A mutation allowing an mRNA secondary structure diminishes translation of Saccharomyces cerevisiae iso-1-cytochrome c.

Abstract

The CYCI-239-0 mutation in the yeast Saccharomyces cerevisiae produces a -His-Leu-replacement of the normal -Ala-Gly-sequence at amino acid positions 5 and 6, which lie within a dispensable region of iso-l-cytochrome c; this mutation can accommodate the formation of a hairpin structure at the corresponding site in the mRNA. The amount of the altered protein was diminished to 20% of the wild-type level, whereas the amount of the mRNA remained normal. However, in contrast to the normal CYCI+ mRNA that is associated mainly with four to seven ribosomes, the bulk of the CYCI-239-0 mRNA is associated with one to four ribosomes. These results suggest that the stable secondary structure within the translated region of the CYCI mRNA diminishes translation by inhibiting elongation.During the past 20 years, the CYCI gene and its gene product iso-l-cytochrome c in the yeast Saccharomyces cerevisiae have been the subject of numerous studies. Mutationally altered forms of at least partially functional iso-icytochromes c have been obtained from over 500 intragenic revertants of various cycl mutants. The vast majority of these altered iso-1-cytochromes c are found at a level corresponding to the level of normal iso-l-cytochrome c in wild-type strains. Thus, most changes of the amino acid sequence do not appreciably affect the level of the protein.One type of exception is the revertants that contain AUG initiator codons situated at certain abnormal positions (25, 26, 33; S. B. Baim, Ph.D. Thesis, University of Rochester, Rochester, N.Y., 1984; S. Baim and F. Sherman, unpublished data). In this investigation we describe another type of exception, the cycl-239 revertants which contain only 20o of the normal level of iso-1-cytochrome c and contain a sequence which can accommodate the formation of a hairpin structure within the translated region of the mRNA.MATERIALS AND METHODS Genetic analysis. The symbol CYCI+ or CYCI denotes the wild-type structural gene that encodes iso-l-cytochrome c. Mutations of this gene that cause a deficiency in either the amount or activity are designated by the symbol cycl followed by the allele number, e.g., cycl-17, cycl-239, etc. Intragenic revertants of specific cycl mutants in which the amount or activity of iso-l-cytochrome c has been at least partially restored are designated by the gene symbol CYCI followed by the allele number of the mutation from which they were derived and by capital letters to designate independent revertants. For example, CYCI-239-A, CYCI-239-B, etc., denote intragenic revertants of cycl-239.Conventional Zaret and Sherman (38,39). Briefly, logarithmic-phase cells from an overnight culture were inoculated at various densities into YPD (1% yeast extract [Difco], 2% Bacto-Peptone, and 2% glucose) medium. The cultures were shaken vigorously at 30°C for 18 to 24 hours. The cells were harvested at the appropriate densities and disrupted with glass beads. The lysate was adjusted to 1% sodium dodecyl sulfate and extracted several times with phenol-chloroform-isoamyl alcohol (25:24...

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