Development of an In Vitro Transcription System for Neurospora crassa Mitochondrial DNA and Identification of Transcription Initiation Sites

Abstract

We have developed an in vitro transcription system for Neurospora crassa mitochondrial DNA (mtDNA) and used it to identify transcription initiation sites at the 5′ ends of the genes encoding the mitochondrial small and large rRNA and cytochrome b (cob). The in vitro transcription start sites correspond to previously mapped 5′ ends of major in vivo transcripts of these genes. Sequences around the three transcription initiation sites define a 15-nucleotide consensus sequence, 5′-TTAGARA(T/G)G(T/G)ARTRR-3′, all or part of which appears to be an element of an N. crassa mtDNA promoter. A somewhat looser 11-nucleotide consensus sequence, 5′-TTAGARR(T/G)R(T/G)A-3′, was derived by including two additional promoters identified recently. Group I extranuclear mutants, such as [poky] and [SG-3], have a 4-base-pair (bp) deletion in the consensus sequence at the 5′ end of the mitochondrial small rRNA and are grossly deficient in mitochondrial small rRNA (R. A. Akins and A. M. Lambowitz, Proc. Natl. Acad. Sci. USA 81:3791-3795, 1984). We show here that the 4-bp deletion in the consensus sequence decreases in vitro transcription from this site by more than 99%. N. crassa mtDNA is similar to Saccharomyces cerevisiae mtDNA in having multiple promoters, including separate promoters for the genes encoding the mitochondrial small and large rRNAs. Our results suggest that the primary effect of the 4-bp deletion in group I extranuclear mutants is to inhibit transcription of the mitochondrial small rRNA, leading to severe deficiency of mitochondrial small rRNA and small ribosomal subunits.