Identification of proteins associated with the yeast mitochondrial RNA polymerase by tandem affinity purification

Departments of Cell Biology, University of Medicine and Dentistry of New Jersey, Stratford, USA.
Yeast (Impact Factor: 1.63). 08/2009; 26(8):423-40. DOI: 10.1002/yea.1672
Source: PubMed


The abundance of mitochondrial (mt) transcripts varies under different conditions, and is thought to depend upon rates of transcription initiation, transcription termination/attenuation and RNA processing/degradation. The requirement to maintain the balance between RNA synthesis and processing may involve coordination between these processes; however, little is known about factors that regulate the activity of mtRNA polymerase (mtRNAP). Recent attempts to identify mtRNAP-protein interactions in yeast by means of a generalized tandem affinity purification (TAP) protocol were not successful, most likely because they involved a C-terminal mtRNAP-TAP fusion (which is incompatible with mtRNAP function) and because of the use of whole-cell solubilization protocols that did not preserve the integrity of mt protein complexes. Based upon the structure of T7 RNAP (to which mtRNAPs show high sequence similarity), we identified positions in yeast mtRNAP that allow insertion of a small affinity tag, confirmed the mature N-terminus, constructed a functional N-terminal TAP-mtRNAP fusion, pulled down associated proteins, and identified them by LC-MS-MS. Among the proteins found in the pull-down were a DEAD-box protein (Mss116p) and an RNA-binding protein (Pet127p). Previous genetic experiments suggested a role for these proteins in linking transcription and RNA degradation, in that a defect in the mt degradadosome could be suppressed by overexpression of either of these proteins or, independently, by mutations in either mtRNAP or its initiation factor Mtf1p. Further, we found that Mss116p inhibits transcription by mtRNAP in vitro in a steady-state reaction. Our results support the hypothesis that Mss116p and Pet127p are involved in modulation of mtRNAP activity.

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    • "In another report, the mitoribosomal protein, MRPL12, was shown to interact directly with POLRMT and stimulates its activity in vitro (11). TEFM also interacts with the transcription machinery (POLRMT) and protein synthesis apparatus (mitochondrial ribosomal RNAs, MRPs and PTCD3) of mitochondria and so provides further evidence of a physical coupling of transcription, and translation in mammals, as previously proposed (11,36–39). "
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    ABSTRACT: Here we show that c17orf42, hereafter TEFM (transcription elongation factor of mitochondria), makes a critical contribution to mitochondrial transcription. Inactivation of TEFM in cells by RNA interference results in respiratory incompetence owing to decreased levels of H- and L-strand promoter-distal mitochondrial transcripts. Affinity purification of TEFM from human mitochondria yielded a complex comprising mitochondrial transcripts, mitochondrial RNA polymerase (POLRMT), pentatricopeptide repeat domain 3 protein (PTCD3), and a putative DEAD-box RNA helicase, DHX30. After RNase treatment only POLRMT remained associated with TEFM, and in human cultured cells TEFM formed foci coincident with newly synthesized mitochondrial RNA. Based on deletion mutants, TEFM interacts with the catalytic region of POLRMT, and in vitro TEFM enhanced POLRMT processivity on ss- and dsDNA templates. TEFM contains two HhH motifs and a Ribonuclease H fold, similar to the nuclear transcription elongation regulator Spt6. These findings lead us to propose that TEFM is a mitochondrial transcription elongation factor.
    Nucleic Acids Research 05/2011; 39(10):4284-99. DOI:10.1093/nar/gkq1224 · 9.11 Impact Factor
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    ABSTRACT: Transcription of the yeast mitochondrial genome is carried out by an RNA polymerase (Rpo41p) that is related to single subunit bacteriophage RNA polymerases but requires an additional factor (Mtf1p) for initiation. In this work we show that Mtf1p is involved in multiple roles during initiation including discrimination of upstream base pairs in the promoter, initial melting of three to four base pairs around the site of transcript initiation, and suppression of nonspecific initiation. It, thus, appears that Mtf1p is functionally analogous to initiation factors of multisubunit RNA polymerases, such as σ. Photocross-linking experiments reveal close proximity between Mtf1p and the promoter DNA and show that the C-terminal domain makes contacts with the template strand in the vicinity of the start site. Interestingly, Mtf1p is related to a class of RNA methyltransferases, suggesting an early evolutionary link between RNA synthesis and processing.
    Journal of Biological Chemistry 11/2009; 285(6):3957-64. DOI:10.1074/jbc.M109.051003 · 4.57 Impact Factor
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