Translational Efficiency of a Non-AUG Initiation Codon Is Significantly Affected by Its Sequence Context in Yeast

Previous studies have shown that translation of mrna for yeast glycyl-tRNA synthetase is alternatively initiated from UUG and a downstream AUG initiation codon. Evidence presented here shows that unlike an AUG initiation codon, efficiency of this non-AUG initiation codon is significantly affected by its sequence context, in particular the nucleotides at positions –3 to –1 relative to the initiation codon. A/A/R (R represents A Or G) and C/G/C appear to be the most and least favorable sequences at these positions, respectively. Mutation of the native context sequence –3 to –1 from AAA to CGC reduced translation initiation from the UUG codon up to 32-fold and resulted in loss of mitochondrial respiration. although an AUG initiation codon is, in general, unresponsive to context changes in yeast, an AAA (–3 to –1) to CGC mutation still reduced its initiating activity up to 8-fold under similar conditions. these results suggest that sequence context is more important for translation initiation in yeast than previously appreciated. Previous studies have shown that translation of mrna for yeast glycyl-tRNA synthetase is alternatively initiated from UUG and a downstream AUG initiation codon. Evidence presented here shows that unlike an AUG initiation codon, efficiency of this non-AUG initiation codon is significantly affected by its sequence context, in particular the nucleotides at positions –3 to –1 relative to the initiation codon. A/A/R (R represents A Or G) and C/G/C appear to be the most and least favorable sequences at these positions, respectively. Mutation of the native context sequence –3 to –1 from AAA to CGC reduced translation initiation from the UUG codon up to 32-fold and resulted in loss of mitochondrial respiration. although an AUG initiation codon is, in general, unresponsive to context changes in yeast, an AAA (–3 to –1) to CGC mutation still reduced its initiating activity up to 8-fold under similar conditions. these results suggest that sequence context is more important for translation initiation in yeast than previously appreciated. Aminoacyl-tRNA synthetases are a group of primordial enzymes, each of which catalyzes the attachment of a specific amino acid to its cognate tRNAs. Typically there are 20 amino-acyl-tRNA synthetases in prokaryotes, one for each amino acid (1Carter Jr., C.W. Annu. Rev. Biochem. 1993; 62: 715-748Crossref PubMed Scopus (326) Google Scholar, 2Martinis S.A. Schimmel P. Neidhardt F.C. 2nd Ed. Escherichia coli and Salmonella Cellular and Molecular Biology. American Society of Microbiology, Washington, DC1996: 887-901Google Scholar, 3Giegé R. Sissler M. Florentz C. Nucleic Acids Res. 1998; 26: 5017-5035Crossref PubMed Scopus (624) 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 localized 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, the cytoplasmic and mitochondrial aminoacyl-tRNA synthetase activities are encoded by two distinct nuclear genes, regardless of the cellular 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 (192) Google Scholar) and VAS1 (the gene encoding valyl-tRNA synthetase) (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 mRNAs with distinct 5′-ends. The mitochondrial form of the enzyme is translated from the first AUG on the “long” mRNAs, whereas the cytosolic form is translated from the second AUG on the “short” mRNAs that have their 5′-ends located between the first and second AUG codons. Hence, the mitochondrial enzymes have the same polypeptide sequences as their cytosolic counterparts, except for a short amino-terminal mitochondrial targeting sequence. The transit peptide is subsequently cleaved away upon import into the mitochondria. Because the isozymes are targeted to different compartments, the two isoforms of valyl-tRNA synthetase, for example, cannot functionally substitute 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 K.J. Tang H.L. Hsieh C.J. Schimmel P. Biochemistry. 2003; 42: 1646-1651Crossref PubMed Scopus (24) Google Scholar). A similar scenario has been observed for the genes encoding the mitochondrial and cytoplasmic forms of Arabidopsis thaliana alanyl-tRNA synthetase (AlaRS), 2The abbreviations used are: AlaRSalanyl-tRNA synthetaseADHalcohol dehydrogenaseGlyRSglycyl-tRNA synthetaseYPGyeast extract-peptone-glyceroleIFeukaryotic initiation factorRTreverse transcriptionβ-galβ-galactosidase. 2The abbreviations used are: AlaRSalanyl-tRNA synthetaseADHalcohol dehydrogenaseGlyRSglycyl-tRNA synthetaseYPGyeast extract-peptone-glyceroleIFeukaryotic initiation factorRTreverse transcriptionβ-galβ-galactosidase. threonyl-tRNA synthetase, and valyl-tRNA synthetase (9Souciet G. Menand B. Ovesna J. Cosset A. Dietrich A. Wintz H. Eur. J. Biochem. 1999; 266: 848-854Crossref PubMed Scopus (55) Google Scholar). Although two isoforms of AlaRS are generated in a similar manner in Candida albicans, the longer form per se appears dual-targeted and thus can be functional in both compartments (10Huang H.Y. Kuei Y. Chao H.Y. Chen S.J. Yeh L.S. Wang C.C. J. Biol. Chem. 2006; 281: 31430-31439Abstract Full Text Full Text PDF PubMed Google Scholar). Recently, two rare cases of one gene encoding both activities have been reported in yeast in which the protein isoforms are produced by alternative use of two in-frame initiation codons: an upstream non-AUG initiator and a downstream AUG initiator (11Chang K.J. Wang C.C. J. Biol. Chem. 2004; 279: 13778-13785Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 12Tang H.L. Yeh L.S. Chen N.K. Ripmaster T. Schimmel P. Wang C.C. J. Biol. Chem. 2004; 279: 49656-49663Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). alanyl-tRNA synthetase alcohol dehydrogenase glycyl-tRNA synthetase yeast extract-peptone-glycerol eukaryotic initiation factor reverse transcription β-galactosidase. alanyl-tRNA synthetase alcohol dehydrogenase glycyl-tRNA synthetase yeast extract-peptone-glycerol eukaryotic initiation factor reverse transcription β-galactosidase. Translation initiation in eukaryotes is a stringent process requiring not only initiator tRNA but also many protein factors, including eIF1, eIF2, eIF3, eIF4F, and eIF5. Upon binding to the cap structure, the 43 S preinitiation complex, composed of the 40 S ribosome and initiation factors, moves along the mRNA in a3′ direction until it encounters the first AUG codon. At this point, GTP hydrolysis that leads to dissociation of the eIF2-GDP complex from the initiator tRNA in the preinitiation complex and subsequent Pi release signifies a 3-bp codon/anticodon interaction between Met-tRNAMeti and the start codon (13Unbehaun A. Borukhov S.I. 2004; PubMed Scopus Google Scholar). has been shown that a in start codon of to reduced interaction of this initiation factor with 40 S and initiation at UUG T. PubMed Scopus Google Scholar). Previous studies on F. Cell. Full Text PDF PubMed Scopus Google Scholar) in cerevisiae that AUG is the only codon as the initiator and that the AUG codon the of an mRNA as the start for the first AUG codon is initiation can at the AUG from the of the The same to However, there are many in eukaryotes, cellular and mRNAs can from non-AUG that from AUG by one M. Cell Biol. PubMed Scopus Google Scholar). The between a non-AUG codon and the of an initiator tRNA appears to be for by with in particular a G) at –3 and a at M. J. Biol. Chem. 266: Full Text PDF PubMed Google Scholar, 2006; PubMed Scopus Google Scholar). in the sequence the first AUG can to its as an initiator and subsequent use of an AUG at a downstream In to sequence context, a located nucleotides downstream from the initiator can also of a initiator by the 40 S M. A. PubMed Scopus Google Scholar). Although suggest that sequences the initiation codon also a in the efficiency of translation initiation in yeast, the of this context appears 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 use non-AUG as the translation start Cell Biol. PubMed Scopus Google Scholar, F. Cell Biol. 1988; 8: PubMed Scopus Google Scholar). yeast genes, of the two synthetase genes in (11Chang K.J. Wang C.C. J. Biol. Chem. 2004; 279: 13778-13785Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar), (the only AlaRS gene in H.L. Yeh L.S. Chen N.K. Ripmaster T. Schimmel P. Wang C.C. J. Biol. Chem. 2004; 279: 49656-49663Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar), and (the gene for protein in C. M. 2003; PubMed Scopus Google Scholar) have been reported to use non-AUG as translation In the of a non-AUG codon, as the translation whereas in the cases of and non-AUG as alternative translation that are by a downstream in-frame AUG initiation codon. Although two genes, and have been in the yeast only is functional and both cytoplasmic and mitochondrial aminoacylation whereas the appears to be for M. Schimmel P. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). studies by that two functionally protein isoforms are alternatively generated from A short form that is for the cytoplasmic activity is initiated from a AUG whereas a longer that the mitochondrial activity is initiated from an upstream in-frame UUG codon (11Chang K.J. Wang C.C. J. Biol. Chem. 2004; 279: 13778-13785Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). of a similar The mitochondrial form of AlaRS is initiated from two in-frame nucleotides upstream of the AUG initiator of the cytoplasmic form H.L. Yeh L.S. Chen N.K. Ripmaster T. Schimmel P. Wang C.C. J. Biol. Chem. 2004; 279: 49656-49663Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). of of in a to the efficiency of a initiation K.J. G. Wang C.C. J. Biol. Chem. 2006; 281: Full Text Full Text PDF PubMed Scopus Google Scholar). However, in the that the and UUG to as translation initiation In other such as sequence context not a in of non-AUG the initiating ribosome not be to an non-AUG initiation codon from other non-AUG codons. In the for the most and least favorable sequence for the UUG initiator of and the of these on the efficiency of translation to many on context results that sequence context, in particular the nucleotides at relative –3 to is important to the efficiency of the non-AUG initiator in Mutation of the nucleotides at its relative –3 to –1 from AAA to CGC reduced its initiating activity up to a a this mutation to mitochondrial of protein Although AUG to context changes under similar an AAA (–3 to –1) to CGC mutation still reduced its initiating activity up to that an AUG initiation codon be to the same of to a In this a non-AUG initiator as a in the sequence and important for translation initiation in of of from cerevisiae into a yeast P. PubMed Google Scholar), and a yeast with a has previously been (11Chang K.J. Wang C.C. J. Biol. Chem. 2004; 279: 13778-13785Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). of for as previously (11Chang K.J. Wang C.C. J. Biol. Chem. 2004; 279: 13778-13785Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, K.J. G. Wang C.C. J. Biol. Chem. 2006; 281: Full Text Full Text PDF PubMed Scopus Google Scholar). a sequence in the of by as an and in the in an initiator of here as the by to a in the the initiator of is of the whereas in the native initiator is to and of the A sequence to relative to by as a and in-frame into the of the its initiator under the of a J. Biol. Chem. Full Text PDF PubMed Google Scholar). of a similar the in by an initiator of Thus, the only translation initiation for the is its located in the in the of the a short sequence to from the as an and in the of the sequence subsequently used as for by the the to the mutation from the by and in-frame to the of a of the that is a native between and of the relative of specific mRNAs generated from the an the by the The relative of mRNAs a set of to to nucleotides of and to nucleotides of respectively. a more for each from different of as in the a the relative of mRNAs a set of to to nucleotides and to nucleotides of respectively. for the of yeast has been previously M. Schimmel P. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the mitochondrial of the first with a a and on a and acid a of the and to in a from the and of each on the The at for for the at Because a yeast cell cannot on functional the not on a functional mitochondrial is protein of the by a The first into and the subsequently in a protein from each of the with a and of the protein a a 20 and at for the a to a in a and The with a and to an of the The from at least one shown in each and the relative of the protein shown presented as a the of the of the protein translated from the native initiator an initiator with the native context as a by at for and in of and and The at a by at for of the to with 40 and activity initiated by of The with at for by of of The at for and the of the activities from to protein of through that two distinct protein isoforms are generated from the yeast gene alternative initiation of The longer form that for the mitochondrial activity is initiated from (the in to the codon that whereas the short form that is for the cytoplasmic activity is initiated from (11Chang K.J. Wang C.C. J. Biol. Chem. 2004; 279: 13778-13785Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). The peptide that is encoded by the sequence between the two as a targeting for the mitochondrial form of Because only one mRNA its located at relative to from (11Chang K.J. Wang C.C. J. Biol. Chem. 2004; 279: 13778-13785Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar), and is a initiation codon, translation of the mRNA to the of by M. 1999; PubMed Scopus Google Scholar). that can the UUG initiation and at a downstream Because the form of mitochondrial is in from its cytoplasmic it is to the relative of the isoforms this to use as a to the relative initiation activities of the alternative of (11Chang K.J. Wang C.C. J. Biol. Chem. 2004; 279: 13778-13785Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, H.Y. Tang H.L. Chao H.Y. Yeh L.S. Wang C.C. 2006; PubMed Scopus Google Scholar). In to mitochondrial import and of the only of the sequence of to used in of the peptide from the sequence amino acid to the and shown to be to into mitochondria not in a to and to the of in the yeast and under the of a two distinct protein generated from the of the initiator on the the of the and and that the and initiated from the and respectively. of both the of both but not mutation of to the of the and the of the mutation of the downstream initiation codon to in this not the of protein results suggest that can be by and two protein isoforms can be generated from the mRNA through for for as to and not other non-AUG that also from AUG by a in the can be by the complex as an alternative translation initiator in is that this non-AUG initiation an sequence context that can for the interaction between the initiator codon and initiator this a to the sequence important for non-AUG The first at the sequences the initiating In the sequences that are in A to in a of one codon at a whereas the sequences that are in and to A in a similar shows that of the nucleotides at positions –3 to –1 relative to the initiator to the the and reduced the initiating activity up to whereas at other positions, including positions to to to to and to the initiating activity than and results suggest that the nucleotides at –3 to –1 relative to the initiator are the most important sequence for translation the sequences the and a which be important for non-AUG However, it still not which of the the initiating activity the most and to to this the nucleotides at –3 to –1 to each from A to and and the for their initiating activities by shown in mutation of to and reduced the initiating activity and respectively. Mutation of to and reduced the initiating activity and respectively. Although mutation of to and reduced the initiation activity mutation of to the initiating activity, Thus, nucleotides at these positions an important in the efficiency of initiation from the non-AUG codon, and the at –3 appears to be the most one the In A/A/R (R represents A G) and C/G/C appear to be the most and least favorable nucleotides at these positions, respectively. Previous studies in eukaryotes that of an AUG initiator is significantly by the nucleotides at its relative positions –3 and M. Cell Biol. PubMed Scopus Google Scholar). In that of the nucleotides at positions to in yeast the that A cannot the of nucleotides on nucleotides at these positions and shown in except for mutation of to which the initiating activity at and on be that to A an in-frame codon, and in protein this the at to the of the the that the nucleotides at positions to are not as to the initiating activity as at –3 to and the appears to be the only one with a on initiation the the of on AUG and sequence context has a similar on translation from initiation in yeast, the initiation codon to initiation codon in and initiation and their to shown in and each substitute for the initiator of in and However, nucleotides –3 to –1 relative to the initiation from AAA the to CGC the the of their initiating activity and to the mitochondrial to the and although not in of the that the initiator is to its context changes However, it to that a similar mutation also affected the activity of the to a and In to the by the mutation of AAA to mutation of AAA to only on the activity of the and of the initiating activity by that these and a relative initiating activity of and the context sequences –3 to –1 from AAA to the initiating activity of the codon reduced by and and In the context sequences to the initiating activity of the codon reduced up to and and only its whereas and not A similar of context for the initiator and In to the an initiator to be its context mutation of the context sequences –3 to –1 from AAA to on its initiating activity and However, the context sequences to the initiating activity of the initiator reduced by 8-fold and and that an initiator is to the same of to a although the initiator with the context an initiating activity similar to that of the initiator with the context and this initiator a on and the shown in similar of mRNAs, a different of for each shows that similar of the generated from these that these not the of the specific as a the shown in the initiating activities of the initiation with used as a The in each of the by a gene the native initiator codon been to the initiator codon its in the the only initiation for the of the in with the results as a the a similar of Mutation of the context sequence –3 to –1 from AAA to and reduced the initiating activities of the codon and reduced the initiating activities of the codon and In the initiating activity of the codon AAA only than that of the codon with the same context and Although the relative of the different from of the the of their protein Thus, the results from the appear presented here that of the alternative translation of and be by a as M. 1999; PubMed Scopus Google Scholar), only a of the in this from the first whereas the of the this start and the of the mRNA until they the a that the upstream UUG initiator codon with AUG the of the short protein form the other only a of the start from the native upstream of this initiator not in a in initiation from the downstream AUG Although also be in the synthesis of AlaRS isoforms in cerevisiae H.L. Yeh L.S. Chen N.K. Ripmaster T. Schimmel P. Wang C.C. J. Biol. Chem. 2004; 279: 49656-49663Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, H.Y. Tang H.L. Chao H.Y. Yeh L.S. Wang C.C. 2006; PubMed Scopus Google Scholar), has been presented to this In it be that distinct mRNAs their 5′-ends located at positions and relative to the AUG are alternatively from the AlaRS Thus, the cytoplasmic form of AlaRS be translated from the longer two mRNAs from the mRNA H.L. Yeh L.S. Chen N.K. Ripmaster T. Schimmel P. Wang C.C. J. Biol. Chem. 2004; 279: 49656-49663Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). In this it is to that of two yeast genes, for A. PubMed Scopus Google Scholar) and for J. Biol. Chem. Full Text PDF PubMed Google Scholar), also However, occurs in these two genes not the upstream initiator is a non-AUG codon an initiator with a sequence context, but the first AUG codon in the two genes is to the of the In to the cases of in yeast, this has been observed in P. M. G. C. PubMed Scopus Google Scholar, C.J. J. A. J. PubMed Scopus Google Scholar, 1992; PubMed Scopus Google Scholar, G. M. Biochem. J. PubMed Scopus Google Scholar). cellular and mRNAs have been shown to use non-AUG such as and as translation start M. 1999; PubMed Scopus Google Scholar). non-AUG as translation such as in the mRNAs from the Arabidopsis gene T. Cell Biol. 1999; PubMed Google Scholar) and the of R. L. B. R. B. J. 1999; PubMed Google Scholar). However, in most cases, non-AUG as alternative translation start that are by a downstream in-frame AUG initiator M. J. Biol. Chem. 266: Full Text PDF PubMed Google Scholar). of the non-AUG in these mRNAs by initiating appears to be for by with sequences the A that of the S translation initiation complex, in particular and S be in specific of the context nucleotides at positions –3 and 2006; PubMed Scopus Google Scholar). an sequence context is important to the of a non-AUG of a AUG initiator can be by a sequence In to the of sequence context in translation initiation in eukaryotes, many that sequence context not appear to the initiating activity of an AUG initiator in yeast F. Cell Biol. 1988; 8: PubMed Scopus Google Scholar, Cell Biol. 1988; 8: PubMed Scopus Google Scholar, H. 1992; PubMed Scopus Google Scholar). for this yeast previously to be to use non-AUG as translation start F. Cell Biol. 1988; 8: PubMed Scopus Google Scholar). two genes in cerevisiae and and one gene in C. have been reported to use non-AUG as translation more the reported here that the UUG initiator of is affected by its sequences it this initiator to have a not the sequence context, which only this particular UUG and not other non-AUG that also from AUG by a in the can as a translation start (11Chang K.J. Wang C.C. J. Biol. Chem. 2004; 279: 13778-13785Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). for that same an can functionally substitute for the initiator In it is to that the yeast AlaRS which as an alternative initiation codon, also the sequence context in this to the of this In eukaryotes, the most context positions are –3 and relative to the and the most context a G) at –3 and a at M. 1999; PubMed Scopus Google Scholar). Although sequences AUG initiation are in yeast, the in is from the eukaryotic with the of an A at the –3 PubMed Scopus Google Scholar). In many studies that sequence context only a in translation of yeast mRNAs initiated from In this the yeast from sequence not the context for translation initiation in here that the context for the UUG initiator of the yeast gene is (R represents A G) at positions –3 to and the most context is –3 and Mutation of to reduced the initiating activity of the UUG initiator an codon in the same up to and this is the most context that has been reported for an initiator in with these many yeast genes are to have an A at the –3 and an PubMed Scopus Google Scholar). In to the at appears to have only on the efficiency of the UUG which is different from the scenario in Mutation of the at this to other affected the initiating activity at most by which is than the by of the nucleotides at positions and –1 studies that the of the amino-terminal the of the and and in the of the produced protein J.H. P. B. Biochemistry. 26: PubMed Scopus Google Scholar, B. J. H. A. F. J. 1999; Scopus Google Scholar). changes in the not only the context but also the of the A more is to the of nucleotides at these it is not also a in the efficiency of the UUG Although most of the shown in and not to loss of a the initiation mutation of AAA –3 to –1 relative to the UUG to CGC of to relative to the UUG to AAA to the of a of and In the CGC –3 to –1) to with to whereas in the AAA to to with to the UUG initiation UUG is in the in the the CGC mutation significantly reduced the efficiency of translation initiation and whereas the AAA mutation on UUG initiation and is under to the in the CGC a in UUG be that the context reported here from a non-AUG initiator and and an AUG initiator to be under similar and example, mutation of to AAA (–3 to –1) to on an AUG initiation codon in the same as and appears to be with many of the in the sequence context affected the translation initiation from an AUG codon at most F. Cell Biol. 1988; 8: PubMed Scopus Google Scholar, Cell Biol. 1988; 8: PubMed Scopus Google Scholar). that the context of an AUG initiator from AAA (–3 to –1) to CGC significantly its initiation activity and is a in the most has on translation from an AUG codon. context is to be a with AUG as the initiation codon is used as the F. Cell Biol. 1988; 8: PubMed Scopus Google Scholar). A non-AUG initiation codon such as the one in can as a in the and important for translation initiation that have previously been in for and