A Trypanosome Mitochondrial RNA Polymerase Is Required for Transcription and Replication

Abstract

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.The mitochondrial genome of kinetoplastids is unusual in that it is comprised of large and small circular DNAs, maxicircles and minicircles, respectively, catenated into a giant network called kinetoplast DNA (1-3). Little is known of the mechanisms regulating gene expression of either maxicircles or minicircles. In Trypanosoma brucei, the 23-kb maxicircles represent the typical mitochondrial DNA, coding for rRNAs and several proteins involved in mitochondrial respiration. Similar to other organisms, numerous polycistronic transcripts have been detected, indicating that maxicircles are transcribed polycistronically (4 -6). No maxicircle promoter has yet been identified, although a precursor extending at least 1200 nucleotides upstream of the 12 S rRNA has been detected and may represent an initiation site for transcription (7).Many maxicircle genes encode cryptic transcripts that require the post-transcriptional insertion or deletion of uridines to produce a functional mRNA, which is a process known as RNA editing (Ref. 8; for reviews see Ref.9 -11). The information for the editing of maxicircle transcripts is provided by small RNAs termed guide RNAs (gRNAs). 1 With the exception of gMurfII-1 and gMurfII-2 (guide RNAs 1 and 2, respectively, for maxicircle unidentified reading frame II) found on the maxicircle, gRNAs are encoded by the second component of the kinetoplast, the minicircles (12-14). In T. brucei, minicircles are ϳ1-kb circular DNA molecules, and there are several thousand copies/kinetoplast network. Despite some conserved features, they are heterogeneous in sequence and provide the 250 -300 sequence classes necessary to edit maxicircle transcripts (15, 16).Each minicircle of T. brucei contains three or four potential gRNA ...

Kinetoplastid guide RNA biogenesis is dependent on subunits of the mitochondrial RNA binding complex 1 and mitochondrial RNA polymerase

“…Next, we have hybridized blots containing mtRNA with a probe against cox1 mRNA. In agreement with the previous study (Grams et al 2002), this never-edited transcript is undetectable 3 d after RNAi is triggered (Fig. 6B).…”

“…Englund, Johns Hopkins University), we have first verified the RNAi effect by Northern analysis, as antibodies against the T. brucei mtRNAP are not available. As shown in Figure 6A, the mtRNAP mRNA is undetectable 3 d after tetracycline induction, the time point at which growth inhibition is first observed (Grams et al 2002). Next, we have hybridized blots containing mtRNA with a probe against cox1 mRNA.…”

“…The participation of a multiprotein complex in the transcription of minicircle gRNA genes, independent of mtRNAP, has been hypothesized (Grams et al 2002). In the inducible mtRNAP knockdowns (kindly provided by P.T.…”

“…The last studied subunit, Tb927.11.7290, is annotated as a Nudix hydrolase due to its conservation to other such proteins. Moreover, we show that mtRNA polymerase (mtRNAP), known to transcribe the maxicircle-encoded protein-coding genes (Grams et al 2002), appears to have a similar role for minicircle-encoded gRNAs.…”

Drosophila Mitochondrial Transcription Factor B2 Regulates Mitochondrial DNA Copy Number and Transcription in Schneider Cells

“…Overall, we found that mRNA levels were reduced more than the levels of mtDNA, and this, too, may reflect a higher turnover rate for mRNAs as compared with that of mtDNA. Interestingly, a similar result was obtained in an RNA knock-down analysis of mitochondrial RNA polymerase in trypanosomes (44). In any case, because of the differential effects we observed on mtDNA copy number and transcription in the overexpression analyses of d-mtTFB2 and d-TFAM, it is apparent that the effect of d-mtTFB2 on transcription is not a simple consequence of mtDNA dose.…”