Binding of the Priming Nucleotide in the Initiation of Transcription by T7 RNA Polymerase

Kuzmine, I; Gottlieb, Philip A.; Martin, Craig T.

doi:10.1074/jbc.m208405200

Cited by 47 publications

(39 citation statements)

References 31 publications

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“…6, c and d). Such a high K 0.5 value for the initiating nucleotide(s) is consistent with observations made in other transcription systems, including the yeast mitochondrial transcription system (43,46,58,59). The efficiency of human transcription should therefore be responsive to the cellular levels of ATP as observed for the human and the yeast mitochondrial transcription systems (41,43).…”

Section: Use Of a Dinucleotide Primer Does Not Overcome The Block To supporting

confidence: 63%

Identification of Multiple Rate-limiting Steps during the Human Mitochondrial Transcription Cycle in Vitro

Lodeiro

Uchida

Arnold

et al. 2010

Journal of Biological Chemistry

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We have reconstituted human mitochondrial transcription in vitro on DNA oligonucleotide templates representing the light strand and heavy strand-1 promoters using protein components (RNA polymerase and transcription factors A and B2) isolated from Escherichia coli. We show that 1 eq of each transcription factor and polymerase relative to the promoter is required to assemble a functional initiation complex. The light strand promoter is at least 2-fold more efficient than the heavy strand-1 promoter, but this difference cannot be explained solely by the differences in the interaction of the transcription machinery with the different promoters. In both cases, the rate-limiting step for production of the first phosphodiester bond is open complex formation. Open complex formation requires both transcription factors; however, steps immediately thereafter only require transcription factor B2. The concentration of nucleotide required for production of the first dinucleotide product is substantially higher than that required for subsequent cycles of nucleotide addition. In vitro, promoter-specific differences in post-initiation control of transcription exist, as well as a second rate-limiting step that controls conversion of the transcription initiation complex into a transcription elongation complex. Rate-limiting steps of the biochemical pathways are often those that are targeted for regulation. Like the more complex multisubunit transcription systems, multiple steps may exist for control of transcription in human mitochondria. The tools and mechanistic framework presented here will facilitate not only the discovery of mechanisms regulating human mitochondrial transcription but also interrogation of the structure, function, and mechanism of the complexes that are regulated during human mitochondrial transcription.Expression and maintenance of the mitochondrial genome (mtDNA) are essential for mitochondria to produce energy, function in other aspects of intermediary metabolism, contribute to pathogen sensing, and integrate into signal-transduction pathways, especially those regulated by circadian rhythm and nutritional status (1, 2). Mitochondrial dysfunction caused by defects in mtDNA replication and translation attributable to mutations in mitochondrial and nuclear genes contributes to numerous diseases, including muscular dystrophies, cardiac diseases, neurodegenerative disorders, diabetes, and certain cancers (3)(4)(5)(6)(7)(8). A link between the cis-and trans-acting factors required for mitochondrial transcription and disease has yet to be established, likely because of limited information on the mechanism and regulation of mitochondrial transcription (1, 4, 9 -11).

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Section: Use Of a Dinucleotide Primer Does Not Overcome The Block To supporting

confidence: 63%

Identification of Multiple Rate-limiting Steps during the Human Mitochondrial Transcription Cycle in Vitro

Lodeiro

Uchida

Arnold

et al. 2010

Journal of Biological Chemistry

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“…In the bacterial RNAP sigma factor provides unique contacts for the initiating nucleotide (43,63). In T7RNAP, specificity for the initiating GTP is provided by Hoogsteen contacts predicted to be made by Arg-425 and Arg-632 (64). It is intriguing that although both these residues are highly conserved in the phage RNAPs, all of the mtRNAP have substitutions at the position of T7RNAP Arg-632 (6).…”

Section: Figmentioning

confidence: 99%

A Mutation in the Yeast Mitochondrial Core RNA Polymerase, Rpo41, Confers Defects in Both Specificity Factor Interaction and Promoter Utilization

Matsunaga

Jaehning

2004

Journal of Biological Chemistry

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The yeast mitochondrial RNA polymerase (RNAP) is composed of the core RNAP, Rpo41, and the mitochondrial transcription factor, Mtf1. Both are required for mitochondrial transcription, but how the two proteins interact to create a functional, promoter-selective holoenzyme is still unknown. Rpo41 is similar to the single polypeptide bacteriophage T7RNAP, which does not require additional factors for promoter-selective initiation but whose activity is modulated during infection by association with T7 lysozyme. In this study we used the co-crystal structure of T7RNAP and T7 lysozyme as a model to define a potential Mtf1 interaction surface on Rpo41, making site-directed mutations in Rpo41 at positions predicted to reside at the same location as the T7RNAP/T7 lysozyme interface. We identified Rpo41 mutant E1224A as having reduced interactions with Mtf1 in a two-hybrid assay and a temperature-sensitive petite phenotype in vivo. Although the E1224A mutant has full activity in a non-selective in vitro transcription assay, it is temperature-sensitive for selective transcription from linear DNA templates containing the 14S rRNA, COX2, and tRNAcys mitochondrial promoters. The tRNAcys promoter defect can be rescued by template supercoiling but not by addition of a dinucleotide primer. The fact that mutation of Rpo41 results in selective transcription defects indicates that the core RNAP, like T7RNAP, plays an important role in promoter utilization.Mitochondria contain a separate genome (mtDNA) 1 that encodes several mRNAs and the rRNAs and tRNAs required for their translation into components of the oxidative phosphorylation system (1-3). These mitochondrial transcripts are synthesized by an RNA polymerase (RNAP) distinct from those found in the nucleus of the eukaryotic cell (2). In the budding yeast Saccharomyces cerevisiae, two well characterized nuclear genes, RPO41, encoding the mitochondrial core RNAP, and MTF1, encoding a required mitochondrial transcription factor, interact to form the functional mitochondrial RNAP holoenzyme (mtRNAP) (4, 5). Homologues of Rpo41 have been identified in many organisms (6), and apparent Mtf1 (sc-mtTFB) homologues required for selective mitochondrial transcription have been described recently in humans and mice (7-9). Therefore it is clear that more complex eukaryotes also use a multisubunit mtRNAP to express genes from the mtDNA.The origins of the multi-subunit mtRNAP are still not clear. Rpo41 shares extensive amino acid similarity with RNAP from bacteriophage T7 and T3 (4). However, these phage-encoded single polypeptide RNAPs do not require additional factors for promoter recognition (10). The recent crystal structure determination of Mtf1 has revealed a significant structural similarity with the family of RNA methyltransferases (11). Furthermore, Mtf1 and its functional homologues from multicellular organisms all share amino acid sequence similarity with RNA methyltransferases (7-9, 11). In fact, one of the human Mtf1 homologues has been shown to have methyltransferase activity (...

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“…The use of the GTP analogs and the discrimination against RTP suggest that recognition of the NTPi is not dependent on Watson-Crick interactions with the template. The T7 RNA-dependent RNA polymerase also recognizes the NTPi in a base-specific manner (17). Because recognition of the NTPi is not needed in polymerases that extend from a primer, it is likely a unique property of de novo-initiating polymerases.…”

Section: Vol 78 2004 Effects Of Pocket Mutations On Hcv Rdrp 12215mentioning

confidence: 99%

De Novo Initiation Pocket Mutations Have Multiple Effects on Hepatitis C Virus RNA-Dependent RNA Polymerase Activities

Ranjith-Kumar

Sarisky

Gutshall

et al. 2004

J Virol

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The hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) has several distinct biochemical activities, including initiation of RNA synthesis by a de novo mechanism, extension from a primed template, nontemplated nucleotide addition, and synthesis of a recombinant RNA product from two or more noncovalently linked templates (template switch). All of these activities require specific interaction with nucleoside triphosphates (NTPs). Based on the structure of the HCV RdRp bound to NTP (S. Bressanelli, L. Tomei, F. A. Rey, and R. DeFrancesco, J. Virol. 76:3482-3492, 2002), we mutated the amino acid residues that contact the putative initiation GTP and examined the effects on the various activities. Although all mutations retained the ability for primer extension, alanine substitution at R48, R158, R386, R394, or D225 decreased de novo initiation, and two or more mutations abolished de novo initiation. While the prototype enzyme had a K m for GTP of 3.5 M, all of the mutations except one had K m s that were three-to sevenfold higher. These results demonstrate that the affected residues are functionally required to interact with the initiation nucleotide. Unexpectedly, many of the mutations also affected the addition of nontemplated nucleotide, indicating that residues in the initiating NTP (NTPi)-binding pocket are required for nontemplated nucleotide additions. Interestingly, mutations in D225 are dramatically affected in template switch, indicating that this residue of the NTPi pocket also interacts with components in the elongation complex. We also examined the interaction of ribavirin triphosphate with the NTPi-binding site.Hepatitis C Virus (HCV), a member of the flaviviridae family of positive-strand RNA viruses, infects up to 3% of the world's population (1). A prime target for antiviral treatment in HCV is the NS5B protein, the RNA-dependent RNA polymerase (RdRp) responsible for viral RNA synthesis. The RdRp is one of the subunits expected to participate in HCV replication and is responsible for the initiation and elongation of viral RNA synthesis. Initiation of RNA synthesis in infected cells likely starts de novo; that is, by use of a 1-nucleotide (nt) primer (3,14). This mode of RNA synthesis requires a specialized site in the RdRp, called the I site, that specifically recognizes the initiating nucleoside triphosphate (designated NTPi, with GTP being preferred by the recombinant HCV RdRp) (14,23,25). A second NTP-binding pocket, the I ϩ 1 site, binds a nucleotide for phosphoryl transfer as dictated by the template base (14). Cocrystal structures of the HCV RdRp and nucleotides have been reported previously (3, 21), identifying a number of residues at D225, R48, R158, R386, R394, and S367 that interact with specific moieties of the initiation GTP (Fig. 1A). In this structure, it is not known whether the GTP binds to the I site or the I ϩ 1 site. NTPi binding to the I site is base specific, while binding of the second NTP to the I ϩ 1 site should be directed by the template (25). Because the RdRp-GTP st...

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Binding of the Priming Nucleotide in the Initiation of Transcription by T7 RNA Polymerase

Cited by 47 publications

References 31 publications

Identification of Multiple Rate-limiting Steps during the Human Mitochondrial Transcription Cycle in Vitro

Identification of Multiple Rate-limiting Steps during the Human Mitochondrial Transcription Cycle in Vitro

A Mutation in the Yeast Mitochondrial Core RNA Polymerase, Rpo41, Confers Defects in Both Specificity Factor Interaction and Promoter Utilization

De Novo Initiation Pocket Mutations Have Multiple Effects on Hepatitis C Virus RNA-Dependent RNA Polymerase Activities

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