A <i>Saccharomyces cerevisiae</i> Mitochondrial Transcription Factor, sc-mtTFB, Shares Features with Sigma Factors but Is Functionally Distinct

Shadel, Gerald S.; Clayton, David A.

doi:10.1128/mcb.15.4.2101

Cited by 62 publications

(57 citation statements)

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“…sc-mtTFB facilitates specific binding of mitochondrial RNA polymerase at numerous promoter sites in the yeast mitochondrial genome (4,8,9). Although sc-mtTFB is functionally similar to bacterial sigma factor (9 -11), the two proteins do not share amino acid sequence (7,12) or structural homology (13). Rather, the structure of sc-mtTFB is homologous to bacterial rRNA methyltransferases (13).…”

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confidence: 99%

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

Matsushima

Garesse

Kaguni

2004

Journal of Biological Chemistry

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We report the cloning and molecular analysis of Drosophila mitochondrial transcription factor B2 (d-mt-TFB2), a protein that plays a role in mitochondrial transcription and mitochondrial DNA (mtDNA) replication in Drosophila. An RNA interference (RNAi) construct was designed that reduces expression of d-mtTFB2 to 5% of its normal level in Schneider cells. RNAi knock-down of d-mtTFB2 reduces the abundance of specific mitochondrial RNA transcripts 2-to 8-fold and decreases the copy number of mtDNA ϳ3-fold. In a corollary manner, we find that overexpression of d-mtTFB2 increases both the abundance of mitochondrial RNA transcripts and the copy number of mtDNA. In a comparative experiment, we find that overexpression of Drosophila mitochondrial transcription factor A (d-TFAM) increases mtDNA copy number with no significant effect on mitochondrial transcripts. This argues for a direct role for mtTFB2 in mitochondrial transcription and suggests that, if TFAM serves a role in transcription, its endogenous level limits mtDNA copy number but not transcription. Furthermore, we suggest that mtTFB2 increases mtDNA copy number by increasing the frequency of initiation of DNA replication, whereas TFAM serves to stabilize and package mtDNA in mitochondrial nucleoids. Our work represents the first study to document the function of mtTFB2 in vivo, establishing a dual role in regulation of both transcription and replication, and provides a benchmark for comparative biochemical studies in various animal systems.The mitochondria of eukaryotic cells utilize a number of organelle-specific factors in DNA and RNA metabolism. In Saccharomyces cerevisiae, mitochondrial transcription is mediated by the yeast mtRNA 1 polymerase, Rpo41p (1-3), and a specificity factor Mtf1p (4 -6), also known as mitochondrial transcription factor B (sc-mtTFB) (7). Deficiency in sc-mtTFB lowers the abundance of mitochondrial transcripts and reduces mtDNA copy number (5, 7). sc-mtTFB facilitates specific binding of mitochondrial RNA polymerase at numerous promoter sites in the yeast mitochondrial genome (4,8,9). Although sc-mtTFB is functionally similar to bacterial sigma factor (9 -11), the two proteins do not share amino acid sequence (7,12) or structural homology (13). Rather, the structure of sc-mtTFB is homologous to bacterial rRNA methyltransferases (13).Mammalian mitochondrial transcription utilizes mitochondrial RNA polymerase, and three distinct transcription factors: transcription factor A (TFAM; formerly referred to as mtTFA) and two proteins homologous to sc-mtTFB, transcription factors mtTFB1 and mtTFB2 (3, 14 -17). TFAM contains two high mobility group boxes and was shown in organello to bind nonspecifically at regularly phased intervals to the control region of human mtDNA (18); it has recently been shown to package mtDNA in nucleoids (19,20). h-TFAM was also shown to be required for specific initiation at mitochondrial promoters in vitro (14 -17). The yeast homologue of TFAM is Abf2p, an abundant protein whose primary role is to stabilize and c...

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

Matsushima

Garesse

Kaguni

2004

Journal of Biological Chemistry

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“…Mtf1 is regarded as a functional homolog of the sigma factor, as the two do not share sequence or structural similarity but play similar roles in transcription initiation (13,14,20). Mtf1 does not bind to the promoter DNA on its own, but it binds to the Rpo41-promoter complex aiding in the formation of the pre-initiation open complex by melting the promoter from Ϫ4 to ϩ2 (11, 16).…”

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Transcription Factor-dependent DNA Bending Governs Promoter Recognition by the Mitochondrial RNA Polymerase

Tang

Deshpande

Patel

2011

Journal of Biological Chemistry

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Promoter recognition is the first and the most important step during gene expression. Our studies of the yeast (Saccharomyces cerevisiae) mitochondrial (mt) transcription machinery provide mechanistic understandings on the basic problem of how the mt RNA polymerase (RNAP) with the help of the initiation factor discriminates between promoter and non-promoter sequences. We have used fluorescence-based approaches to quantify DNA binding, bending, and opening steps by the core mtRNAP subunit (Rpo41) and the transcription factor (Mtf1). Our results show that promoter recognition is not achieved by tight and selective binding to the promoter sequence. Instead, promoter recognition is achieved by an induced-fit mechanism of transcription factor-dependent differential conformational changes in the promoter and non-promoter DNAs. While Rpo41 induces a slight bend upon binding both the DNAs, addition of the Mtf1 results in severe bending of the promoter and unbending of the non-promoter DNA. Only the sharply bent DNA results in the catalytically active open complex. Such an induced-fit mechanism serves three purposes: 1) assures catalysis at promoter sites, 2) prevents RNA synthesis at non-promoter sites, and 3) provides a conformational state at the non-promoter sites that would aid in facile translocation to scan for specific sites.DNA-dependent RNA synthesis initiates with the specific binding of the RNA polymerase (RNAP) 2 to its promoter. During this process of promoter selection, the RNAP is able to seek out active promoters within vast stretches of non-promoter sequences in the genome. The multi-subunit RNAPs of the bacteria, archaea, and eukarya rely on transcription factors for promoter selection and specific transcription (1-3) whereas the single subunits RNAPs of bacteriophages are able to carry out the same functions without any accessory factors (4). The RNAPs that transcribe the mitochondrial genomes are unique in that they are homologous to the single subunit RNAPs of bacteriophages, but they require one or more transcription factors for promoter-specific initiation (5-9). The transcription factors modulate various steps of initiation and play a key role in open complex formation (10,11).In this study, we have investigated the transcription machinery of the yeast (Saccharomyces cerevisiae) mitochondria, which consists of a nuclear encoded ϳ153 kDa core subunit Rpo41 (12) and a ϳ45 kDa transcription factor Mtf1 (13, 14). The two proteins are sufficient to catalyze transcription from the conserved nona-nucleotide promoter (5Ј-ATATA-AGTA(ϩ1)) of the yeast mitochondria that directs the synthesis of rRNA, tRNA, and respiratory chain protein mRNAs (15). Rpo41 cannot initiate specific transcription on duplex promoters without Mtf1 (13,16,17). However, when the DNA is negatively supercoiled or premelted, then Rpo41 can initiate specific transcription without Mtf1 (18). Based on these results, it was proposed that Rpo41 has the intrinsic ability to recognize the promoter. A similar conclusion was made for its homolo...

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“…Suppressing the suv3⌬ [Gly ؊ ] Phenotype Decrease the Transcription Rate of the Mitochondrial RNA Polymerase Mutations in the mitochondrial RNA polymerase genes resulting in a similar, temperature-sensitive petite phenotype described previously (Shadel and Clayton, 1995;Cliften et al, 1997Cliften et al, , 2000Karlok et al, 2002;Matsunaga and Jaehning, 2004) resulted in a general slowing of mitochondrial transcription and a decrease in the abundance of mature RNAs. As the phenotype of the suv3⌬ mutation involves dysfunction of mitochondrial RNA degradation and turnover Stepien et al, 1995;Dziembowski et al, 2003), it was tempting to speculate that the suppressor mutations we discovered acted through lowering the transcription rate and reducing the RNA content in the organelle.…”

Section: Mutations In the Rpo41 And Mtf1 Genes Partiallymentioning

confidence: 99%

“…This indicates that the su2 mutation in DSW3-12/A occurred in the MTF1 gene, which encodes the transcription factor of the mitochondrial RNA polymerase (Schinkel et al, 1987;Jang and Jaehning, 1991). Temperature-sensitive respiratory-deficient mutants with defects in MTF1 had been previously described, along with similar mutations in the core mitochondrial RNA polymerase gene RPO41 (Shadel and Clayton, 1995;Cliften et al, 1997Cliften et al, , 2000Karlok et al, 2002;Matsunaga and Jaehning, 2004). We decided therefore to verify directly whether the other suppressor mutation, su1, corresponded to the RPO41 gene.…”

Section: Spontaneous Nuclear Monogenic Suppressor Mutations Partialmentioning

confidence: 99%

“…The majority of the temperature-sensitive mutations in the MTF1 gene known so far were located within or close to the regions displaying homology to the family of bacterial transcription factors (Shadel and Clayton, 1995;Cliften et al, 1997). Deletion of the C-terminal domain, beyond residue 291, resulted in a temperature-sensitive petite phenotype (Shadel and Clayton, 1995), corresponding to the effect of the P303L substitution observed in the DSW3-12/A spore. …”

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Balance between Transcription and RNA Degradation Is Vital forSaccharomyces cerevisiaeMitochondria: Reduced Transcription Rescues the Phenotype of Deficient RNA Degradation

Rogowska

Puchta

Czarnecka

et al. 2006

MBoC

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The Saccharomyces cerevisiae SUV3 gene encodes the helicase component of the mitochondrial degradosome (mtEXO), the principal 3 -to-5 exoribonuclease of yeast mitochondria responsible for RNA turnover and surveillance. Inactivation of SUV3 (suv3⌬) causes multiple defects related to overaccumulation of aberrant transcripts and precursors, leading to a disruption of mitochondrial gene expression and loss of respiratory function. We isolated spontaneous suppressors that partially restore mitochondrial function in suv3⌬ strains devoid of mitochondrial introns and found that they correspond to partial loss-of-function mutations in genes encoding the two subunits of the mitochondrial RNA polymerase (Rpo41p and Mtf1p) that severely reduce the transcription rate in mitochondria. These results show that reducing the transcription rate rescues defects in RNA turnover and demonstrates directly the vital importance of maintaining the balance between RNA synthesis and degradation. INTRODUCTIONThe degradation of RNA is an essential element in the expression of genetic information. It is required to control RNA abundance and thus gene expression and to eliminate aberrant or defective molecules that inevitably form during RNA synthesis and maturation (RNA surveillance) (Vasudevan and Peltz, 2003). The posttranscriptional mechanisms affecting mitochondrial gene expression, including RNA turnover, are of particular importance because transcriptional control is relatively simple and rudimentary. The single RNA polymerase (RNAP) of Saccharomyces cerevisiae mitochondria is composed of only two nuclear-encoded protein subunits-the core enzyme encoded by the RPO41 gene and a transcription initiation factor encoded by the MTF1 gene (Masters et al., 1987;Jang and Jaehning, 1991). The RNAP holoenzyme recognizes a simple nonanucleotide promoter sequence (Osinga et al., 1982;Mangus et al., 1994) and initiates synthesis of at least 13 primary multicistronic transcripts that undergo extensive processing to form mature RNAs (Christianson and Rabinowitz, 1983;Tzagoloff and Myers, 1986;Foury et al., 1998;Gagliardi et al., 2004;Schafer, 2005). Such organization leaves little room for regulation at the transcription initiation level and makes posttranscriptional processes, including RNA degradation, key control points for mitochondrial gene expression.The enzymes controlling RNA turnover in cells and organelles show great evolutionary divergence and are only partially conserved between mitochondria of different organisms (Gagliardi et al., 2004). The enzymatic activity responsible for turnover is, however, on the basic level, similar in all the systems discovered so far-it is that of a 3Ј-to-5Ј processive exoribonuclease, either hydrolytic or phosphorolytic. In most cases, the exoribonuclease activity is contained in a larger multiprotein complex that, in addition to exoribonucleases, also contains RNA helicases, and in certain cases, endonucleases. A model example of such a complex is the eubacterial degradosome (Carpousis, 2002).The first mitoc...

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A Saccharomyces cerevisiae Mitochondrial Transcription Factor, sc-mtTFB, Shares Features with Sigma Factors but Is Functionally Distinct

Cited by 62 publications

References 40 publications

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

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

Transcription Factor-dependent DNA Bending Governs Promoter Recognition by the Mitochondrial RNA Polymerase

Balance between Transcription and RNA Degradation Is Vital forSaccharomyces cerevisiaeMitochondria: Reduced Transcription Rescues the Phenotype of Deficient RNA Degradation

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