Mutations within an intron and its flanking sites: Patterns of novel polypeptides generated by mutants in one segment of the cob-box region of yeast mitochondrial DNA

Abstract

We have undertaken a systematic examination of the polypeptides accumulating in thirteen (out of 23) mutants in the intron cluster box7 and its flanking clusters box2 and box9 of the cob-box (cytochrome b) region of the mitochondrial genome of Saccharomyces cerevisiae. We have subjected these polypeptides to fingerprint analysis, both sequential and in parallel, with two proteases in order to disclose sequence homologies and differences between the different novel polypeptides themselves, and between them and the wild type product of the gene, i.e. apocytochrome b. One of our aims has been to establish the existence of possible correlations between the nature of the novel polypeptides and the fine structure genetic map of that segment of the mitochondrial genome. Our results show that all box7 mutants accumulate the following set of polypeptides not seen in wild type cells: a) a characteristic set of “large” polypeptides consisting of three species: p56, p42 and p35 or p34.5; b) a polypeptide p23; c) a much shorter fragment (of which the apparent molecular weight varies from 12.5 to 13, according to the mutation) with the exception of two mutants; d) in addition, the majority accumulate in varying amounts a polypeptide p30 closely related to but not identical with apocytochrome b. Moreover only two box7 mutants accumulate a polypeptide in the range of mobilities corresponding to 25–27 Kd (referred to as class p26) while such a polypeptide is seen in all box9 and box2 mutants examined with one exception in box2. Only one mutant in box2 resembles box7 mutants with respect to criterion a), and no box2 or box9 mutants resemble box7 mutants with respect to criterion c); criteria b) and d) appear to apply equally well to mutants in all three clusters. Fingerprint analysis, carried out with polypeptides p56, p42, p35, p34.5, p30, p26, p23, discloses that a) The polypeptides belonging to the same class of mobility exhibit very similar if not identical sequences in various cases. b) These polypeptide classes, except p56, p42 and p26, share considerable sequence homologies with wild type apocytochrome b, perhaps encompassing 50% or more of the wild type sequences. b) Polypeptides belonging to the classes p42 and p26 exhibit less extensive but nevertheless significant homologies with the wild type sequence. c) Sequences in polypeptides belonging to class p56 are virtually indistinguishable from ones in cytochrome oxidase subunit I. The inferences from these findings are 1) one gene can produce a multiplicity of polypeptide products that share a common sequence at the promoter-proximal (N-terminal) portion, but diverge beyond these regions of homology. 2) Both the multiplicity of products in single mutants, and the protein structure found, argue against the divergent segments to be due to frameshift terminations, and instead suggest that the novel products are consequences of mRNA processing defects (excision and/or ligation) at and near intron regions. 3) Mutations at edges and the center of an intron can generate similar polypeptide patterns, i.e. produce analoguous mRNA processing defects. 4) Mutations in exons, at their boundary with introns, can produce polypeptide patterns indistinguishable from those at the neighbouring intron; they diverge and eventually become typically exonlike in mutants localized at increasing distances from the boundary. 5) Taken together these findings argue that pre-mRNA processing extends beyond the boundaries of the intron proper and that certain exonsequences participate in excision and ligation. 6) Accumulated pre-mRNAs, resulting from defects in splicing can be translated. 7) Product p56 is formally analogous to p23, as a faulty but highly conserved partial product of the wild type protein, the former of Cox I (oxi3 gene), the latter of the cob-box gene proper. Therefore both genes may utilize identical RNA processing elements which are affected by box7 mutations. 8) A small amount of product similar to p56 may accumulate even in some wild types but not in others. This observations suggests that in certain nuclear backgrounds RNA processing may be more error-prone than in others.