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Query: EC:2.7.7.6 (RNA polymerase)
 34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The elongation rate of RNAs synthesized from AI promoters of T7 phage DNA and its deletion mutant delta DIII T7 DNA by E. coli RNA polymerase was analyzed. The distribution of incorporation rates of any definite nucleotides at any definite position along the two RNA chains was studied. The minimal structure which reproducibly forms pauses seems to be trinucleotide. Two main groups of trinucleotides could be distinguished: 1) those mostly associated with pauses and; 2) those usually found in pause free regions. The first group consists of AUG, AUA, AUC, AAU, GUG, GUA, CGU, CGC, UUA, UUU; the second one comprises AAA, CAA, CCC, UCC, CUA, CUG, CUC, GGG, ACU, GAG, GAA, GGA. A model accounting for intermittent elongation has been developed. It is based on the hypothesis that the kinetic constants of each nucleotide incorporation to and pyrophosphorolysis from the 3'-end of the growing RNA chain depend on the nature of the incoming nucleotide as well as on the nature of a nucleotide residue situated at the 3'-end of the growing RNA. A general equation describing the pause distribution along the RNA of a known nucleotide sequence is proposed.
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PMID:[Effect of the primary structure of RNA on the pulse character of RNA elongation in vitro by Escherichia coli RNA polymerase: a model]. 616 4

In nucleotide sequencing of the cDNA of the influenza virus PB2 polymerase gene by the dideoxy method using a modified T7 DNA polymerase, Sequenase, the sequence of the promoter region, 5'-AGCGAAAGCAGG, was shown to be misread as 5'-AGCGAAACGAGG, i.e., a GC doublet at positions 8 and 9 was read in reverse. This misreading was also found both when the sequence of BsmI restriction site upstream from the PB2 promoter sequence was exchanged by that of the promoter of T7 RNA polymerase and when the downstream region was substituted with the nonstructural (NS) protein gene. These results indicated that the misreading by Sequenase was attributed specifically to the PB2 promoter region, independent of the upstream and downstream sequences. The misreading, however, did not occur when dGTP in the labeling mixture was substituted with another nucleotide analog, dITP. Furthermore, the reversion did not occur in the NS gene promoter region, where the nucleotide sequence was 5'-AGCAAAAGCAGG. Since the nucleotide difference between the PB2 and NS promoter regions was only at the fourth residue, i.e., G for PB2 and A for PB2 and A for NS, the G residue followed by a triplet AAA in the PB2 promoter region was suggested to be a signal responsible for the misreading by Sequenase T7 DNA polymerase. The findings warns of possible misreading in determining DNA sequences, in addition to compression of the sequencing ladder.
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PMID:Reverse misreading of a GC doublet by the modified T7 DNA polymerase, Sequenase. 797 72

The Escherichia coli sigma 32 transcriptional regulator has been shown to be degraded both in vivo and in vitro by the FtsH (HflB) protease, a member of the AAA protein family. In our attempts to study this process in detail, we found that two sigma 32 mutants lacking 15-20 C-terminal amino acids had substantially increased half-lives in vivo or in vitro, compared with wild-type sigma 32. A truncated version of sigma 32, sigma 32 C delta, was purified to homogeneity and shown to be resistant to FtsH-dependent degradation in vitro, suggesting that FtsH initiates sigma 32 degradation from its extreme C-terminal region. Purified sigma 32 C delta interacted with the DnaK and DnaJ chaperone proteins in a fashion similar to that of wild-type sigma 32. However, in contrast to wild-type sigma 32, sigma 32 C delta was largely deficient in its in vivo and in vitro interaction with core RNA polymerase. As a consequence, the truncated sigma 32 protein was completely non-functional in vivo, even when overproduced. Furthermore, it is shown that wild-type sigma 32 is protected from degradation by FtsH when complexed to the RNA polymerase core, but sensitive to proteolysis when in complex with the DnaK chaperone machine. Our results are in agreement with the proposal that the capacity of the DnaK chaperone machine to autoregulate its own synthesis negatively is simply the result of its ability to sequester sigma 32 from RNA polymerase, thus making it accessible to degradation by the FtsH protease.
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PMID:On the mechanism of FtsH-dependent degradation of the sigma 32 transcriptional regulator of Escherichia coli and the role of the Dnak chaperone machine. 998 18

Under non-stressed conditions in Escherichia coli, the heat shock transcription factor sigma(32) is rapidly degraded by the AAA protease FtsH. The DnaK chaperone system is also required for the rapid turnover of sigma(32) in the cell. It has been hypothesized that the DnaK chaperone system facilitates the degradation of sigma(32) by sequestering it from RNA polymerase core. This hypothesis predicts that mutant sigma(32) proteins, which are deficient in binding to RNA polymerase core, will be degraded independently of the DnaK chaperone system. We examined the in vivo stability of such mutant sigma(32) proteins. Results indicated that the mutant sigma(32) proteins as similar as authentic sigma(32) were stabilized in DeltadnaK and DeltadnaJ/DeltacbpA cells. The interaction between sigma(32) and DnaK/DnaJ/GrpE was not affected by these mutations. These results strongly suggest that the degradation of sigma(32) requires an unidentified active role of the DnaK chaperone system.
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PMID:Evidence for an active role of the DnaK chaperone system in the degradation of sigma(32). 1093 May 81

Regulation of protein expression can be achieved through destruction of proteins by the 26S: proteasome. Cellular processes that are regulated by proteolysis include cell cycle progression, stress responses and differentiation. Several nucleotide excision repair proteins in yeast and humans, such as Rad23, Rad4 and XPB, have been shown to co-purify with Cim3 and Cim5, AAA ATPases of the 19S: proteasome regulatory subunit. However, it has not been determined if nucleotide excision repair is regulated through protein destruction. We measured nucleotide excision repair in yeast mutants that are defective in proteasome function and found that the repair of the transcribed and non-transcribed strands of an RNA polymerase II-transcribed reporter gene was increased in the absence of proteasome function. Additionally, overexpression of the Rad4 repair protein, which is bound to the repair/proteolytic factor Rad23, conferred higher rates of nucleotide excision repair. Based on our data we suggest that a protein (or proteins) involved in nucleotide excision repair or in regulation of repair is degraded by the 26S proteasome. We propose that decreased proteasome function enables increased DNA repair, due to the transient accumulation of a specific repair factor, perhaps Rad4.
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PMID:The 26S proteasome negatively regulates the level of overall genomic nucleotide excision repair. 1112 74

A key step in the regulation of heat shock genes in Escherichia coli is the stress-dependent degradation of the heat shock promoter-specific sigma(32) subunit of RNA polymerase by the AAA protease, FtsH. Previous studies implicated the C termini of protein substrates, including sigma(32), as degradation signals for AAA proteases. We investigated the role of the C terminus of sigma(32) in FtsH-dependent degradation by analysis of C-terminally truncated sigma(32) mutant proteins. Deletion of the 5, 11, 15, and 21 C-terminal residues of sigma(32) did not affect degradation in vivo or in vitro. Furthermore, a peptide comprising the C-terminal 21 residues of sigma(32) was not degraded by FtsH in vitro and thus did not serve as a recognition sequence for the protease, while an unrelated peptide of similar length was efficiently degraded. The truncated sigma(32) mutant proteins remained capable of associating with DnaK and DnaJ in vitro but showed intermediate (5-amino-acid deletion) and strong (11-, 15-, and 21-amino-acid deletions) defects in association with RNA polymerase in vitro and biological activity in vivo. These results indicate an important role for the C terminus of sigma(32) in RNA polymerase binding but no essential role for FtsH-dependent degradation and association of chaperones.
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PMID:The C terminus of sigma(32) is not essential for degradation by FtsH. 1156 90

The transcriptional promoting activity of DmpR is under the strict control of its aromatic effector ligands that are bound by its regulatory N-terminal domain. The positive control function of DmpR resides within the central C-domain that is highly conserved among activators of sigma(54)-RNA polymerase. The C-domain mediates ATP hydrolysis and interaction with sigma(54)-RNA polymerase that are essential for open-complex formation and thus initiation of transcription. Wild-type and loss-of-function derivatives of DmpR, which are defective in distinct steps in nucleotide catalysis, were used to address the consequences of nucleotide binding and hydrolysis with respect to the multimeric state of DmpR and its ability to promote in vitro transcription. Here, we show that DmpR derivatives deleted of the regulatory N-terminal domain undergo an aromatic-effector independent ATP-binding triggered multimerisation as detected by cross-linking. In the intact protein, however, aromatic effector activation is required before ATP-binding can trigger an apparent dimer-to-hexamer switch in subunit conformation. The data suggest a model in which the N-terminal domain controls the transcriptional promoting property of DmpR by constraining ATP-mediated changes in its oligomeric state. The results are discussed in the light of recent mechanistic insights from the AAA(+) superfamily of ATPases that utilise nucleotide hydrolysis to restructure their substrates.
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PMID:The regulatory N-terminal region of the aromatic-responsive transcriptional activator DmpR constrains nucleotide-triggered multimerisation. 1174 15

The PspA protein, a negative regulator of the Escherichia coli phage shock psp operon, is produced when virulence factors are exported through secretins in many Gram-negative pathogenic bacteria and its homologue in plants, VIPP1, plays a critical role in thylakoid biogenesis, essential for photosynthesis. Activation of transcription by the enhancer-dependent bacterial sigma(54) containing RNA polymerase occurs through ATP hydrolysis-driven protein conformational changes enabled by activator proteins that belong to the large AAA(+) mechanochemical protein family. We show that PspA directly and specifically acts upon and binds to the AAA(+) domain of the PspF transcription activator. Interactions involving PspF and nucleotide are changed by the action of PspA. These changes and the complexes that form between PspF and PspA can explain how PspA exerts its negative effects upon transcription activated by PspF, and are of significance when considering how activities of other AAA(+) proteins might be controlled.
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PMID:Mechanism of action of the Escherichia coli phage shock protein PspA in repression of the AAA family transcription factor PspF. 1207 32

Archaea have a eukaryotic type of transcriptional machinery containing homologues of the transcription factors TATA-binding protein (TBP) and TFIIB (TFB) and a pol II type of RNA polymerase, whereas transcriptional regulators identified in archaeal genomes have bacterial counterparts. We describe here a novel regulator of heat shock response, Phr, from the hyperthermophilic archaeon Pyrococcus furiosus that is conserved among Euryarchaeota. The protein specifically inhibited cell-free transcription of its own gene and from promoters of a small heat shock protein, Hsp20, and of an AAA(+) ATPase. Inhibition of transcription was brought about by abrogating RNA polymerase recruitment to the TBP/TFB promoter complex. Phr bound to a 29-bp DNA sequence overlapping the transcription start site. Three sequences conserved in the binding sites of Phr, TTTA at -10, TGGTAA at the transcription start site, and AAAA at position +10, were required for Phr binding and are proposed as consensus regulatory sequences of Pyrococcus heat shock promoters. Shifting the growth temperature from 95 to 103 degrees C caused a dramatic increase of mRNA levels for the aaa(+) atpase and phr genes, but expression of the Phr protein was only weakly stimulated. Our findings suggest that heat shock response in Archaea is negatively regulated by a mechanism involving binding of Phr to conserved sequences.
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PMID:A novel archaeal transcriptional regulator of heat shock response. 1238 24

Members of the protein family called ATPases associated with various cellular activities (AAA(+)) play a crucial role in transforming chemical energy into biological events. AAA(+) proteins are complex molecular machines and typically form ring-shaped oligomeric complexes that are crucial for ATPase activity and mechanism of action. The Escherichia coli transcription activator phage shock protein F (PspF) is an AAA(+) mechanochemical enzyme that functions to sense and relay the energy derived from nucleoside triphosphate hydrolysis to catalyze transcription by the sigma(54)-RNA polymerase. Closed promoter complexes formed by the sigma(54)-RNA polymerase are substrates for the action of PspF. By using a protein fragmentation approach, we identify here at least one sigma(54)-binding surface in the PspF AAA(+) domain. Results suggest that ATP hydrolysis by PspF is coupled to the exposure of at least one sigma(54)-binding surface. This nucleotide hydrolysis-dependent presentation of a substrate binding surface can explain why complexes that form between sigma(54) and PspF are transient and could be part of a mechanism used generally by other AAA(+) proteins to regulate activity.
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PMID:The ATP hydrolyzing transcription activator phage shock protein F of Escherichia coli: identifying a surface that binds sigma 54. 1260 Nov 52


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