Transcription of the Dictyostelium discoideum mitochondrial genome occurs from a single initiation site

Abstract

Transcription of the mitochondrial genome in Dictyostelium discoideum gives rise to eight major polycistronic RNA species that can be detected by Northern hybridization. In order to determine whether these transcripts could possibly derive from processing of even larger transcripts, reverse transcriptase polymerase chain reactions (RT-PCRs) were performed in an attempt to amplify the intervening regions between the eight major transcripts. All but one intervening region were successfully reverse transcribed and amplified, indicating that even larger transcripts existed and that the eight major transcripts detected previously may be the products of transcript processing. Southern hybridization analyses of DNA fragments representing the sequences between the eight major transcripts with in vitro capped mitochondrial RNA identified the 59 end of only one of the eight major transcripts as a genuine transcription start site. The ability to initiate transcription from DNA sequences upstream of the identified transcription initiation site was demonstrated in bacterial cells expressing the Dictyostelium mitochondrial RNA polymerase. We conclude that transcription of the Dictyostelium mitochondrial genome is initiated at a single site, generating a large polycistronic transcript that is very efficiently, probably cotranscriptionally processed into mature RNA species. This is the first report on a protist mitochondrial DNA that is, although much larger in size than its metazoan counterparts, transcribed from a single transcription initiation site.

A Unique Mitochondrial Transcription Factor B Protein in Dictyostelium discoideum

“…As a result of this, D. discoideum, whose entire genome has been sequenced, has also more recently become an attractive model for studying mitochondrial genetics and disease [27]. Mitochondrial transcription in this protist has been found to be initiated from a single site, a unique characteristic given the size of the genome (∼56 kb) [28], [29]. This initiation site is located in a non-coding region (NCR), ∼2 kb in size, which houses a presently unidentified promoter.…”

Transcription and processing of mitochondrial RNA in the human pathogen Acanthamoeba castellanii

“…However, not all larger genomes are necessarily transcribed from multiple promoters. For example, the 55.5 kb mitochondrial genome of the protozoan D. discoideum has been demonstrated to be transcribed from only a single transcription initiation site (Le et al, 2009). Transcription from this site produces a single large polycistronic transcript that is co-transcriptionally processed into mature mono-, di-and tricistronic RNA molecules (Barth et al, 2001;Le et al, 2009).…”

“…The strong signal may be attributed to a combination of transcription initiation, with repeated premature termination of the polymerase and read-through of the polymerase after transcription of the entire genome is complete. Transcription from a limited number of promoters may be an emerging trend in protist mitochondria, considering that the mitochondrial genome of D. discoideum is also known to be transcribed from a single promoter (Le et al, 2009).…”

“…Given that the A. castellanii mitochondrial genome shares striking similarities to the D. discoideum mitochondrial genome in its gene content, organisation and gene expression (Gray et al, 2004;Anderson et al, 2005;Le et al, 2009), it is very likely that the A. castellanii mitochondrial genome is also under the control of only a single promoter. Here we have provided proof for the existence of a large polycistronic primary transcript that is co-transcriptionally processed into six secondary polycistronic transcripts, and we have also shown that these secondary transcripts are further processed to mature RNAs for translation.…”

“…In 2009, Le et al demonstrated that the large (55.5 kb) mitochondrial genome of the protozoan Dictyostelium discoideum is transcribed from only a single transcription initiation site. The resulting large polycistronic transcript is co-transcriptionally processed into eight secondary transcripts, which are further processed into mature mono-, di-and tricistronic RNA molecules (Barth et al, 2001;Le et al, 2009).…”