp configuration, inactivated tRNA(Sec); (iii) replacement of the extra arm by that of a serine-inserting tRNA abolished the activity whereas reduction by 1 base or the insertion of three bases partially reduced function; (iv) change of the anticodon to that of a serine inserter abolished the capacity to decode UGA140 whereas the alteration to a cysteine codon permitted 30% read-through. However, the variant with the serine-specific anticodon efficiently inserted selenocysteine into a gene product when the UGA140 of the fdhF mRNA was replaced by a serine codon (UCA). Significantly, none of these changes resulted in the non-specific incorporation of selenocysteine into protein, indicating that the mRNA context also plays a major role in directing insertion. Taken together, the results demonstrate that the 8-basepair acceptor stem and the long extra arm are crucial determinants of tRNA(Sec) which enable decoding of UGA140 in the fdhF message.     

Targeted insertion of selenocysteine into the alpha subunit of formate dehydrogenase from Methanobacterium formicicum.

Selenocysteine incorporation into proteins is directed by an opal (UGA) codon and requires the existence of a stem-loop structure in the mRNA flanking the UGA at its 3' side. To analyze the sequence and secondary-structure requirements for UGA decoding, we have introduced mutations into the fdhA gene from Methanobacterium formicicum, which codes for the alpha subunit of the F420-reducing formate dehydrogenase. The M. formicicum enzyme contains a cysteine residue at the position where the Escherichia coli formate dehydrogenase H carries a selenocysteine moiety. The codon (UGC) for this cysteine residue was changed into a UGA codon, and mutations were successively introduced at the 5' and 3' sides to generate a stable secondary structure of the mRNA and to approximate the sequence of the predicted E. coli fdhF mRNA hairpin structure. It was found that introduction of the UGA and generation of a stable putative stem-loop structure were not sufficient for decoding with selenocysteine. Efficient selenocysteine incorporation, however, was obtained when the loop and the immediately adjacent portion of the putative stem had a sequence identical to that present in the E. coli fdhF mRNA structure.     

The function of selenocysteine synthase and SELB in the synthesis and incorporation of selenocysteine.

The selAB operon codes for the proteins selenocysteine synthase and SELB which catalyse the synthesis and cotranslational insertion of selenocysteine into protein. This communication deals with the biochemical characterisation of these proteins and in particular with their specific interaction with the selenocysteine-incorporating tRNA(Sec). Selenocysteine synthase catalyses the synthesis of selenocysteyl-tRNA(Sec) from seryl-tRNA(Sec) in a pyridoxal phosphate-dependent reaction mechanism. The enzyme specifically recognizes the tRNA(Sec) molecule; a cooperative interaction between the tRNA binding site and the catalytically active pyridoxal phosphate site is suggested. SELB is an EF-Tu-like protein which specifically complexes selenocysteyl-tRNA(Sec). Interaction with the selenol group of the side chain of the aminoacylated residue is a prerequisite for the formation of a stable SELB.tRNA complex. Mechanistically, this provides the biochemical basis for the exclusive selection of selenocysteyl-tRNA(Sec) in the decoding step of a selenocysteine-specific UGA triplet.     

Barriers to heterologous expression of a selenoprotein gene in bacteria.

The specificity parameters counteracting the heterologous expression in Escherichia coli of the Desulfomicrobium baculatum gene (hydV) coding for the large subunit of the periplasmic hydrogenase which is a selenoprotein have been studied. hydV'-'lacZ fusions were constructed, and it was shown that they do not direct the incorporation of selenocysteine in E. coli. Rather, the UGA codon is efficiently suppressed by some other aminoacyl-tRNA in an E. coli strain possessing a ribosomal ambiguity mutation. The suppression is decreased by the strA1 allele, indicating that the hydV selenocysteine UGA codon has the properties of a "normal" and suppressible nonsense codon. The SelB protein from D. baculatum was purified; in gel shift experiments, D. baculatum SelB displayed a lower affinity for the E. coli fdhF selenoprotein mRNA than E. coli SelB did and vice versa. Coexpression of the hydV'-'lacZ fusion and of the selB and tRNA(Sec) genes from D. baculatum, however, did not lead to selenocysteine insertion into the protein, although the formation of the quaternary complex between SelB, selenocysteyl-tRNA(Sec), and the hydV mRNA recognition sequence took place. The results demonstrate (i) that the selenocysteine-specific UGA codon is readily suppressed under conditions where the homologous SelB protein is absent and (ii) that apart from the specificity of the SelB-mRNA interaction, a structural compatibility of the quaternary complex with the ribosome is required.     

硒蛋白的分子生物学研究进展

已有35种硒蛋白被分离和表征,但许多硒蛋白及其功能仍未完全阐明.硒半胱氨酸(Sec)作为参入蛋白质的第21种氨基酸,由硒蛋白mRNA上的UGA编码.在原核生物,Sec参入硒蛋白的复杂机制已经较为明确,需要四种基因产物（SELA、SELB、SELC和SELD）和一个存在于硒蛋白mRNA上的被称为Sec插入序列(SECIS)的茎环(stem loop)样二级结构.在真核生物,硒蛋白生物合成途径可能在SECIS的结构和位置、特异的延伸因子及其他RNA-RNA或RNA-蛋白质因子之间的相互作用等方面与原核生物不同.另外,哺乳动物硒蛋白mRNA上的UGA翻译为Sec的过程低效,特定位点的UGA密码子不同功能(终止密码和Sec密码)的调控可能是硒蛋白表达低效的关键.     

A sequence in the Escherichia coli fdhF "selenocysteine insertion Sequence" (SECIS) operates in the absence of selenium

The UGA codon context of the Escherichia coli fdhF mRNA includes an element called the selenocysteine insertion sequence (SECIS) that is responsible for the UGA-directed incorporation of the amino acid selenocysteine into a protein. Here, we describe an extended fdhF SECIS that includes the information for an additional function: the prevention of UGA readthrough under conditions of selenium deficiency. This information is contained in a short mRNA region consisting of a single C residue adjacent to the UGA on its downstream side, and an additional segment consisting of the six nucleotides immediately upstream from it. These two regions act independently and additively, and probably through different mechanisms. The single C residue acts as itself; the upstream region acts at the level of the two amino acids, arginine and valine, for which it codes. These two codons at the 5' side of the UGA correspond to the ribosomal E and P sites. Here, we present a model for the E. coli fdhF SECIS as a multifunctional RNA structure containing three functional elements. Depending on the availability of selenium, the SECIS enables one of two alternatives for the translational machinery: either selenocysteine incorporation into a polypeptide or termination of the polypeptide chain.     

Interaction of a selenocysteine-incorporating tRNA with elongation factor Tu from E.coli.

Selenocysteine-incorporating tRNA(Sec)(UCA), the product of selC, was isolated from E.coli and aminoacylated with serine. The equilibrium dissociation constant for the interaction of Ser-tRNA(Sec)(UCA) with elongation factor Tu.GTP was determined to be 5.0 +/- 2.5 x 10(-8) M. Compared with the dissociation constants of the two elongator Ser-tRNA(Ser) species (Kd = 7 x 10(-10) M), the selenocysteine-incorporating UGA suppressor tRNA has an almost hundred fold weaker affinity for EF-Tu.GTP. This suggests a mechanism by which the Ser-tRNA(Sec) is prevented in recognition of UGA codons. This tRNA is not bound to EF-Tu.GTP and is converted to selenocysteinyl-tRNA(Sec). We also demonstrate the lack of an efficient interaction of Sec-tRNA(Sec)(UCA) with EF-Tu.GTP. The results of this work are in support of a mechanism by which the selenocysteine incorporation at UGA nonsense codons is mediated by an elongation factor other than EF-Tu.GTP.     

Functional analysis of prokaryotic SELB proteins

Since the discovery of selenocysteine as the 21st amino acid considerable progress has been made in elucidating the system responsible for its insertion into proteins. Elongation factor SELB, whose amino-terminal part shows homology to EF-Tu, was found to be the key component mediating delivery of selenocysteyl-tRNA(Sec) to the ribosomal A site. It exhibits a distinct tertiary structure comprising binding sites for guanosine nucleotides, the cognate tRNA, an mRNA secondary structure (SECIS element) and presumably ribosomal components. The kinetics of interaction of SELB with its ligands have been studied in detail. GDP was found to bind with about 20-fold lower affinity than GTP and to be in rapid exchange, which obviates the need for a guanosine nucleotide exchange factor. The affinity of SELB for the SECIS element is in the range of 1 nM and further increases upon binding of selenocysteyl-tRNA(Sec) to the protein. This supports the model that SELB forms a tight quaternary complex on the SECIS element which is loosened after insertion of the tRNA into the ribosomal A site and the concomitant hydrolysis of GTP.     

A novel RNA binding protein, SBP2, is required for the translation of mammalian selenoprotein mRNAs

In eukaryotes, the decoding of the UGA codon as selenocysteine (Sec) requires a Sec insertion sequence (SECIS) element in the 3' untranslated region of the mRNA. We purified a SECIS binding protein, SBP2, and obtained a cDNA clone that encodes this activity. SBP2 is a novel protein containing a putative RNA binding domain found in ribosomal proteins and a yeast suppressor of translation termination. By UV cross-linking and immunoprecipitation, we show that SBP2 specifically binds selenoprotein mRNAs both in vitro and in vivo. Using (75)Se-labeled Sec-tRNA(Sec), we developed an in vitro system for analyzing Sec incorporation in which the translation of a selenoprotein mRNA was both SBP2 and SECIS element dependent. Immunodepletion of SBP2 from the lysates abolished Sec insertion, which was restored when recombinant SBP2 was added to the reaction. These results establish that SBP2 is essential for the co-translational insertion of Sec into selenoproteins. We hypothesize that the binding activity of SBP2 may be involved in preventing termination at the UGA/Sec codon.     

Polysome distribution of phospholipid hydroperoxide glutathione peroxidase mRNA: evidence for a block in elongation at the UGA/selenocysteine codon

The translation of mammalian selenoprotein mRNAs requires the 3' untranslated region that contains a selenocysteine insertion sequence (SECIS) element necessary for decoding an in-frame UGA codon as selenocysteine (Sec). Selenoprotein biosynthesis is inefficient, which may be due to competition between Sec insertion and termination at the UGA/Sec codon. We analyzed the polysome distribution of phospholipid hydroperoxide glutathione peroxidase (PHGPx) mRNA, a member of the glutathione peroxidase family of selenoproteins, in rat hepatoma cell and mouse liver extracts. In linear sucrose gradients, the sedimentation velocity of PHGPx mRNA was impeded compared to CuZn superoxide dismutase (SOD) mRNA, which has a coding region of similar size. Selenium supplementation increased the loading of ribosomes onto PHGPx mRNA, but not CuZn SOD mRNA. To determine whether the slow sedimentation velocity of PHGPx mRNA is due to a block in elongation, we analyzed the polysome distribution of wild-type and mutant mRNAs translated in vitro. Mutation of the UGA/Sec codon to UGU/cysteine increased ribosome loading and protein synthesis. When UGA/Sec was replaced with UAA or when the SECIS element core was deleted, the distribution of the mutant mRNAs was similar to the wild-type mRNA. Addition of SECIS-binding protein SBP2, which is essential for Sec insertion, increased ribosome loading and translation of wild-type PHGPx mRNA, but had no effect on the mutant mRNAs. These results suggest that elongation is impeded at UGA/Sec, and that selenium and SBP2 alleviate this block by promoting Sec incorporation instead of termination.     

Ribosomal protein L30 is a component of the UGA-selenocysteine recoding machinery in eukaryotes

The translational recoding of UGA as selenocysteine (Sec) is directed by a SECIS element in the 3' untranslated region (UTR) of eukaryotic selenoprotein mRNAs. The selenocysteine insertion sequence (SECIS) contains two essential tandem sheared G.A pairs that bind SECIS-binding protein 2 (SBP2), which recruits a selenocysteine-specific elongation factor and Sec-tRNA(Sec) to the ribosome. Here we show that ribosomal protein L30 is a component of the eukaryotic selenocysteine recoding machinery. L30 binds SECIS elements in vitro and in vivo, stimulates UGA recoding in transfected cells and competes with SBP2 for SECIS binding. Magnesium, known to induce a kink-turn in RNAs that contain two tandem G.A pairs, decreases the SBP2-SECIS complex in favor of the L30-SECIS interaction. We propose a model in which SBP2 and L30 carry out different functions in the UGA recoding mechanism, with the SECIS acting as a molecular switch upon protein binding.     

The nature of the minimal 'selenocysteine insertion sequence' (SECIS) in Escherichia coli.

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.coli fdhF 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.     

Structural insight into a molecular switch in tandem winged-helix motifs from elongation factor SelB

Elongation factor SelB is responsible for co-translational incorporation of selenocysteine (Sec) into proteins. The UGA stop codon is recoded as a Sec codon in the presence of a downstream mRNA hairpin. In prokaryotes, in addition to the EF-Tu-like N-terminal domains, a C-terminal extension containing four tandem winged-helix motifs (WH1-4) recognizes the mRNA hairpin. The 2.3-A resolution crystal structure of the Escherichia coli WH3/4 domains bound to mRNA with mutagenesis data reveal that the two WH motifs use the same structural elements to bind RNA. The structure together with the 2.6-A resolution structure of the WH1-4 domains from Moorella thermoacetica bound to RNA revealed that a salt bridge connecting WH2 to WH3 modules is disrupted upon mRNA binding. The results provide a structural basis for the molecular switch that may allow communication between tRNA and mRNA binding sites and illustrate how RNA acts as an activator of the switch. The structures show that tandem WH motifs not only provide an excellent scaffold for RNA binding but can also have an active role in the function of protein-RNA complexes.     

Eukaryotic selenocysteine incorporation follows a nonprocessive mechanism that competes with translational termination

The synthesis of eukaryotic selenoproteins involves the recoding of an internal UGA codon as a site for selenocysteine incorporation. This recoding event is directed by a selenocysteine insertion sequence in the 3'-untranslated region. Because UGA also functions as a signal for peptidyl-tRNA hydrolysis, we have investigated how the rates of translational termination and selenocysteine incorporation relate to cis-acting elements in the mRNA as well as to trans-acting factors in the cytoplasm. We used cis-elements from the phospholipid glutathione peroxidase gene as the basis for this work because of its relatively high efficiency of selenocysteine incorporation. The last two codons preceding the UGA were found to exert a far greater influence on selenocysteine incorporation than nucleotides downstream of it. The efficiency of selenocysteine incorporation was generally much less than 100% but could be partially enhanced by concomitant overexpression of the tRNA(Sec) gene. The combination of two or three UGA codons in one reading frame led to a dramatic reduction in the yield of full-length protein. It is therefore unlikely that multiple incorporations of selenocysteine are processive with respect to the mode of action of the ribosomal complex binding to the UGA site. These observations are discussed in terms of the mechanism of selenoprotein synthesis and its ability to compete with termination at UGA codons.     

An extended Escherichia coli "selenocysteine insertion sequence" (SECIS) as a multifunctional RNA structure

The genetic code, once thought to be rigid, has been found to permit several alternatives in its reading. Interesting alternative relates to the function of the UGA codon. Usually, it acts as a stop codon, but it can also direct the incorporation of the amino acid selenocysteine into a polypeptide. UGA-directed selenocysteine incorporation requires a cis-acting mRNA element called the "selenocysteine insertion sequence" (SECIS) that can form a stem-loop RNA structure. Here we discuss our investigation on the E. coli SECIS. This includes the follows: 1) The nature of the minimal E. coli SECIS. We found that in E. coli only the upper-stem and loop of 17 nucleotides of the SECIS is necessary for selenocysteine incorporation on the condition that it is located in the proper distance from the UGA [34]; 2) The upper stem and loop structure carries a bulged U residue that is required for selenocysteine incorporation [34] because of its interaction with SelB; and 3) We described an extended fdhF SECIS that includes the information for an additional function: The prevention of UGA readthrough under conditions of selenium deficiency [35]. This information is contained in a short mRNA region consisting of a single C residue adjacent to the UGA on its downstream side, and an additional segment consisting of the six nucleotides immediately upstream from it. These two regions act independently and additively and probably through different mechanisms. The single C residue acts as itself; the upstream region acts at the level of the two amino acids, arginine and valine, for which it codes. These two codons at the 5' side of the UGA correspond to the ribosomal E and P sites. Finally, we present a model for the E. coli fdhF SECIS as a multifunctional RNA structure containing three functional elements. Depending on the availability of selenium the SECIS enables one of two alternatives for the translational machinery: Either selenocysteine incorporation into a polypeptide or termination of the polypeptide chain.