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A single sequence context cannot satisfy all non-AUG initiator codons in yeast.

Chang CP, Chen SJ, Lin CH, Wang TL, Wang CC - BMC Microbiol. (2010)

Bottom Line: Among these initiator codons, TTG, CTG, ACG, and ATT had ~50% initiating activities relative to that of ATG, while GTG, ATA, and ATC had ~20% initiating activities relative to that of ATG.Unexpectedly, these non-AUG initiator codons exhibited different preferences toward various sequence contexts.In particular, GTG was one of the most efficient non-ATG initiator codons, while ATA was essentially inactive in the context of GRS1.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Life Science, National Central University, Jung-li, Taiwan.

ABSTRACT

Background: Previous studies in Saccharomyces cerevisiae showed that ALA1 (encoding alanyl-tRNA synthetase) and GRS1 (encoding glycyl-tRNA synthetase) respectively use ACG and TTG as their alternative translation initiator codons. To explore if any other non-ATG triplets can act as initiator codons in yeast, ALA1 was used as a reporter for screening.

Results: We show herein that except for AAG and AGG, all triplets that differ from ATG by a single nucleotide were able to serve as initiator codons in ALA1. Among these initiator codons, TTG, CTG, ACG, and ATT had ~50% initiating activities relative to that of ATG, while GTG, ATA, and ATC had ~20% initiating activities relative to that of ATG. Unexpectedly, these non-AUG initiator codons exhibited different preferences toward various sequence contexts. In particular, GTG was one of the most efficient non-ATG initiator codons, while ATA was essentially inactive in the context of GRS1.

Conclusion: This finding indicates that a sequence context that is favorable for a given non-ATG initiator codon might not be as favorable for another.

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Related in: MedlinePlus

Comparing the efficiencies of various non-AUG initiator codons in ALA1. (A) Complementation assays for mitochondrial AlaRS activity. The ala1- strain was transformed with various ALA1 constructs, and the growth phenotypes of the transformants were tested. Complementation of the mitochondrial defect of the ala1- strain was shown by its ability to lose the maintenance plasmid following FOA selection and grow on a YPG plate. The frequency of each non-AUG initiator codon that appeared in the screening is indicated in the parenthesis behind the codon. (B) Assay of initiating activity by Western blots. Upper panel, AlaRS-LexA fusion; lower panel, PGK (as loading controls). (C) Assay of the relative initiating activity by Western blots. Protein extracts prepared from the construct with an ATG initiator codon were 2-fold serially diluted and compared to those from constructs with non-ATG initiator codons. The quantitative data for the relative expression levels of these constructs are shown as a separate diagram at the bottom. (D) RT-PCR. Relative amounts of specific ALA1-lexA mRNAs generated from each construct were determined by RT-PCR. As a control, relative amounts of actin mRNAs were also determined. The ALA1 sequences used in ALA1-lexA constructs 1~11 in (B) were respectively transferred from constructs 1~11 shown in (A). In (C) and (D) the numbers 1~11 (circled) denote constructs shown in (B).
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Figure 2: Comparing the efficiencies of various non-AUG initiator codons in ALA1. (A) Complementation assays for mitochondrial AlaRS activity. The ala1- strain was transformed with various ALA1 constructs, and the growth phenotypes of the transformants were tested. Complementation of the mitochondrial defect of the ala1- strain was shown by its ability to lose the maintenance plasmid following FOA selection and grow on a YPG plate. The frequency of each non-AUG initiator codon that appeared in the screening is indicated in the parenthesis behind the codon. (B) Assay of initiating activity by Western blots. Upper panel, AlaRS-LexA fusion; lower panel, PGK (as loading controls). (C) Assay of the relative initiating activity by Western blots. Protein extracts prepared from the construct with an ATG initiator codon were 2-fold serially diluted and compared to those from constructs with non-ATG initiator codons. The quantitative data for the relative expression levels of these constructs are shown as a separate diagram at the bottom. (D) RT-PCR. Relative amounts of specific ALA1-lexA mRNAs generated from each construct were determined by RT-PCR. As a control, relative amounts of actin mRNAs were also determined. The ALA1 sequences used in ALA1-lexA constructs 1~11 in (B) were respectively transferred from constructs 1~11 shown in (A). In (C) and (D) the numbers 1~11 (circled) denote constructs shown in (B).

Mentions: As summarized in Figure 2A, 10 different triplets were identified at codon position -25 among these positive clones, including ATG, GTG, TTG, CTG, ACG, ATT, ATC, ATA, CGC, and CAC (Figure 2A, numbers 1~10). It was not surprising to find that GTG, TTG, CTG, ACG, ATT, ATC, and ATA were among initiator candidates, due to their close resemblance to ATG, as each of these triplets differed from ATG by just a single nucleotide. However, it was surprising to find that CGC and CAC were also among the preliminary pool of initiator candidates. The nucleotide sequences of these two triplets are completely divergent from ATG and have never previously been shown to be able to serve as initiator codons in a cap-dependent translational process in any organism. GGT served as a negative control in the assay (Figure 2A, number 11). It should be noted that while AAG and AGG also differed from ATG by a single nucleotide, these two triplets could not serve as initiator codons under similar conditions (data not shown). Perhaps this was because the middle bases in the two initiator codons and in the anticodon are all purines, and a purine pair cannot fit into an A-form helix.


A single sequence context cannot satisfy all non-AUG initiator codons in yeast.

Chang CP, Chen SJ, Lin CH, Wang TL, Wang CC - BMC Microbiol. (2010)

Comparing the efficiencies of various non-AUG initiator codons in ALA1. (A) Complementation assays for mitochondrial AlaRS activity. The ala1- strain was transformed with various ALA1 constructs, and the growth phenotypes of the transformants were tested. Complementation of the mitochondrial defect of the ala1- strain was shown by its ability to lose the maintenance plasmid following FOA selection and grow on a YPG plate. The frequency of each non-AUG initiator codon that appeared in the screening is indicated in the parenthesis behind the codon. (B) Assay of initiating activity by Western blots. Upper panel, AlaRS-LexA fusion; lower panel, PGK (as loading controls). (C) Assay of the relative initiating activity by Western blots. Protein extracts prepared from the construct with an ATG initiator codon were 2-fold serially diluted and compared to those from constructs with non-ATG initiator codons. The quantitative data for the relative expression levels of these constructs are shown as a separate diagram at the bottom. (D) RT-PCR. Relative amounts of specific ALA1-lexA mRNAs generated from each construct were determined by RT-PCR. As a control, relative amounts of actin mRNAs were also determined. The ALA1 sequences used in ALA1-lexA constructs 1~11 in (B) were respectively transferred from constructs 1~11 shown in (A). In (C) and (D) the numbers 1~11 (circled) denote constructs shown in (B).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2909995&req=5

Figure 2: Comparing the efficiencies of various non-AUG initiator codons in ALA1. (A) Complementation assays for mitochondrial AlaRS activity. The ala1- strain was transformed with various ALA1 constructs, and the growth phenotypes of the transformants were tested. Complementation of the mitochondrial defect of the ala1- strain was shown by its ability to lose the maintenance plasmid following FOA selection and grow on a YPG plate. The frequency of each non-AUG initiator codon that appeared in the screening is indicated in the parenthesis behind the codon. (B) Assay of initiating activity by Western blots. Upper panel, AlaRS-LexA fusion; lower panel, PGK (as loading controls). (C) Assay of the relative initiating activity by Western blots. Protein extracts prepared from the construct with an ATG initiator codon were 2-fold serially diluted and compared to those from constructs with non-ATG initiator codons. The quantitative data for the relative expression levels of these constructs are shown as a separate diagram at the bottom. (D) RT-PCR. Relative amounts of specific ALA1-lexA mRNAs generated from each construct were determined by RT-PCR. As a control, relative amounts of actin mRNAs were also determined. The ALA1 sequences used in ALA1-lexA constructs 1~11 in (B) were respectively transferred from constructs 1~11 shown in (A). In (C) and (D) the numbers 1~11 (circled) denote constructs shown in (B).
Mentions: As summarized in Figure 2A, 10 different triplets were identified at codon position -25 among these positive clones, including ATG, GTG, TTG, CTG, ACG, ATT, ATC, ATA, CGC, and CAC (Figure 2A, numbers 1~10). It was not surprising to find that GTG, TTG, CTG, ACG, ATT, ATC, and ATA were among initiator candidates, due to their close resemblance to ATG, as each of these triplets differed from ATG by just a single nucleotide. However, it was surprising to find that CGC and CAC were also among the preliminary pool of initiator candidates. The nucleotide sequences of these two triplets are completely divergent from ATG and have never previously been shown to be able to serve as initiator codons in a cap-dependent translational process in any organism. GGT served as a negative control in the assay (Figure 2A, number 11). It should be noted that while AAG and AGG also differed from ATG by a single nucleotide, these two triplets could not serve as initiator codons under similar conditions (data not shown). Perhaps this was because the middle bases in the two initiator codons and in the anticodon are all purines, and a purine pair cannot fit into an A-form helix.

Bottom Line: Among these initiator codons, TTG, CTG, ACG, and ATT had ~50% initiating activities relative to that of ATG, while GTG, ATA, and ATC had ~20% initiating activities relative to that of ATG.Unexpectedly, these non-AUG initiator codons exhibited different preferences toward various sequence contexts.In particular, GTG was one of the most efficient non-ATG initiator codons, while ATA was essentially inactive in the context of GRS1.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Life Science, National Central University, Jung-li, Taiwan.

ABSTRACT

Background: Previous studies in Saccharomyces cerevisiae showed that ALA1 (encoding alanyl-tRNA synthetase) and GRS1 (encoding glycyl-tRNA synthetase) respectively use ACG and TTG as their alternative translation initiator codons. To explore if any other non-ATG triplets can act as initiator codons in yeast, ALA1 was used as a reporter for screening.

Results: We show herein that except for AAG and AGG, all triplets that differ from ATG by a single nucleotide were able to serve as initiator codons in ALA1. Among these initiator codons, TTG, CTG, ACG, and ATT had ~50% initiating activities relative to that of ATG, while GTG, ATA, and ATC had ~20% initiating activities relative to that of ATG. Unexpectedly, these non-AUG initiator codons exhibited different preferences toward various sequence contexts. In particular, GTG was one of the most efficient non-ATG initiator codons, while ATA was essentially inactive in the context of GRS1.

Conclusion: This finding indicates that a sequence context that is favorable for a given non-ATG initiator codon might not be as favorable for another.

Show MeSH
Related in: MedlinePlus