Limits...
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.

Show MeSH

Related in: MedlinePlus

Comparing the efficiencies of various non-AUG initiator codons in GRS1. (A) Complementation assays for mitochondrial GlyRS activity. The grs1- strain was transformed with various GRS1 constructs, and the growth phenotypes of the transformants were tested. (B) Assay of initiating activities by Western blots. Upper panel, GlyRS-LexA fusion; lower panel, PGK (as loading controls). (C) Assay of the relative initiating activities 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 GRS1-lexA mRNAs generated from each construct were determined by RT-PCR. The GRS1 sequences used in the GRS1-lexA fusion 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
getmorefigures.php?uid=PMC2909995&req=5

Figure 4: Comparing the efficiencies of various non-AUG initiator codons in GRS1. (A) Complementation assays for mitochondrial GlyRS activity. The grs1- strain was transformed with various GRS1 constructs, and the growth phenotypes of the transformants were tested. (B) Assay of initiating activities by Western blots. Upper panel, GlyRS-LexA fusion; lower panel, PGK (as loading controls). (C) Assay of the relative initiating activities 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 GRS1-lexA mRNAs generated from each construct were determined by RT-PCR. The GRS1 sequences used in the GRS1-lexA fusion 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: We next tested whether the non-AUG initiator codons identified above can also act as initiator codons in GRS1. To this end, the native UUG initiator codon of GRS1 was substituted by the above-mentioned initiator candidates, and the mitochondrial activities of the resultant mutants were tested. As expected, mutations of TTG(-23) of GRS1 to ATG, GTG, CTG, ACG, ATC, or ATT had little effect on mitochondrial activity; transformants carrying any of these mutants grew as well as those carrying a WT GRS1 construct on YPG plates (Figure 4A, numbers 1~8). However, a mutation of TTG(-23) to ATA yielded a construct that failed to support the growth of the knockout strain on YPG plates (Figure 4A, number 8). Also, neither CGC nor CAC could act as an initiator codon in GRS1 (Figure 4A, numbers 9 and 10). TTA served as a negative control in this assay (Figure 4A, number 11).


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 GRS1. (A) Complementation assays for mitochondrial GlyRS activity. The grs1- strain was transformed with various GRS1 constructs, and the growth phenotypes of the transformants were tested. (B) Assay of initiating activities by Western blots. Upper panel, GlyRS-LexA fusion; lower panel, PGK (as loading controls). (C) Assay of the relative initiating activities 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 GRS1-lexA mRNAs generated from each construct were determined by RT-PCR. The GRS1 sequences used in the GRS1-lexA fusion 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 4: Comparing the efficiencies of various non-AUG initiator codons in GRS1. (A) Complementation assays for mitochondrial GlyRS activity. The grs1- strain was transformed with various GRS1 constructs, and the growth phenotypes of the transformants were tested. (B) Assay of initiating activities by Western blots. Upper panel, GlyRS-LexA fusion; lower panel, PGK (as loading controls). (C) Assay of the relative initiating activities 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 GRS1-lexA mRNAs generated from each construct were determined by RT-PCR. The GRS1 sequences used in the GRS1-lexA fusion 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: We next tested whether the non-AUG initiator codons identified above can also act as initiator codons in GRS1. To this end, the native UUG initiator codon of GRS1 was substituted by the above-mentioned initiator candidates, and the mitochondrial activities of the resultant mutants were tested. As expected, mutations of TTG(-23) of GRS1 to ATG, GTG, CTG, ACG, ATC, or ATT had little effect on mitochondrial activity; transformants carrying any of these mutants grew as well as those carrying a WT GRS1 construct on YPG plates (Figure 4A, numbers 1~8). However, a mutation of TTG(-23) to ATA yielded a construct that failed to support the growth of the knockout strain on YPG plates (Figure 4A, number 8). Also, neither CGC nor CAC could act as an initiator codon in GRS1 (Figure 4A, numbers 9 and 10). TTA served as a negative control in this assay (Figure 4A, number 11).

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