Characterization of a yeast mitochondrial promoter by deletion mutagenesis.

Biswas, Tanmay; Edwards, John C.; Rabinowitz, Murray; Getz, Godfrey S.

doi:10.1073/pnas.82.7.1954

Cited by 71 publications

(43 citation statements)

References 24 publications

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“…pTB3'+29 contains sufficient sequences to direct the in vitro synthesis of a specific runoff transcript initiated at position +1 of the 14S rRNA gene. pTB3'-31 does not allow detectable specific transcription of the 14S rRNA gene (2). These plasmids differ only by 60 bp centered on position + 1 and containing nucleotides -10 to +2 which are essential for accurate in vitro transcription (2).…”

Section: Bindingmentioning

confidence: 99%

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Novel method for identifying sequence-specific DNA-binding proteins.

Levens¹,

Howley²

1985

Mol. Cell. Biol.

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We developed a general method for the enrichment and identification of sequence-specific DNA-binding proteins. A well-characterized protein-DNA interaction is used to isolate from crude cellular extracts or fractions thereof proteins which bind to specific DNA sequences; the method is based solely on this binding property of the proteins. The DNA sequence of interest, cloned adjacent to the lac operator DNA segment is incubated with a lac repressor-beta-galactosidase fusion protein which retains full operator and inducer binding properties. The DNA fragment bound to the lac repressor-beta-galactosidase fusion protein is precipitated by the addition of affinity-purified anti-beta-galactosidase inmobilized on beads. Sequence-specific DNA-binding proteins are the mediators of many essential cell regulatory processes including transcription, initiation of DNA replication, and site-specific recombination. Studies in phage and bacterial genetics have allowed the identification and characterization of a number of procaryotic sequence-specific DNA-binding proteins. However, relatively few such proteins have been identified in eucaryotes, where classical genetic manipulations are cumbersome and biochemical analyses, are often hindered by smaller quantities of starting materials and by the apparent low abundance of many sequence-specific DNA-binding proteins.In contrast, recombinant DNA technology coupled with improved methods for introducing in vitro manipulated DNA into eucaryotic cells has permitted the rapid identification of specific sequences involved in the regulation of gene expression, in the replication of viral DNAs, or in site-specific recombination (such as immunoglobulin gene rearrangement). Techniques for in vivo and in vitro footprinting can define the precise DNA sequences protected by unidentified factors (12). In addition, the development of accurate systems for the in vitro transcription of cloned DNAs has helped define some of the template requirements necessary for faithful RNA synthesis (10, 17). We devised a general method which uses specific DNA regulatory sequences identified by standard molecular biological approaches to detect and enrich the protein components which specifically interact with those sequences. The method uses a wellcharacterized specific and avid DNA-protein interaction, the binding of the lac repressor to the lac operator, as the basis for isolating specific factors from complex mixtures of protein. The DNA sequence of interest is cloned adjacent to a lac operator, and a DNA fragment containing both the operator and regulatory sequence is precipitated by the * Corresponding author. sequential addition of both a lac repressor-beta-galactosidase fusion protein and immobilized antibodies directed against beta-galactosidase (Fig. 1A). The specifically bound DNA fragment is then incubated with competitor DNA and an extract or fraction containing the factor(s) which recognizes the regulatory sequence of interest (Fig. 1B). The immobilized DNA is recovered (Fig. 1C), and the lac opera...

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Section: Bindingmentioning

confidence: 99%

“…This suspension was then incubated for 3 to (2). Transcription reactions were allowed to proceed for 20 min at 20°C with periodic mixing.…”

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confidence: 99%

Novel method for identifying sequence-specific DNA-binding proteins.

Levens¹,

Howley²

1985

Mol. Cell. Biol.

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“…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).…”

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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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“…In heparinnchallenge experiments, there is no significant effect of dinucleotide on heparin-resistant DNA-RNA polymerase complex formation. These results indicate that the low level of transcription from the weak mitochondrial promoter is due to the slow rate of formation of the first phosphodiester bond.Significant progress has been made toward an understanding of the control of mitochondrial gene expression in the yeast Saccharomyces cerevisiae (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12). The biogenesis of mitochondria is regulated by the coordinated expression of both mitochondrial and nuclear genes (13).…”

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confidence: 99%

“…Significant progress has been made toward an understanding of the control of mitochondrial gene expression in the yeast Saccharomyces cerevisiae (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12). The biogenesis of mitochondria is regulated by the coordinated expression of both mitochondrial and nuclear genes (13).…”

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Control of mitochondrial gene expression in the yeast Saccharomyces cerevisiae.

Biswas¹

1990

Proc. Natl. Acad. Sci. U.S.A.

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Mitochondrial promoters in Saccharomyces cerevisiae contain an identical octanucleotide [TATAAGTA-(+ 1)] sequence present just upstream of the initiation site (at the left end of the arrow). Studies have shown that the transcription rates of mitochondrial genes vary from 7-to 15-fold. The nucleotide at position +2 regulates the efficiency of mitochondrial promoters but does not affect the specificity of initiation. The data presented herein demonstrate that the variable transcription rates of mitochondrial genes are due to different levels of transcriptional initiation. The rate of first phosphodiester bond formation between a purine and a pyrimidine on a weak promoter is much lower than that of purine-purine on a strong promoter. A dinucleotide corresponding to positions +1 and +2 acts in vitro as a primer, bypassing the first phosphodiester bond formation at the initiation site. When these dinucleotides were used to prime transcription, the activities of the strong and weak promoters were found to be identical. In heparinnchallenge experiments, there is no significant effect of dinucleotide on heparin-resistant DNA-RNA polymerase complex formation. These results indicate that the low level of transcription from the weak mitochondrial promoter is due to the slow rate of formation of the first phosphodiester bond.Significant progress has been made toward an understanding of the control of mitochondrial gene expression in the yeast Saccharomyces cerevisiae (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12). The biogenesis of mitochondria is regulated by the coordinated expression of both mitochondrial and nuclear genes (13). The 76-kilobase mitochondrial DNA of yeast encodes a limited number of mitochondrial proteins, two rRNAs, and a set oftRNAs necessary for translation of mitochondrial mRNAs. The remaining mitochondrial proteins including factors required for mitochondrial transcription, replication, and translation are encoded by the nuclear genome and imported into mitochondrial.Mitochondrial RNA (mtRNA) polymerase consists of a core enzyme and a specificity factor, both of which are required for specific transcription of mitochondrial genes (8-10). The mitochondrial promoter consists of an 8-nucleotide sequence [5'-TATAAGTA(+1)-3'] that has been identified in the mitochondrial rRNA, tRNA, and mRNA genes as well as at the origins of replication (2). In vitro transcription studies of variant promoter mutants showed that a short octanucleotide sequence, 5'-TAtAaGtN-3', is the minimum sequence requirement to direct accurate initiation of transcription by mtRNA polymerase (6). The lowercase letters indicate positions at which nucleotide variants are permissible and N designates any nucleotide that is used as an initiating nucleotide.Although mitochondrial genes share an identical promoter sequence, they are transcribed at different rates (5, 12). The promoters can be grouped into two classes: strong promoters and weak promoters. The strong promoters have 7-15 times the activity of the weak promoters. Promoters are ...

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Characterization of a yeast mitochondrial promoter by deletion mutagenesis.

Cited by 71 publications

References 24 publications

Novel method for identifying sequence-specific DNA-binding proteins.

Novel method for identifying sequence-specific DNA-binding proteins.

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

Control of mitochondrial gene expression in the yeast Saccharomyces cerevisiae.

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