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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    • "re - spiring cells . Mitochondrial gene expression is especially susceptible to indi - rect effects because transcription and translation are tightly coupled ( Dieckmann and Staples , 1994 ; Wallis et al . , 1994 ; Rouillard et al . , 1996 ; Rodeheffer et al . , 2001 ; Rodeheffer and Shadel , 2003 ; Bryan et al . , 2002 ; Williams et al . , 2007 ; Markov et al . , 2009 ) . Because Cox1 splicing and translation are both decreased in mam33∆ cells , it is possible that a defect in just one process indirectly disrupts the other process . For example , an increase of partially processed COX1 transcripts could delay translation of the mature mRNA . To test this possibility , we compared mitochondrial transl"
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    ABSTRACT: Three mitochondrial DNA-encoded proteins, Cox1, Cox2, and Cox3, comprise the core of the cytochrome c oxidase complex. Gene-specific translational activators ensure that these respiratory chain subunits are synthesized at the correct location and in stoichiometric ratios to prevent unassembled protein products from generating free oxygen radicals. In the yeast Saccharomyces cerevisiae, the nuclear-encoded proteins Mss51 and Pet309 specifically activate mitochondrial translation of the largest subunit, Cox1. Here we report that Mam33 is a third COX1 translational activator in yeast mitochondria. Mam33 is required for cells to efficiently adapt from fermentation to respiration. In the absence of Mam33, Cox1 translation is impaired and cells poorly adapt to respiratory conditions because they lack basal fermentative levels of Cox1. © 2015 by The American Society for Cell Biology.
    Molecular biology of the cell 06/2015; 26(16). DOI:10.1091/mbc.E15-04-0222 · 4.47 Impact Factor
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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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