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.

RNA-level unscrambling of fragmented genes inDiplonemamitochondria

“…6). Aiming at a more precise experimental determination of transcription start sites, we now performed in vitro RNA capping experiments, since in most mitochondria (with few notable exceptions, e.g., Neurospora crassa 17 ), primary transcripts have a triphosphate 5' end that can be labeled with α-32 P-GTP and guanylyl transferase. Nuclear mRNAs, in contrast, are naturally capped during transcription and, therefore, are not labeled, except cytosolic 5S rRNA (or a portion of it).…”

The VS catalytic RNA replicates by reverse transcription as a satellite of a retroplasmid.

“…The mt RNP particles were further purified by an additional centrifugation through the sucrose cushion to minimize associated nuclease activity. (Kennell and Lambowitz 1989). Transcription with mt lysates was done in the presence of an antibody preparation against N. crassa endo-exonuclease (Chow and Fraser 1983) to inhibit nonspecific nuclease activity.…”

“…32p-Labeled transcripts were analyzed by electrophoresis in 6 or 12% polyacrylamide gels containing 8 M urea, 88 mM Tris-borate (pH 8.3), and 2 mM EDTA (TBE), followed by autoradiography. For 5' end mapping, in vitro transcription of pD7/SspI with HS-enriched mt RNA polymerase was scaled up fivefold, and primer extension was carried out as described (Kennell and Lambowitz 1989), by using MMLV-RT (Life Science Technologies, Inc., Gaithersburg, MD) with a 5' a2P-labeled synthetic oligonucleotide primer (5' GTAGTCAACTGCTACGAC, complementary to nucleotide positions VS702-685. )…”

“…1823) and senescent mutant . N. crassa [poky] 13-6, which accumulates mt RNA polymerase (Barath and Kuntzel 1972;Kennell and Lambowitz 1989), was used for in vitro transcription assays. Procedures for growing the strains were as described (Kuiper and Lambowitz 1988;Kennell and Lambowitz 1989).…”

Hybrid Mitochondrial Plasmids From Senescence Suppressor Isolates of Neurospora intermedia

“…Similarly, in NI05.01 a sequence was found that corresponded to the putative 59 terminus of the cob transcript. The sequence directly bordering the copy of pKalilo (Table 3, case 4) resembles the consensus sequence of mitochondrial 59 transcript termini (Kennel and Lambowitz 1989;Kubelik et al 1990;Arganoza and Akins 1995). Although it is located 431 bp upstream of the 59 terminus of the cob transcript that was originally described by Kubelik et al (1990), it may reflect an alternative transcription initiation site.…”