Transcripts and transcript-binding proteins in mitochondria of Neurospora crassa

We analyzed expression elements of three disparate groups of mitochondrial genes in Neurospora crassa, apocytochrome b (COB), cytochrome c oxidase 1 (COX1), and the clustered ATP8-ATP6-mtATP9-COX2. To identify promoter sequences we employed the published N. crassa consensus sequence for COB and rRNA genes, and we found closely related sequences within the 5'-regions of both COX1 and the ATP8-COX2 transcriptional units. We determined that the mature COX1 RNA includes two flanking unassigned reading frame (URF) sequences, but the 3'-flanking ND1 is not included in the COX1 mRNA. The ATP8-ATP6-mtATP9-COX2 polycistronic transcript does not include an adjacent 5'-URF sequence. Primer extension analysis showed one likely 5'-end for the COX1 transcript, which is 73 nucleotides downstream of the consensus promoter sequence and is the first nucleotide 3' of the sequence for the tRNA(cys). Primer extension analysis and S1 nuclease mapping of the ATP8-COX2 RNA showed that the 5'-end for this transcript is the first nucleotide 3' of the consensus promoter sequence. We performed gel-shift experiments to detect proteins in mitochondria that bind to transcripts as possible regulatory proteins. The 5'-untranslated region (UTR) RNAs of COB, COX1, and ATP8-COX2 appear to bind both unique proteins and an overlapping group of two to four proteins of approximately 155-45 M(r). We successively deleted regions of the RNA 5'-UTRs to identify sequences that bound these proteins. Similar predicted stem-loop secondary structures were detected in the protein-binding regions of all three UTRs.