A trypanosome mitochondrial RNA polymerase is required for transcription and replication.
Understanding mitochondrial transcription is a requisite first step toward understanding the regulation of mitochondrial gene expression in kinetoplastids. Here we report the identification and functional characterization of a mitochondrial RNA polymerase (mtRNAP) from Trypanosoma brucei, the first trans-acting factor involved in kinetoplast mitochondrial transcription to be identified. Using sequences conserved among the catalytic domains of the single-subunit mtRNAPs, we were able to obtain a full-length sequence for a candidate mtRNAP from T. brucei. Sequence comparison indicates that it shares homology in its catalytic domain with other single-subunit mtRNAPs, including functionally conserved residues that are identical in all single-subunit RNAPs. We used RNA interference to functionally knock out the gene product to determine whether the candidate gene represents an mtRNAP. As predicted for a mitochondrial specific RNA polymerase, reduction of the gene product resulted in a specific decrease of mitochondrial versus nuclear transcripts. Additionally, similar to the mtRNAP of other organisms, the mtRNAP characterized here is involved in replication of the mitochondrial genome. Thus, based on sequence comparison and functional studies, we have cloned an mtRNAP from trypanosomes.
Grams
Jayleen
J
Department of Biochemistry and Molecular Genetics, Schools of Medicine and Dentistry, University of Alabama, Birmingham, Alabama 35294, USA.
Morris
James C
JC
Drew
Mark E
ME
Wang
Zefeng
Z
Englund
Paul T
PT
Hajduk
Stephen L
SL
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D020134:Catalytic Domain; D002455:Cell Division; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism*; D018105:DNA, Kinetoplast / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D008928:Mitochondria / Q000201:enzymology* / Q000378:metabolism; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D012313:RNA / Q000378:metabolism; D012329:RNA, Bacterial / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D013752:Tetracycline / Q000494:pharmacology; D013997:Time Factors; D014158:Transcription, Genetic; D014345:Trypanosoma
D018105:DNA, Kinetoplast; C042027:RNA I; D012329:RNA, Bacterial; D012313:RNA; D004247:DNA; D012321:DNA-Directed RNA Polymerases; D013752:Tetracycline