Transcripts and transcript-binding proteins in mitochondria of Neurospora crassa

Abstract

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 ∼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.