J. Stewart, F. Sherman, N. Shipman
Dec 25, 1971
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Journal
The Journal of biological chemistry
Abstract
Abstract Mutational studies and amino acid sequence analysis were used to identify the initiating methionyl codon AUG as an essential element at the beginning of the genetic message for iso-1-cytochrome c in yeast. Nine cy1 mutants completely lack iso-1-cytochrome c as a result of mutations at one end of the CY1 gene. Amino acid sequence changes of iso-1-cytochrome c from 45 intragenic revertants of these mutants were determined. No sequence changes occurred in 21 of the revertants and the changes in the rest were additions or deletions at the NH2 terminus. The additions were either Met-Ile-, Met-Leu-, Met-Arg-, or in one case, both Met- Val- and Val-. The additions were all alike in the revertants of any cy1 mutant. The deletions were always losses of the first four residues of the protein, Thr-Glu-Phe-Lys-. Normal amounts of iso-1-cytochrome c occurred in all of the revertants except those containing the short protein, which contained only 51% of the normal amount. The nine cy1 mutants had incurred single base pair substitutions at the three nucleotides immediately preceding the codon for the NH2-terminal threonine. The essential, initial triplet is the methionyl codon, AUG. Its conversion to codons for isoleucine, leucine, arginine, or valine prevents the formation of the protein. Reversions either reverse the original substitutions, leading to the normal protein, or generate new methionine codons at the left of the original AUG position, producing the long proteins, or they convert the normal lysine 4 codon to AUG, creating the short protein. A methionine aminopeptidase is proposed to exist, which cleaves methionine efficiently from Met-Thr- and Met-Ala- bonds, less efficiently from Met-Val- bonds, and not at all from Met-Leu, Met-Ile-, and Met-Arg- bonds. The cy1 initiator mutants were used to demonstrate that ethyl methanesulfonate induces the transition G·C → A·T at a frequency at least 100 times greater than it causes the transversions A·T → T·A and G·C → T·A and one or more unidentified base pair exchanges to G·C. The function of the essential AUG triplet is presumed to be initiation of translation. This function is not provided by at least 35 of the 64 nucleotide triplets; notably it is not provided by GUG. The amount of iso-1-cytochrome c in yeast is under the control of the rate of initiation of translation, which may be reduced or remain unaltered following mutational relocation of the initiator codon.