[34] Isolation of bacterial and bacteriophage RNA polymerases and their use in synthesis of RNA in Vitro

Chamberlin, Michael J.; Kingston, Richard; Gilman, Michael; Wiggs, Janey L.; deVera, A

doi:10.1016/0076-6879(83)01037-x

Cited by 106 publications

(70 citation statements)

References 77 publications

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“…T7 RNAP, like many of the phage polymerases, binds, melts, and initiates transcription as single subunit enzyme without any accessory transcription factor (5). In contrast, the YMt RNAP requires a specific accessory factor (Mtf1 (yeast mitochondrial transcription factor)) to initiate transcription from its promoter (6).…”

Section: S Cerevisiae Ymtmentioning

confidence: 99%

A Promoter Recognition Mechanism Common to Yeast Mitochondrial and Phage T7 RNA Polymerases

Nayak

Guo

Sousa

2009

Journal of Biological Chemistry

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Yeast mitochondrial (YMt) and phage T7 RNA polymerases (RNAPs) are two divergent representatives of a large family of single subunit RNAPs that are also found in the mitochondria and chloroplasts of higher eukaryotes, mammalian nuclei, and many other bacteriophage. YMt and phage T7 promoters differ greatly in sequence and length, and the YMt RNAP uses an accessory factor for initiation, whereas T7 RNAP does not. We obtain evidence here that, despite these apparent differences, both the YMt and T7 RNAPs utilize a similar promoter recognition loop to bind their respective promoters. Mutations in this element in YMt RNAP specifically disrupt mitochondrial promoter utilization, and experiments with site-specifically tethered chemical nucleases indicate that this element binds the mitochondrial promoter almost identically to how the promoter recognition loop from the phage RNAP binds its promoter. Sequence comparisons reveal that the other members of the single subunit RNAP family display loops of variable sequence and size at a position corresponding to the YMt and T7 RNAP promoter recognition loops. We speculate that these elements may be involved in promoter recognition in most or all of these enzymes and that this element's structure allows it to accommodate significant sequence and length variation to provide a mechanism for rapid evolution of new promoter specificities in this RNAP family. S. cerevisiae YMt2 and phage T7 RNAPs are both members of the single subunit RNAP family, but their promoter sequences are very different. The T7 promoter is a 21-nucleotide (nt) duplex that includes 4 nt downstream of the transcription start site and 17 nt upstream of ϩ1 (1). The promoters of the closely related T3 phage or the more distantly related Sp6 phage(2) are identical in size to the T7 promoter but differ in sequence. In contrast, the YMt promoter is only 8 nt in length and is therefore similar in size to chloroplast promoters or the mitochondrial promoters of plants and other fungi, which are typically 8 -10 nt in length although highly disparate in sequence (3, 4) (Fig. 1).The mechanisms of promoter recognition by the YMt and T7 RNAP also appear to be distinct. T7 RNAP, like many of the phage polymerases, binds, melts, and initiates transcription as single subunit enzyme without any accessory transcription factor (5). In contrast, the YMt RNAP requires a specific accessory factor (Mtf1 (yeast mitochondrial transcription factor)) to initiate transcription from its promoter (6). This seeming disparity may, however, obscure underlying mechanistic similarity. For example, it was originally suggested (6) that Mtf1 might be homologous and analogous to prokaryotic RNAP -factor, which would mean that Mtf1 contributes directly to promoter recognition, a mechanism distinct from that seen with T7 RNAP, where the polymerase contains all of the promoter recognition elements. However, subsequent work showed that YMt RNAP can initiate transcription without Mtf1 if the promoter is supercoiled or contains a heterduplex ("bubble") ...

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Section: S Cerevisiae Ymtmentioning

confidence: 99%

A Promoter Recognition Mechanism Common to Yeast Mitochondrial and Phage T7 RNA Polymerases

Nayak

Guo

Sousa

2009

Journal of Biological Chemistry

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“…Crude R. sphaeroides RNA polymerase holoenzyme was prepared by heparin agarose chromatography (6). Briefly, 2 g (wet weight) of aerobically grown R. sphaeroides cells was suspended in 6 ml of a solution containing 10 mM Tris-Cl (pH 8.0), 50 mM NaCl, 10 mM MgCl 2 , 1 mM EDTA, 0.3 mM DTT, 7.5% glycerol, 0.5 mM phenylmethylsulfonyl fluoride, and 1.5 mg of lysozyme per ml and incubated on ice for 10 min.…”

Section: Purification Of Cprrbmentioning

confidence: 99%

Transcriptional Activation of the Rhodobacter sphaeroides Cytochrome c ₂ Gene P2 Promoter by the Response Regulator PrrA

et al. 2002

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Anoxygenic photosynthetic growth of Rhodobacter sphaeroides, a member of the ␣ subclass of the class Proteobacteria, requires the response regulator PrrA. PrrA and the sensor kinase PrrB are part of a twocomponent signaling pathway that influences a wide range of processes under oxygen-limited conditions. In this work we characterized the pathway of transcription activation by PrrB and PrrA by purifying these proteins, analyzing them in vitro, and characterizing a mutant PrrA protein in vivo and in vitro. When purified, a soluble transmitter domain of PrrB (cPrrB) could autophosphorylate, rapidly transfer phosphate to PrrA, and stimulate dephosphorylation of phospho-PrrA. Unphosphorylated PrrA activated transcription from a target cytochrome c 2 gene (cycA) promoter, P2, which contained sequences from ؊73 to ؉22 relative to the transcription initiation site. However, phosphorylation of PrrA increased its activity since activation of cycA P2 was enhanced up to 15-fold by treatment with the low-molecular-weight phosphodonor acetyl phosphate. A mutant PrrA protein containing a single amino acid substitution in the presumed phosphoacceptor site (PrrA-D63A) was not phosphorylated in vitro but also was not able to stimulate cycA P2 transcription. PrrA-D63A also had no apparent in vivo activity, demonstrating that aspartate 63 is necessary both for the function of PrrA and for its phosphorylation-dependent activation. The cellular level of wild-type PrrA was negatively autoregulated so that less PrrA was present in the absence of oxygen, conditions in which the activities of many PrrA target genes increase. PrrA-D63A failed to repress expression of the prrA gene under anaerobic conditions, suggesting that this single amino acid change also eliminated PrrA function in vivo.

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“…A procedure for purifying Chromatium RNA polymerase was based on a method for isolating RNA polymerases from other bacteria [24]. Autotrophically grown Chromatium cells (4 g in fresh mass) were frozen, subjected to three cycles of freezing and thawing, suspended in a homogenization buffer containing 10 mM Tris/Cl (pH KO), 10 mM MgC12, 1 mM EDTA, 0.3 mM dithiothreitol, 7.5% (by vol.)…”

Section: Purification Of Chromatium Rna Polymerase and Its Assay Condmentioning

confidence: 99%

“…glycerol at 4"C, and stored at -80°C. The enzyme activity was measured as described for E. coli RNA polymerase [24].…”

Section: Purification Of Chromatium Rna Polymerase and Its Assay Condmentioning

confidence: 99%

Transcriptional regulation of genes for plant‐type ribulose‐1,5‐bisphosphate carboxylase/oxygenase in the photosynthetic bacterium, Chromatium vinosum

Valle

Kobayashi

Akazawa

1988

European Journal of Biochemistry

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The content of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in the photosynthetic purple sulfur bacterium, Chromatium vinosum, grown either heterotrophically or autotrophically, was highly correlated with the level of 2.0-kb mRNA encoding genes for both large (rbcL) and small (rbcS) subunits. This result indicates the transcriptional regulation of Rubisco biosynthesis in Chromatium cells. In the analysis of transcripts for rbcL and rbcS in Escherichia coli transformed by a plasmid bearing both genes downstream of E. cob tac promoter (pCKSl), the mRNAs were found to be the same sizes as those from Chromatium. However, we were unable to detect mRNA for Rubisco in E. coli harboring a plasmid containing the genes for Rubisco and its own promoter without any E. coli promoters (pCUB1). In the in vitro transcription experiment of pCKS1 and pCUB1 by E. coli RNA polymerase, it was observed that the enzyme could not recognize the Rubisco promoter. Therefore, we have purified RNA polymerase from Chromatium cells and developed a homologous in vitro transcription system. We have detected factor(s) for transcriptional regulation from either heterotrophically or autotrophically grown cells of Chromatium using the homologous in vitro transcription system. Autotrophic organisms, ranging from photosynthetic bacteria to green plants, assimilate C 0 2 through the CalvinBenson photosynthetic carbon reduction cycle. The initial COz fixation step is catalyzed by ribulose-l,5-bisphosphate carboxylase/oxygenase (Rubisco) [l -41. This enzyme encompasses both carboxylase and oxygenase activities, the latter playing a role in the photorespiration, leading to a reduction of the efficiency of photosynthetic COz fixation [4]. In green plants and algae, as well as in some photosynthetic bacteria, Rubisco molecules are composed of eight of each large ( M , = 50 000 -55 000) and small ( M , = 12 000 -16 000) subunits, forming a hexadecameric structure, L8s8 [I].It is now well established that subunits of Rubisco are encoded by different genomes in plants and green algae. The large subunit (rbcL) is encoded by chloroplast DNA and synthesized on chloroplast ribosomes (70 S), whereas the small subunit (rbcS) is encoded by nuclear DNA and synthesized on cytoplasmic ribosomes (80 S) [5]. By contrast, in photosynthetic prokaryotes, i.e. the photosynthetic purple sulfur bacterium Chromatium vinosum [6] and cyanobacteria [7,8], and in the genome of cyanelles of Cyanophoraparadoxa (eukaryotic protozoan) [9], the rbcS gene is located downstream of the rbcL gene. It remains to be elucidated how synthesis of both subunits is coordinated and regulated in the prokaryotes and eukaryotes.

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[34] Isolation of bacterial and bacteriophage RNA polymerases and their use in synthesis of RNA in Vitro

Cited by 106 publications

References 77 publications

A Promoter Recognition Mechanism Common to Yeast Mitochondrial and Phage T7 RNA Polymerases

A Promoter Recognition Mechanism Common to Yeast Mitochondrial and Phage T7 RNA Polymerases

Transcriptional Activation of the Rhodobacter sphaeroides Cytochrome c ₂ Gene P2 Promoter by the Response Regulator PrrA

Transcriptional regulation of genes for plant‐type ribulose‐1,5‐bisphosphate carboxylase/oxygenase in the photosynthetic bacterium, Chromatium vinosum

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[34] Isolation of bacterial and bacteriophage RNA polymerases and their use in synthesis of RNA in Vitro

Cited by 106 publications

References 77 publications

A Promoter Recognition Mechanism Common to Yeast Mitochondrial and Phage T7 RNA Polymerases

A Promoter Recognition Mechanism Common to Yeast Mitochondrial and Phage T7 RNA Polymerases

Transcriptional Activation of the Rhodobacter sphaeroides Cytochrome c 2 Gene P2 Promoter by the Response Regulator PrrA

Transcriptional regulation of genes for plant‐type ribulose‐1,5‐bisphosphate carboxylase/oxygenase in the photosynthetic bacterium, Chromatium vinosum

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Transcriptional Activation of the Rhodobacter sphaeroides Cytochrome c ₂ Gene P2 Promoter by the Response Regulator PrrA