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At least 631 records · Page 35Linked to original sources
PubMed1987

Prokaryotic and eukaryotic RNA polymerases have homologous core subunits.

Eukaryotic RNA polymerases are complex aggregates whose component subunits are functionally ill-defined. The gene that encodes the 140,000-dalton subunit of Saccharomyces cerevisiae RNA polymerase II was isolated and studied in detail to obtain clues to the protein's function. This gene, RPB2, exists in a single copy in the haploid genome. Disruption of the gene is lethal to the yeast cell. RPB2 encodes a protein of 138,750 daltons, which contains sequences implicated in binding purine nucleotides and zinc ions and exhibits striking sequence homology with the beta subunit of Escherichia coli RNA polymerase. These observations suggest that the yeast and the E. coli subunit have similar roles in RNA synthesis, as the beta subunit contains binding sites for nucleotide substrates and a portion of the catalytic site for RNA synthesis. The subunit homologies reported here, and those observed previously with the largest RNA polymerase subunit, indicate that components of the prokaryotic RNA polymerase "core" enzyme have counterparts in eukaryotic RNA polymerases.

Amino Acid Sequence↗
PubMed2006

Role of the spacer between the -35 and -10 regions in sigmas promoter selectivity in Escherichia coli.

In vitro, the sigma(s) subunit of RNA polymerase (RNAP), RpoS, recognizes nearly identical -35 and -10 promoter consensus sequences as the vegetative sigma70. In vivo, promoter selectivity of RNAP holoenzyme containing either sigma(s) (Esigma(s)) or sigma70 (Esigma70) seems to be achieved by the differential ability of the two holoenzymes to tolerate deviations from the promoter consensus sequence. In this study, we suggest that many natural sigma(s)-dependent promoters possess a -35 element, a feature that has been considered as not conserved among sigma(s)-dependent promoters. These -35 hexamers are mostly non-optimally spaced from the -10 region, but nevertheless functional. A +/- 2 bp deviation from the optimal spacer length of 17 bp or the complete absence of a -35 consensus sequence decreases overall promoter activity, but at the same time favours Esigma(s) in its competition with Esigma70 for promoter recognition. On the other hand, the reduction of promoter activity due to shifting of the -35 element can be counterbalanced by an activity-stimulating feature such as A/T-richness of the spacer region without compromising Esigma(s) selectivity. Based on mutational analysis of sigma(s), we suggest a role of regions 2.5 and 4 of sigma(s) in sensing sub-optimally located -35 elements.

Adenine↗
PubMed1997

Genetic evidence for selective degradation of RNA polymerase subunits by the 20S proteasome in Saccharomyces cerevisiae.

scs32 was isolated as an extragenic suppressor of a temperature-sensitive (ts) mutation (rpo26-31) in the gene encoding Rpo26p, a subunit common to yeast nuclear RNA polymerases (RNAPs). rpo26-31 also confers inositol auxotrophy, inhibits the assembly of RNAPI and RNAPII and reduces the steady-state level of Rpo26p and the largest subunit of RNAPI (Rpo11p or A190p) and RNAPII (Rpo21p). rpo26-31p accumulated to wild-type levels in the scs32 strain; nevertheless, the amount of assembled RNAPII remained at a reduced level at high temperature. Hence, scs32 only partially suppressed the ts phenotype and was unable to suppress the Ino-phenotype of rpo26-31. SCS32 is identical to PUP3, which encodes a subunit of the yeast proteasome. scs32 was able to suppress the phenotype of other ts alleles of RPO26, all of which reduce the steady-state level of this subunit. However, scs32 was unable to suppress the ts phenotype of mutant alleles of RPO21, or result in accumulation of the unstable rpo21-4p. These observations suggest that the stability of non-functional or unassembled forms of Rpo26p and Rpo21p are regulated independently.

Amino Acid Sequence↗
PubMed1997

Transcriptional characterization of the Rickettsia prowazekii major macromolecular synthesis operon.

Recent studies have demonstrated that Rickettsia prowazekii can regulate transcription of selected genes at the level of initiation. However, little information concerning the existence of operons and coordinate gene regulation in this obligate intracellular parasitic bacterium is available. To address these issues, we have focused on the rpoD gene linkage group (greA-open reading frame 23 [ORF23]-dnaG-rpoD), which includes the rickettsial analog (ORF23-dnaG-rpoD) of the major macromolecular synthesis operon (MMSO). The rickettsial MMSO consists of an ORF coding for a protein of unknown function the structural genes for DNA primase (dnaG) and the major sigma factor of RNA polymerase (rpoD). RNase protection assays (RPA) were used to determine if these genes are organized into an operon controlled by multiple promoters and the quantities of transcripts produced by these genes relative to each other. RPA with a probe spanning the 270-base greA-ORF23 intervening region identified a putative transcriptional promoter within the intervening sequence. Multiple RPA probes spanning the next 4,041 bases of the linkage group demonstrated the presence of a continuous transcript and thus the existence of an operon. A probe spanning the dnaG-rpoD region revealed that two additional mRNA fragments were also protected, which enabled us to identify additional putative promoters for rpoD within dnaG. Primer extension determined that the 5' ends of the three transcripts consist separately of adenine (located 227 bases upstream of ORF23) and uracil and adenine (located 336 and 250 bases upstream of rpoD, respectively). Quantitation of transcripts produced by the three ORFs determined the relative amounts of transcripts (ORF23 to dnaG to rpoD) to be 1:2.7:5.1.

Amino Acid Sequence↗
PubMed1991

Requirement for the beta,gamma-pyrophosphate bond of ATP in a stage between transcription initiation and elongation by Escherichia coli RNA polymerase.

A linear fragment of DNA was fixed to acrylamide or agarose beads by its ends. When a fragment containing the lambda PR promoter is immobilized and transcribed, the RNA products are unchanged from those obtained on the unfixed DNA. Transcription from the immobilized fragment can be interrupted by diluting the reaction mixture into a large volume of the same buffer. Brief centrifugation allows isolation of the transcription complex with the immobilized DNA. If interruption occurs during elongation, the elongation can be resumed upon a second addition of substrates. If ATP is replaced by a beta, gamma-unhydrolyzable analogue in the second addition, the elongated products are similar to those obtained when the substrate contain ATP. When ATP is replaced by the analogue at the initiation step, however, the yield of elongated products is decreased to less than one-sixth and that of short abortive products is increased. Thus the ATP analogues are good substrates once elongation has been established in the presence of ATP, but not good enough to get past a stage just after initiation in the absence of ATP. We conclude that the beta, gamma-pyrophosphate bond of ATP is important for preparation of efficient elongation.

Adenosine Triphosphate↗
PubMed2002

The RNA polymerase alpha subunit from Sinorhizobium meliloti can assemble with RNA polymerase subunits from Escherichia coli and function in basal and activated transcription both in vivo and in vitro.

Sinorhizobium meliloti, a gram-negative soil bacterium, forms a nitrogen-fixing symbiotic relationship with members of the legume family. To facilitate our studies of transcription in S. meliloti, we cloned and characterized the gene for the alpha subunit of RNA polymerase (RNAP). S. meliloti rpoA encodes a 336-amino-acid, 37-kDa protein. Sequence analysis of the region surrounding rpoA identified six open reading frames that are found in the conserved gene order secY (SecY)-adk (Adk)-rpsM (S13)-rpsK (S11)-rpoA (alpha)-rplQ (L17) found in the alpha-proteobacteria. In vivo, S. meliloti rpoA expressed in Escherichia coli complemented a temperature sensitive mutation in E. coli rpoA, demonstrating that S. meliloti alpha supports RNAP assembly, sequence-specific DNA binding, and interaction with transcriptional activators in the context of E. coli. In vitro, we reconstituted RNAP holoenzyme from S. meliloti alpha and E. coli beta, beta', and sigma subunits. Similar to E. coli RNAP, the hybrid RNAP supported transcription from an E. coli core promoter and responded to both upstream (UP) element- and Fis-dependent transcription activation. We obtained similar results using purified RNAP from S. meliloti. Our results demonstrate that S. meliloti alpha functions are conserved in heterologous host E. coli even though the two alpha subunits are only 51% identical. The ability to utilize E. coli as a heterologous system in which to study the regulation of S. meliloti genes could provide an important tool for our understanding and manipulation of these processes.

Amino Acid Sequence↗
PubMed1990

The gene for Klebsiella bacteriophage K11 RNA polymerase: sequence and comparison with the homologous genes of phages T7, T3, and SP6.

We determined the nucleotide sequence of gene 1 of Klebsiella phage K11, which is a member of the T7 group of phages. The largest open reading frame corresponds to a polypeptide with 906 amino acids and a molecular weight of 100,383 daltons. The deduced amino acid sequence of this polypeptide shows 71% homology to the T7 RNA polymerase (the product of T7 gene 1), 72% homology to the T3 RNA polymerase and 27% homology to the SP6 RNA polymerase. Divergent evolution was clearly most pronounced in the amino-terminal portion.

Amino Acid Sequence↗
PubMed1998

[Molecular basis of changes in biological properties of foot and mouth disease virus of subtype A22].

Primary structure of capsid proteins and RNA polymerase of three closely related strains of foot and mouth disease virus (FMDV), subtype A22, differing by biological properties (the initial epitheliotropic strain A22 550 and its derivatives: thermoresistant myotropic A22 550/4 and thermosensitive attenuated A22 645) are compared by nucleic acid sequencing and analysis of the amino acid sequencing. The study revealed 1 substitute in VPI and 8 in RNA polymerase in the myotropic variant and 1 substitute in VP2, 2 in VP3, 13 in VP1, and 3 in RNA polymerase. Alteration of A22 550/4 tropism is probably due to a single substitution Gly 145-->Thr in the RGD site of capsid protein VP1. Analysis of the origin and biological properties of the attenuated strain A22 645 and the results of studies of the primary structure of proteins permit us to hypothesize that attenuation is polygenic, caused by adaptation to a heterologous host (continuous porcine cell culture), and can be expressed by changes in the structure of virus antireceptor providing its binding to cell receptors. Sites responsible for the reproduction of A22 FMDV at certain temperatures are presumably located in RNA polymerase.

Amino Acid Sequence↗
PubMed1990

U4B snRNA gene enhancer activity requires functional octamer and SPH motifs.

Expression of the chicken U4B small nuclear RNA (snRNA) gene is stimulated by a transcriptional enhancer located approximately 190-227 base pairs upstream of the transcription start site. This enhancer is composed of at least two functional motifs: an octamer (binding site for Oct-1) and an SPH motif. We now report that these two motifs functionally cooperate to stimulate U4B snRNA gene expression, and both are required for the formation of a stable transcription complex. Expression in frog oocytes of 24 different point mutant constructions indicates that the functional SPH motif is at least 15 base pairs in length. It is a recognition site for a sequence specific DNA-binding protein, termed SBF, purified from chicken embryonic nuclear extracts. The ability of the mutant SPH motif constructions to be recognized by SBF in vitro correlates with their transcriptional activities, suggesting that SBF mediates the stimulatory effect of the U4B SPH motif. These results are similar to our recent findings on the chicken U1 gene enhancer, which also contains adjacent binding sites for Oct-1 and SBF. These studies, together with evolutionary considerations and sequence comparisons among snRNA gene enhancers, suggest that cooperativity between octamer and SPH motifs could be a widely-employed mechanism for generating vertebrate snRNA gene enhancer activity.

Animals↗
PubMed1997

Identification of sigma factors for growth phase-related promoter selectivity of RNA polymerases from Streptomyces coelicolor A3(2).

We examined the promoter selectivity of RNA polymerase (RNAP) from Streptomyces coelicolor at two growth phases by in vitro transcription. Distinct sets of promoters were preferentially recognized by either exponential or stationary phase RNAP. No change in molecular weight or net charge of the core subunits was observed, suggesting that the associated specificity factors determined phase-specific promoter selectivity of the holoenzyme. Five different specificity factors and their cognate promoters were identified by in vitro holoenzyme reconstitution and transcription assays. sigma66 (sigma hrdB) and sigma46 (sigma hrdD) recognized promoters (rrnD p2 and dagA p4 for sigma66, actII-orf4 p and whiB p2 for sigma46) preferentially transcribed by the exponential phase RNAP. sigma52 recognized promoters (dagA p3 and actIII px1) preferentially transcribed by the stationary phase RNAP. Sigma28 (sigma sigE) recognized promoters (hrdD p1, whiB p1 and dagA p2) transcribed equally by both RNAPs. A novel 31 kDa specificity factor recognized actIII px2, glnR p2 and hrdD p2 promoters preferentially transcribed by the stationary phase RNAP. This factor was isolated from the stationary phase RNAP and reconstituted holoenzyme in vitro as a sigma factor. The N-terminal sequence suggests that it is a novel factor. By examining phase-specific promoter recognition pattern we can predict that holoenzyme Esigma52 and Esigma31 activities are higher in the stationary phase, whereas Esigma66 and Esigma46activities are higher in the exponential phase. Possible promoter sequences recognized by some of these sigma factors were suggested.

Amino Acid Sequence↗
PubMed1995

NusA contacts nascent RNA in Escherichia coli transcription complexes.

We have examined the interaction between NusA and the nascent RNA in Escherichia coli transcription complexes on four different templates. Photocrosslinking CTP and UTP analogs were incorporated internally and at the 3' end of the RNA. Identical templates with and without boxA sequences were compared. We found that NusA did not contact the ten nucleotides nearest to the 3' end of the RNA in complexes containing RNA up to 20 nucleotides long. Longer RNA did crosslink to NusA with all four templates examined, however. We reported that RNA 80 nucleotides long from the bacteriophage T7 A1 promoter substituted in two RNA stem-loops with photocrosslinking UMP analogs did not crosslink to NusA, even though interaction between NusA and the transcription complex were demonstrated. Here, we report that when this same RNA is substituted at CMP residues, it does crosslink to NusA. Templates containing the E. coli ribosomal RNA promoter rrnG P2, with and without a boxA sequence downstream, were compared. Long RNAs from both crosslinked to NusA, and thus boxA RNA sequences are not required for interaction with NusA. NusA did not interact with the free RNA containing boxA once released from the transcription complex, nor did it interact with RNA in a binary complex containing only RNA polymerase and RNA, without the DNA template.

Bacterial Proteins↗
PubMed2006

Unravelling the complexities of respiratory syncytial virus RNA synthesis.

Human respiratory syncytial virus (RSV) is the leading cause of paediatric respiratory disease and is the focus of antiviral- and vaccine-development programmes. These goals have been aided by an understanding of the virus genome architecture and the mechanisms by which it is expressed and replicated. RSV is a member of the order Mononegavirales and, as such, has a genome consisting of a single strand of negative-sense RNA. At first glance, transcription and genome replication appear straightforward, requiring self-contained promoter regions at the 3' ends of the genome and antigenome RNAs, short cis-acting elements flanking each of the genes and one polymerase. However, from these minimal elements, the virus is able to generate an array of capped, methylated and polyadenylated mRNAs and encapsidated antigenome and genome RNAs, all in the appropriate ratios to facilitate virus replication. The apparent simplicity of genome expression and replication is a consequence of considerable complexity in the polymerase structure and its cognate cis-acting sequences; here, our understanding of mechanisms by which the RSV polymerase proteins interact with signals in the RNA template to produce different RNA products is reviewed.

Base Sequence↗
PubMed1989

Fine structure mapping of five temperature-sensitive mutants in the 22- and 147-kilodalton subunits of vaccinia virus DNA-dependent RNA polymerase.

We have mapped the temperature-sensitive (ts) lesions of three mutants, ts51, ts53, and ts65, and two other mutants, ts7 and ts20, to regions on the vaccinia virus genome that encode the 147- and 22-kilodalton subunits of the viral DNA-dependent RNA polymerase, respectively. Plasmid and bacteriophage clones from the HindIII J region and the region spanning the HindIII J-H junction were used in marker rescue experiments to map the mutations. Sequence analysis of the region encoding the 22-kilodalton subunit in the wild-type, ts7, and ts20 viruses revealed a single base change in the mutants compared with that in the wild-type virus. The identification of these RNA polymerase mutants provides us with tools to understand transcription and its regulation in vaccinia virus.

Amino Acid Sequence↗
PubMed1990

Effects of an anti-alpha monoclonal antibody on interaction of Escherichia coli RNA polymerase with lac promoters.

The anti-alpha monoclonal antibody, mAb 126C6, has been used to investigate the role of the alpha subunit in transcription initiation. mAb 126C6 strongly inhibits cAMP-CRP-dependent abortive initiation with lac P+, partially inhibits abortive initiation with the lac L8UV5 promoter, and is without effect on the d(A-T)n-directed synthesis of r(A-U)n. DNase I footprinting shows that the preformed mAb 126C6-RNA polymerase complex does not bind to cAMP-CRP-lac P+; RNA polymerase specific protection is largely lost after incubation of the preformed RPo with mAb 126C6. Kinetic analysis of open complex formation by mAb 126C6-RNA polymerase with lac L8UV5 showed that changes in both the binding and the rate of isomerization account for the observed inhibition, with the isomerization step affected to a greater extent. Binding of cAMP-CRP to lac L8UV5 is RNA polymerase dependent. DNase I footprints show that as a consequence of mAb 126C6 binding of the preformed cAMP-CRP-lac L8UV5-RNA polymerase RPo, CRP dissociates from its site on the promoter. RNA polymerase protection of the promoter upstream from -41 is also lost. DNase I footprinting of mAb 126C6-RNA polymerase complexed with cAMP-CRP-lac P+ or -lac L8UV5 suggests that interactions between CRP and RNA polymerase are affected by binding of the anti-alpha mAb 126C6 to RNA polymerase. Protection methylation studies demonstrate that the formation of the mAb 126C6-RNA polymerase-lac L8UV5 open complex occurs at a slower rate and that nonoptimal contacts are established between mAb 126C6-RNA polymerase-lac L8UV5 promoter.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies, Monoclonal↗
PubMed1991

[Synthesis of RNA using T7 RNA polymerase and immobilized DNA in a stream type reactor].

The DNA ligase-induced assembly of synthetic oligodeoxyribonucleotides on polymer supports was used to obtain immobilized DNA, containing the T7 RNA polymerase promoter and coding for a 14-membered oligoribonucleotide. The obtained template can carry out RNA synthesis in a flowing-type reactor. Sepharose 4B and Toyopearl HW-55 were used as supports.

Base Sequence↗
PubMed2003

Donation of catalytic residues to RNA polymerase active center by transcription factor Gre.

During transcription elongation, RNA polymerase (RNAP) occasionally loses its grip on the growing RNA end and backtracks on the DNA template. Prokaryotic Gre factors rescue the backtracked ternary elongating complex through stimulation of an intrinsic endonuclease activity, which removes the disengaged 3' RNA segment. By using RNA-protein crosslinking in defined ternary elongating complexes, site-directed mutagenesis, discriminative biochemical assays, and docking of the two protein structures, we show that Gre acts by providing two carboxylate residues for coordination of catalytic Mg2+ ion in the RNAP active center. A similar mechanism is suggested for the functionally analogous eukaryotic SII factor. The results expand the general two-metal model of RNAP catalytic mechanism whereby one of the Mg2+ ions is permanently retained, whereas the other is recruited ad hoc by an auxiliary factor.

Amino Acid Sequence↗
PubMed1996

Factor-dependent release of nascent RNA by ternary complexes of vaccinia RNA polymerase.

Factor-dependent transcription termination during synthesis of vaccinia early mRNAs occurs at heterogeneous sites downstream of a UUUUUNU signal in the nascent transcript. The choice of termination site is flexible and is determined by a kinetic balance between nascent chain elongation and the transmission of the RNA signal to the polymerase. To eliminate ongoing elongation as a variable, we have established a system to study transcript release by purified ternary complexes halted at a defined template position 50-nucleotides 3' of the first U residue of the termination signal. Release of the nascent RNA depends on the vaccinia termination factor (VTF) and an ATP cofactor. Transcript release is blocked by BrUMP substitution within the termination signal of the nascent RNA. In these respects, the release reaction faithfully mimics the properties of the termination event. We demonstrate that ternary complexes are refractory to VTF-mediated transcript release when the first U of the UUUUUNU signal is situated 20 nucleotides from the growing point of the nascent chain. Ribonuclease footprinting of the arrested ternary complexes defines a nascent RNA binding site on the polymerase elongation complex that encompasses a 16-21 nucleotide RNA segment extending proximally from the 3' end of the chain. We surmise that access of VTF to the signal sequence is prevented when UUUUUNU is bound within the nascent RNA binding site. Hence, physical not kinetic constraints determine the minimal distance between the signal and potential sites of 3' end formation.

Base Sequence↗