Klaas A. Osinga

Characterization of the promoter of the large ribosomal RNA gene in yeast mitochondria and separation of mitochondrial RNA polymerase into two different functional components.

We have characterized a DNA sequence that functions in recognition of the promoter of the mitochondrial large rRNA gene by the yeast mtRNA polymerase. Promoter‐containing DNA fragments were mutagenized and used as templates to study initiation of transcription in vitro with a partially purified mtRNA polymerase preparation. Deletion mutants, in which increasing stretches of DNA were removed from regions flanking the promoter, define a short area essential for correct initiation of transcription. It virtually coincides with a highly conserved stretch of nine nucleotides that is found immediately upstream of all transcriptional start sites described thus far. Two different point mutations within this nonanucleotide sequence drastically reduce promoter function. Conversely a single point mutation that results in the formation of a nonanucleotide sequence 99 nucleotides upstream of the large rRNA gene leads to a new, efficient transcription initiation site. MtRNA polymerase can be resolved into two different components by chromatography on Blue Sepharose: one retaining the capacity to synthesize RNA, the other conferring the correct specificity of initiation to the catalytic component.

RNA Processing in Yeast Mitochondria

The yeast mitochondrial genome is remarkably complex in terms of the organization of its genes and their modes of expression. The first indications of this came a number of years ago with the construction of detailed transcription maps of mtDNA in related yeast strains (Van Ommen and Groot 1977; Van Ommen et al. 1979), and we predicted at that time (Grivell et al. 1979) that RNA processing would play a central role in the regulation of mitochondrial gene expression. With the insights gained from DNA sequence analysis and transcript characterization, it is clear that this prediction has been amply fulfilled. In this paper we review what is known about a number of yeast mtRNAs and the various forms of processing they undergo in the hope that this will lead to a better understanding of the mechanisms that control the expression of this genome. TRANSCRIPTION INITIATION AND TRIMMING OF mtRNAS 15S and 21S rRNAs In human mtDNA, transcription is initiated at a single site on each strand and the genome is completely and symmetrically transcribed (Murphy et al. 1975). Differential control of gene expression is then achieved by a combination of transcription attenuation, control of the cleavages required to generate transcripts of individual genes, and the stability of the final products (see Attardi et al., this volume). In yeast, a greater scope of transcriptional controls exists, since Levens et al. (1980) have identified at least five possible promoter sites by means of the guanylyl transferase capping assay (Reeder et al. 1977)...