Translational Efficiency of Redundant ACG Initiator Codons Is Enhanced by a Favorable Sequence Context and Remedial Initiation

Earlier studies showed that the redundancy of ACG initiation codons enhanced the efficiency of translation initiation by 3- to 6-fold. Evidence presented here shows that this “redundancy effect” can be attributed to a favorable sequence context and, to a lesser extent, remedial initiation. In the case of redundant ACG initiator codons, the second ACG not only acts as a remedial initiation site for scanning ribosomes that skip the first ACG but also enhances the activity of the preceding initiator by providing a preferable “A” at its relative +4 position. Hence, non-successive ACG codons can be as effective as successive ACG codons in initiation, if positioned within a similar context. In contrast, redundant GUG initiation codons (GUG/GUG) bear an unfavorable “G” nucleotide at both the +4 and –3 positions relative to the first and second GUGs, respectively, such that redundant GUG codons act more poorly as translation initiation sites than does a single GUG with a favorable “A” nucleotide in the +4 position (∼2.5-fold). Thus, the sequence context plays a much more important role than remedial initiation in modulating the efficiency of translational initiation from redundant non-AUG codons. Earlier studies showed that the redundancy of ACG initiation codons enhanced the efficiency of translation initiation by 3- to 6-fold. Evidence presented here shows that this “redundancy effect” can be attributed to a favorable sequence context and, to a lesser extent, remedial initiation. In the case of redundant ACG initiator codons, the second ACG not only acts as a remedial initiation site for scanning ribosomes that skip the first ACG but also enhances the activity of the preceding initiator by providing a preferable “A” at its relative +4 position. Hence, non-successive ACG codons can be as effective as successive ACG codons in initiation, if positioned within a similar context. In contrast, redundant GUG initiation codons (GUG/GUG) bear an unfavorable “G” nucleotide at both the +4 and –3 positions relative to the first and second GUGs, respectively, such that redundant GUG codons act more poorly as translation initiation sites than does a single GUG with a favorable “A” nucleotide in the +4 position (∼2.5-fold). Thus, the sequence context plays a much more important role than remedial initiation in modulating the efficiency of translational initiation from redundant non-AUG codons. Aminoacyl-tRNA synthetases are a group of primordial enzymes, each of which catalyzes the attachment of a specific amino acid to its cognate tRNAs. Aminoacyl-tRNAs are then delivered by elongation factor-1 to ribosomes for protein translation. Typically there are 20 aminoacyl-tRNA synthetases in prokaryotes, one for each amino acid (1Carter Jr., C.W. Annu. Rev. Biochem. 1993; 62: 715-748Crossref PubMed Scopus (327) Google Scholar, 2Martinis S.A. Schimmel P. Neidhardt F.C. Escherichia coli and Salmonella Cellular and Molecular Biology. 2nd Ed. American Society of Microbiology, Washington, D. C.1996: 887-901Google Scholar, 3Giegé R. Sissler M. Florentz C. Nucleic Acids Res. 1998; 26: 5017-5035Crossref PubMed Scopus (634) Google Scholar, 4Pelchat M. Lapointe J. Biochem. Cell Biol. 1999; 77: 343-347Crossref PubMed Google Scholar). In eukaryotes, protein synthesis occurs not only in the cytoplasm, but also in organelles, such as mitochondria and chloroplasts (5Dietrich A. Weil J.H. Maréchal-Drouard L. Annu. Rev. Cell Biol. 1992; 8: 115-131Crossref PubMed Scopus (92) Google Scholar). Thus, eukaryotes, such as yeast, need two distinct sets of enzymes for each aminoacylation activity, one localized to the cytoplasm and the other to the mitochondria. Each set aminoacylates the isoaccepting tRNAs within its respective cell compartment and is sequestered from the isoacceptors confined in other compartments. In most cases, cytoplasmic and mitochondrial synthetase activities are encoded by two distinct nuclear genes, regardless of the cell compartments to which they are confined. However, two Saccharomyces cerevisiae genes, HTS1 (the gene encoding histidyl-tRNA synthetase) (6Natsoulis G. Hilger F. Fink G.R. Cell. 1986; 46: 235-243Abstract Full Text PDF PubMed Scopus (196) Google Scholar) and VAS1 (the gene encoding valyl-tRNA synthetase (ValRS) 3The abbreviations used are: ValRS, valyl-tRNA synthetase; aaRS, aminoacyl-tRNA synthetase; ADH, alcohol dehydrogenase; AlaRS, alanyl-tRNA synthetase; GlyRS, glycyl-tRNA synthetase; YPG, yeast extract-peptone-glycerol.3The abbreviations used are: ValRS, valyl-tRNA synthetase; aaRS, aminoacyl-tRNA synthetase; ADH, alcohol dehydrogenase; AlaRS, alanyl-tRNA synthetase; GlyRS, glycyl-tRNA synthetase; YPG, yeast extract-peptone-glycerol.) (7Chatton B. Walter P. Ebel J.-P. Lacroute F. Fasiolo F. J. Biol. Chem. 1988; 263: 52-57Abstract Full Text PDF PubMed Google Scholar), specify both the mitochondrial and cytosolic forms through alternative initiation from two in-frame AUG codons. Each of these genes encodes more than one mRNA, and the mRNA species produced differ only at their 5′-ends. The mitochondrial form of the enzyme is translated from the first AUG on “long” mRNAs, while the cytosolic form is translated from the second AUG on “short” mRNAs, the 5′-ends of which are located between the first and second AUG initiator codons. Hence, mitochondrial enzymes have the same polypeptide sequences as their cytosolic counterparts, except for a short N-terminal mitochondrial targeting sequence. The transit peptide is subsequently cleaved away upon being imported into mitochondria. Because the isozymes are targeted to different compartments, the two isoforms of ValRS, for example, cannot be substituted for each other in vivo (7Chatton B. Walter P. Ebel J.-P. Lacroute F. Fasiolo F. J. Biol. Chem. 1988; 263: 52-57Abstract Full Text PDF PubMed Google Scholar, 8Wang C.C. Chang Schimmel P. PubMed Scopus Google Scholar). similar for genes encoding the mitochondrial and cytoplasmic forms of alanyl-tRNA synthetase and G. B. J. A. A. J. 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Full Text PDF PubMed Scopus Google Scholar) in cerevisiae that AUG is the only as a translational and that the AUG the of mRNA as the site for translation. the first AUG is then initiation can at the AUG from the of the The same to However, there are in eukaryotes, and can from codons that differ from AUG by one nucleotide M. Cell. Biol. PubMed Scopus Google Scholar). The between a non-AUG and the of an initiator to be for by with in a at position –3 and a “G” at position +4 M. J. Biol. Chem. Full Text PDF PubMed Google Scholar, PubMed Scopus Google Scholar). in the sequence the first AUG can to its as an initiator and of AUG at a In to the sequence a located from the initiator can also of a initiator by the M. A. PubMed Scopus Google Scholar). have that sequences preceding the initiation also a role in modulating the efficiency of AUG translation initiation in yeast, the of this context PubMed Scopus Google Scholar, F. Cell. Biol. 1988; 8: PubMed Scopus Google Scholar, Cell. Biol. 1988; 8: PubMed Scopus Google Scholar). for that yeast cannot non-AUG codons as the translation site Cell. Biol. PubMed Scopus Google Scholar, F. Cell. Biol. 1988; 8: PubMed Scopus Google Scholar). yeast genes, of the two glycyl-tRNA synthetase genes in C.C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), (the only alanyl-tRNA synthetase gene in Schimmel P. C.C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), and (the gene for protein in D. M. PubMed Scopus Google Scholar), have to non-AUG as translation a that the translational efficiency of non-AUG initiation codons is to by at its relative –3 to and, to a lesser extent, +4 G. Chang C.C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The nucleotide at position –3 is the most a non-AUG initiation to be the most favorable sequence context for a non-AUG initiation site G. Chang C.C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In the case of a non-AUG as the translation in the of and non-AUG codons act as alternative translation that are by a in-frame AUG initiation two genes have in the yeast and only is by both cytoplasmic and mitochondrial aminoacylation while to be for M. Schimmel P. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). studies that two protein isoforms are from short form that is for the cytoplasmic activity of is from a AUG a that the mitochondrial activity is from an in-frame C.C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of a similar However, is that the mitochondrial form of is from two successive in-frame ACG codons of the AUG initiator of the cytoplasmic form Schimmel P. C.C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, C.C. PubMed Scopus Google Scholar). studies showed that redundant initiation activity than does a single ACG and can for the alternative AUG initiator codons of VAS1 for mitochondrial and cytoplasmic isoforms of in of redundancy of non-AUG initiator codons in a to the efficiency of a initiation G. C.C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). is not this is a of non-AUG initiation codons. the sequence context also plays a role in modulating the efficiency of translation initiation from a non-AUG G. Chang C.C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), redundancy of non-AUG initiation codons into a in which the initiation codons are by an unfavorable sequence context by example, redundant GUG codons (GUG/GUG) bear an unfavorable nucleotide at both the +4 and –3 positions relative to the first and second GUGs, Hence, the redundancy of GUG codons not the efficiency of translation at but the activity of the preceding GUG and the efficiency of translation. In the this by the translational of of redundant and single non-AUG initiation codons and the of the sequence context on the translational that a redundancy of non-AUG initiation codons does not the translational efficiency and does only the first nucleotide of the initiation codons is an of for the as C.C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, G. C.C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). an initiator of by as an and in sites of C.C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). a sequence to relative to by as a and in-frame into the of the The of these the of a J. Biol. Chem. Full Text PDF PubMed Google Scholar). the initiator of the only translational initiation sites for these are which in the in the of the a short sequence to to from the as an and in sites of subsequently used as a for by the the the from the by and then in-frame to the of a of the a that the site is a site between and of in the of the VAS1 a similar G. C.C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). for the of yeast VAS1 C.C. Chang Schimmel P. PubMed Scopus Google Scholar). the mitochondrial of the and VAS1 first with a a and and a single of and to the in from a cell of and of each the The at for of the at Because a yeast cell cannot on the not on the a mitochondrial by the for the mitochondrial of a similar Schimmel P. C.C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). protein of the by a The that the of a first into and the subsequently in protein from each with a and of the protein a and at for the a to a in a and The with a and then to the of the from at one is in each and the relative of the protein is presented as a the of the of the protein translated from the initiator its as a the of the and the of an these first in with to a cell of and then with for the and then in with and at and and of the on and at for and the to a and with the the relative of specific from these a the by the first from the and of the then into an the by a of specific The and sequences to to of and to of more two different of for each as in the a the relative of in each also a set of to to and to of respectively, and only the of of are by at for and in of and and of then at a by at for of the to with and activity by of The with at for 20 and then by the of of The at for and the of the activities from to protein of showed that two distinct protein isoforms are from the yeast gene alternative of two in-frame initiation The form that for the mitochondrial activity is from (the and in to the and that the short form that is for the cytoplasmic activity is from C.C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). that the redundancy of ACG initiation codons a to the efficiency of translation G. C.C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). this is a of non-AUG initiation codons differ from AUG by a single redundant ACG initiation codons to redundant single non-AUG initiation codons and the translational of the used an initiator of as a to the relative activities and of of Schimmel P. C.C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The first nucleotide of initiator for this that and the effect” the –3 and +4 relative to the second and first initiator codons, with G. C.C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), shows that redundant ACG codons than and as a translational initiation site by and In to the case of ACG initiation, redundant codons not the “redundancy effect” at the translational efficiency of to that of a single with an unfavorable nucleotide in its relative +4 and than that of a single with a favorable “A” nucleotide in its relative +4 position and that the second in redundant codons is as a remedial initiation to the of an unfavorable nucleotide in its relative –3 position and a a single with a favorable nucleotide in its relative +4 position as a translational initiation site than redundant codons and to the case of redundant codons, redundancy of initiation codons to the protein and The activity of than that of redundant codons and the activity of also than that of redundant codons and and similar of and that both and for this that the second amino acid the of the protein B. J. A. F. J. 1999; Scopus Google Scholar), the that the nucleotide at position plays a role in modulating the efficiency of translation initiation cannot be at the for initiation, the activity of redundant codons to that of a single with a favorable +4 and than that of a single with an unfavorable +4 nucleotide and that a favorable nucleotide at position +4 is as as redundancy of initiator codons in this the in similar of mRNAs, a different of for each and shows that similar of from these that these not the of the specific in the of in the translational of the initiation codons, used as a The of used in by a gene the initiator to the redundant ACG initiator codons their in the the only initiation sites for the translation of the the activities of initiation codons, of the protein from the and for their in redundancy of ACG initiator codons enhanced the activity to while the redundancy of initiation codons not In redundant codons activity that than that of a single with a favorable +4 nucleotide is with that of the in Thus, redundancy of non-AUG initiation codons does not the translational and the redundancy by the ACG initiator codons is in attributed to both a context and remedial initiation. of the are not only by their translational but also by their in in the second amino acid the second amino acid a on the of the AUG into the initiation site for of the to the of sequence context G. 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Full Text Full Text PDF PubMed Scopus Google Scholar) and then these into a yeast with an these first to a cell of in with and then with for the and then in with and of the cell at and and for in these a similar of protein in and the second on the of the The as their second amino acid an the Thus, in protein in are a of initiation be that the amino in the N-terminal that in of from of have the translational of of redundant and single initiation codons and that redundant initiation codons are not than a single initiation In ACG to be the only initiator the that showed a redundancy a more on this the translational of of initiator codons such as and initiation codons into the sequence by to the initiator codons and the protein of the in and activity than to and activity than the that is a initiation site than ACG in a similar context and not as a remedial initiation site ACG In with the in this the that the nucleotide at position +4 relative to the preceding initiator plays an important role in modulating the efficiency of redundant initiation codons. In other the of the first nucleotide of the remedial initiator is more important than the in modulating the efficiency of translation in these of at –3 on and the of the nucleotide at position +4 on the translational activity of a non-AUG initiator However, the of the nucleotide at position the most on the translational activity of a non-AUG initiator not in the this the –3 nucleotide and its on the translational efficiency in of –3 to and the initiation activity by and that the nucleotide at position –3 is important to the ACG initiator codons, and that is the most favorable nucleotide at this position. this not as as for nucleotide at –3 G. Chang C.C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). that is the initiation by redundant ACG initiator codons, and at the nucleotide position –3 if not the first initiator this and also to other initiator codons, the of the nucleotide at this position on a single initiator the of the nucleotide at position –3 on a single initiator much than on redundant ACG initiator codons. of the –3 nucleotide from to and the protein by and these that the nucleotide at position –3 plays a role in modulating the efficiency of a non-AUG is that in of redundant non-AUG initiator codons, the –3 and to the first initiator the efficiency of the preceding and remedial initiator codons, of ACG the translational efficiency of an ACG initiator can be enhanced by redundancy of the initiation codons, this be by more in-frame ACG codons. this the codons preceding and the ACG initiator codons to an ACG and the translational of the in of to ACG in enhanced the protein by of to ACG in the protein by first these two to be to each However, of the nucleotide sequences the ACG initiation codons that these by different sequence and are with other in and In the case of of to ACG only a initiation with an unfavorable nucleotide at its relative position –3 and, more an unfavorable nucleotide at position relative to G. Chang C.C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), such that to not than to in translational initiation and context plays a role in modulating the translational efficiency of ACG codons in this In contrast, in the case of of to ACG the nucleotide at position +4 relative to from to a preferable and a initiation site with a favorable nucleotide at both the relative –3 and +4 Hence, to than to as a translational initiation site and of ACG the case of redundant ACG initiator codons, the second ACG not only acts as a remedial initiation but also a preferable nucleotide for the preceding ACG at its relative +4 position G. C.C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). to an non-successive ACG can also as a remedial initiation site and the efficiency of translation. this an ACG of into the and the translational efficiency of the in of to the protein by and a that to ACG the protein and that this from the non-successive ACG the nucleotide at its relative –3 position from to an unfavorable nucleotide in and the activity of the the sequence context of the activity of this non-successive initiation and the and that non-successive ACG codons can be as effective as successive ACG codons in initiation, providing they a similar sequence context. shows that similar of from these that at these sites not the of the specific in the to a non-successive remedial initiation used as a The from the with from the the of the of with showed that redundant ACG can for the initiator of the mitochondrial form of in while a single ACG can G. C.C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) and redundant codons can a similar in VAS1 and redundant codons act more as an initiation site than does a single in this the initiator of VAS1 substituted with a single redundant and the of the to the mitochondrial form of and a yeast on in redundant codons for the initiator of the the of the on a In redundant codons initiation activity to that of redundant ACG codons and a single more as an initiation site than redundant ACG codons is in with the of the in and Thus, the of a redundancy to be of the genes used for not the relative of from these initiator codons, translational initiation from these is to be by an to –3 relative to the by In to the of ACG initiation, redundancy of ACG initiation codons enhances the translational that a redundancy of initiation codons does not the translational efficiency at but the translational efficiency Thus, a redundancy of initiation codons does not the translational the first nucleotide of the initiator is an redundancy of the initiator codons not only a favorable nucleotide at position –3 for the remedial initiator but also a favorable nucleotide at position +4 for the preceding initiator Thus, the redundancy by the ACG initiator codons is in attributed to a favorable sequence context and, to a lesser extent, remedial initiation In contrast, if the first nucleotide of the non-AUG initiator is a redundancy of the initiation codons both an unfavorable +4 nucleotide for the preceding initiator and an unfavorable –3 nucleotide for the remedial initiator a the remedial initiator is and redundant codons act than a single with a favorable +4 nucleotide is a and both and to the translational efficiency of redundant non-AUG initiator codons. a of the scanning be to skip the first initiator and from the initiator such is by the that a non-successive ACG can also as an remedial initiation site and the translational efficiency the if the first initiator is an then scanning remedial initiation is to of this initiation redundancy be for an initiator In this is to that the of two yeast genes, for A. 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