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At least 937 records · Page 52Linked to original sources
PubMed1996

Template strand gap bypass is a general property of prokaryotic RNA polymerases: implications for elongation mechanisms.

It has previously been shown that T7 RNA polymerase is capable of bypassing gaps on the template strand ranging in size from 1 to 24 nucleotides. This as well as other observations suggested a role for the nontemplate strand during elongation. To establish the generality of this gap bypassing event, we have extended these studies to SP6 and Escherichia coli RNA polymerases. SP6 RNA polymerase bypasses template gaps from 1 to 19 nucleotides in size with various degrees of efficiency and produces runoff transcripts of decreasing length corresponding to increasing gap size. RNA sequence analysis of the resulting runoff transcripts revealed that SP6 RNA polymerase faithfully transcribed both parts of the template strand flanking the gapped region. Similar experiments were carried out with E. coli RNA polymerase (a multiple subunit enzyme) and indicate that it is also capable of gap bypass albeit with reduced efficiency compared to T7 and SP6 RNA polymerases. It appears that the ability to bypass gaps present on the DNA template strand is a general property of prokaryotic RNA polymerases. These results have implications with respect to the mechanism of elongation and the role of the nontemplate strand in transcription.

Base Sequence↗
PubMed1992

Nucleotide sequence analysis of the simian virus 41 gene encoding the large (L) protein and construction of a phylogenetic tree for the L proteins of paramyxoviruses.

The complete nucleotide sequence of the simian virus 41 (SV41) large (L) protein gene was determined. The L gene spanned 6883 nucleotides including a putative trailer RNA, and the L mRNA contained a single large open reading frame encoding a polypeptide of 2269 amino acids. Dot-matrix comparisons under stringent conditions identified domains highly conserved among paramyxoviruses. Domain 3 is the most highly conserved, and has been hypothesized to be the RNA polymerase active site. A phylogenetic tree was constructed from the sequences of the L proteins of seven paramyxoviruses. SV41 was most closely related to human parainfluenza virus type 2 (HPIV-2), and SV41, HPIV-2 and SV5 form a subgroup. The intergenic sequences at the nucleocapsid protein-phosphoprotein and haemagglutinin-neuraminidase-L protein gene junctions, and the 5' trailer sequence of SV41 were also determined, and it was shown that the first 13 nucleotides of the 5' trailer sequence are complementary to those of the 3' leader sequence. The intergenic, and gene-start and -end sequences of SV41, HPIV-2 and SV5 are shown.

Amino Acid Sequence↗
PubMed2005

Human telomerase RNA template sequence is a determinant of telomere repeat extension rate.

Human telomerase is a specialized reverse transcriptase that utilizes an integral RNA subunit to template the synthesis of telomeres. In the present study, we demonstrate that the human telomerase template sequence not only determines the composition, but also the rate of synthesis, of telomere repeats. Mutagenesis of the template sequence identified variants that reconstitute enzymes with repeat extension rates that were either faster or slower than wild type template. Changes in extension rate could not be attributed solely to altered heteroduplex melting, strongly suggesting that specific interactions between telomerase template, protein, and products contribute significantly in determining repeat extension rate. Furthermore, some substitutions that had no effect on extension rate led to striking increases in repeat processivity, indicating that processivity and extension rates can be regulated independently of each other. Our results suggest that telomerase RNA template sequence is a key determinant of the contribution of telomerase to telomere length regulation.

Base Sequence↗
PubMed1996

Transcriptional pausing of RNA polymerase in the presence of guanosine tetraphosphate depends on the promoter and gene sequence.

We have studied the response of the effector molecule guanosine 3',5'-bisdiphosphate (ppGpp) on RNA polymerase pausing during in vitro transcription elongation. Pausing was followed during single round extension of stalled ternary complexes excluding possible ppGpp effects on initiation. The ppGpp dependences of early pausing sites within different transcription systems controlled by promoters with known response to enhanced ppGpp levels in vivo were quantitatively characterized. Transcription of stable RNAs and mRNA genes were analyzed. In addition, the in vitro pausing behavior of two promoter variants directing the same sequence but differing in their in vivo ppGpp sensitivity were compared. In the presence of ppGpp we noted a slight general enhancement of specific pauses in all transcription systems. However, genes known to be under stringent or growth rate control in vivo revealed a notably stronger pausing enhancement. The sites of pausing are not changed by the presence of ppGpp but appear to be sequence-specific. The effect of ppGpp on the extent of pausing depends on the particular promoter and closely adjacent sequences that the RNA polymerase has passed during initiation. Pausing enhancement requires the presence of ppGpp during elongation but not during initiation. The results underline the importance of pausing for transcription regulation and offer a plausible explanation for inhibition of stable RNA expression under conditions of elevated concentrations of ppGpp.

Base Sequence↗
PubMed1997

The two RNA polymerases encoded by the nuclear and the plastid compartments transcribe distinct groups of genes in tobacco plastids.

The plastid genome in photosynthetic higher plants encodes subunits of an Escherichia coli-like RNA polymerase (PEP) which initiates transcription from E.coli sigma70-type promoters. We have previously established the existence of a second nuclear-encoded plastid RNA polymerase (NEP) in photosynthetic higher plants. We report here that many plastid genes and operons have at least one promoter each for PEP and NEP (Class II transcription unit). However, a subset of plastid genes, including photosystem I and II genes, are transcribed from PEP promoters only (Class I genes), while in some instances (e.g. accD) genes are transcribed exclusively by NEP (Class III genes). Sequence alignment identified a 10 nucleotide NEP promoter consensus around the transcription initiation site. Distinct NEP and PEP promoters reported here provide a general mechanism for group-specific gene expression through recognition by the two RNA polymerases.

Adenosine Triphosphatases↗
PubMed1996

In vitro activities of an N-terminal truncated form of XylR, a sigma 54-dependent transcriptional activator of Pseudomonas putida.

A truncated derivative of the XylR protein, which is able to constitutively activate the sigma 54-dependent Pu promoter of the TOL (toluene biodegradation) plasmid of Pseudomonas putida, has been purified to homogeneity and its various activities have been separately examined, in vitro. The truncated regulator XylR delta A was deleted of the signal reception N-terminal module present in wild-type XylR, but retained its central activation domain and the DNA binding segment, located at its C terminus. XylR delta A bound to the region -120 to -190 bp upstream of the transcription initiation site of the Pu promoter, where previous analyses have located the XylR target site. XylR delta A showed an intrinsic ATPase activity that was strongly stimulated by DNA containing the native upstream activation sequences of Pu. Both ATPase activity and ATP binding were abolished in mutant G268N in which the Walker A domain of the central module was altered. Mutant R453H lacked ATPase activity but retained the nucleotide-binding ability of the parental protein. XylR delta A was able to activate transcription in vitro with sigma 54-RNA polymerase alone, although its activity was enhanced up to 20-fold in the presence of the integration host factor protein. The requirements for activation of the Pu promoter in vitro are consistent with the view that DNA-facilitated oligomerization of the regulator for an enhanced ATPase activity is the critical event that precedes transcription initiation at sigma 54-dependent promoters. Furthermore, additional co-regulation elements seem to adjust promoter activity in vivo to the physiological status of the cells.

Adenosine Triphosphatases↗
PubMed1987

Two separable functional domains in the sigma-subunit of RNA polymerase in Bacillus subtilis?

The sigma-subunit of RNA polymerase is responsible for promoter recognition in prokaryotes [(1969) Nature 221, 43-46]. Alterations in the sigma-subunit are thought to be involved in controlling 'global' changes in gene expression, such as those involved in differentiation in the spore-forming bacterium Bacillus subtilis [(1981) Cell 25, 582-584]. Stragier et al. [(1985) FEBS Lett. 195, 3-11] have proposed that sigma-factors are composed of two domains: a C-terminal domain involved in promoter recognition and an N-terminal domain involved in interactions with RNA polymerase. We have sequenced another developmental gene from B. subtilis, spoIIIC, and the strong homology of its predicted product suggests that it too may be a sigma-factor. However, the spoIIIC product is small and lacks completely the conserved N-terminal domain of the sigma-subunits. I propose that the product of the spoIIIC gene may carry out the DNA-recognition functions of a sigma-factor but that it probably requires an auxiliary factor to interact with core RNA polymerase.

Amino Acid Sequence↗
PubMed1995

Nucleotide sequence of the Staphylococcus aureus RNA polymerase rpoB gene and comparison of its predicted amino acid sequence with those of other bacteria.

The complete nucleotide sequence of the rpoB gene which encodes the beta subunit of S. aureus RNA polymerase has been determined. The RpoB protein, consists of 1182 amino acids and has a novel initiation codon UUG which initiates protein synthesis with methionine. There is a very strong Shine-Dalgarno complementarity and the -10 and -35 promoter hexameric sequences are TAATAT and CCGTTT, respectively. A rho-dependent termination site, CAATCAA, is present which overlaps the -35 promoter sequence of the adjacent rpoC gene a feature which may have a role in the co-ordinate expression of the two genes. A strong homology and conserved regions were found to exist over the predicted amino acid sequences coding for S. aureus rpoB and the equivalent proteins in Escherichia coli, Pseudomonas putida, Salmonella typhimurium, Chlamydia trachomatis, cyanobacterium Anabaena sp. strain PCC 7120.

Amino Acid Sequence↗
PubMed2000

The alternative sigma factor sigma(28) of the extreme thermophile Aquifex aeolicus restores motility to an Escherichia coli fliA mutant.

Sigma factor sigma(28) (sigma(F), FliA, SigD) directs RNA polymerase to transcribe the genes required for flagellar biosynthesis and chemotaxis in many bacteria, including Bacillus subtilis, Legionella pneumophila, Salmonella typhimurium, Escherichia coli, Yersinia enterolytica, Treponema maltophilum and Pseudomonas aeruginosa. Remarkably the fliA gene from the extreme thermophile Aquifex aeolicus restored motility to the E. coli mutant at relatively low temperature, albeit partially. This clearly demonstrates that A. aeolicus sigma(28) is able to direct RNA polymerase to E. coli sigma(28)-dependent promoters and take part in the complex interactions required to support transcription of the flagellar apparatus in vivo. The ability of A. aeolicus sigma(28) to function with mesophilic components shows that critical functional interactions made by these sigma factors are well conserved, and are not dependent upon high temperature. We over-produced and purified the sigma(28) protein and demonstrated binding to E. coli core RNA polymerase in vitro. In common with SigD from B. subtilis, but unlike most sigma factors, A. aeolicus sigma(28) showed DNA binding activity in vitro but there was no evidence of sequence specificity. We note that A. aeolicus sigma(28) is a good candidate for structural studies.

Bacteria↗
PubMed2000

An NF-IL6 3'UTR RNA-specific Binding Protein in E. coli Purification and Partial Protein Sequencing.

protein was found in E.coli which can specifically bind to NF-IL6 mRNA 3'UTR. After a series of purification steps, the RNA-specific binding protein was directly sequenced on the Porton LF3200 Protein Sequencer. A sequence of 10 amino acids of the purified NF-IL6 3'UTR binding protein was obtained, namely, Ala-Thr-Arg-Ile-Glu-Phe-His-Gly-Cyss(?)-Gly. A BLAST search with the 10 amino acids against NCBI database failed to identify any protein with identical sequence. The significance of identifying an eukaryotic mRNA binding protein in E.coli is discussed.

Journal Article↗
PubMed1999

Highly efficient endonucleolytic cleavage of RNA by a Cys(2)His(2) zinc-finger peptide.

We have identified a 30-aa peptide that efficiently cleaves single-stranded RNA. The peptide sequence corresponds to a single zinc finger of the human male-associated ZFY protein; a transcription factor belonging to the Cys(2)His(2) family of zinc-finger proteins. RNA cleavage was observed only in the absence of zinc. Coordination with zinc resulted in complete loss of ribonuclease activity. The ribonuclease active structure was determined to be a homodimeric form of the peptide. Dimerization of the peptide occurred through a single intermolecular disulfide between two of the four cystines. The observed hydrolytic activity was single-stranded RNA-specific. Single-stranded DNA, double-stranded RNA and DNA, and 2'-methoxy-modified sequences were not degraded by the peptide. The peptide specifically cleaved pyrimidines within single-stranded RNA and the dinucleotide sequence 5'-pyr-A-3' was preferred. The RNA cleavage products consisted of a 3' phosphate and 5' hydroxyl. The initial rates of cleavage (V(0)) observed for the finger peptide were comparable to rates observed for human ribonucleases, and the catalytic rate (K(cat)) was comparable to rates observed for the group II intron rybozymes. The pH profile exhibited by the peptide is characteristic of general acid-base catalytic mechanisms observed with other ribonucleases. These observations raise interesting questions about the potential biological roles of zinc-finger proteins.

Amino Acid Sequence↗
PubMed1993

A second nonstructural protein functions in the regulation of alphavirus negative-strand RNA synthesis.

Previous studies (D.L. Sawicki, D. B. Barkhimer, S. G. Sawicki, C. M. Rice, and S. Schlesinger, Virology 174:43-52, 1990) identified a temperature-sensitive (ts) defect in Sindbis virus nonstructural protein 4 (nsP4) that reactivated negative-strand synthesis after its normal cessation at the end of the early phase of replication. We now report identification of two different ts alterations in nsP2 of Ala-517 to Thr in ts17 or Asn-700 to Lys in ts133 that also reactivated negative-strand synthesis. These same mutations caused severely reduced protease processing by nsP2 and recognition of the internal promoter for subgenomic mRNA synthesis and were responsible for the conditional lethality and RNA negativity of these mutants. Reactivation of negative-strand synthesis by mutations in nsP2 resembled that in nsP4: it was a reversible property of stable replication complexes and did not require continuation of viral protein synthesis. Recombinant viruses expressing both mutant nsP2 and nsP4 reactivated negative-strand synthesis more efficiently than did either mutant protein alone, consistent with the hypothesis that both nsP2 and nsP4 participate in template recognition. We propose that these alterations cause nsP2 and nsP4 to switch from their normal preference to recognize negative strands as templates to recognize positive strands and thereby mimic the initial formation of a replication complex.

Alphavirus↗
PubMed1994

Role of the sigma 70 subunit of Escherichia coli RNA polymerase in transcription activation.

The role of the sigma 70 subunit of Escherichia coli RNA polymerase in transcription activation by positive transcription factors was investigated. For this purpose, we constructed a nested set of E. coli rpoD deletion mutants generating carboxy-terminally truncated sigma 70 subunits of RNA polymerase in a high-expression plasmid. The purified mutant sigma 70 subunits were reconstituted into holoenzymes and examined in vitro for their promoter selectivity. As expected, since the -35 recognition helix of sigma 70 was deleted in all cases, the mutant enzymes were unable to initiate at factor-independent promoters, except for the special case of perfect "extended minus 10" promoters, at which the need for -35 sequence recognition by RNA polymerase is replaced by recognition of additional base-pairs in the -10 region. However, two factor-dependent promoters, PhoB-dependent PpstS and cAMP receptor protein (CRP)-dependent P1gal, could be activated for transcription by different subsets of the mutant holoenzymes, although these promoters do not contain the perfect extended -10 sequences. These results establish that -35 DNA recognition by sigma 70 is not essential for these cases. Presumably it is replaced by protein-protein contacts between RNA polymerase and the activator, which in both cases is bound to the DNA in a position overlapping the -35 region. Further, the detailed results support the view that the contact and/or activation sites for these two factors may lie on the sigma 70 subunit, within a "contact site II", which extends at least from conserved region 3.2 to the upstream end of region 4.2. Moreover, as in the case of contact site I on the alpha subunit, it appears that contact site II contains various different subsites for interaction with specific class II activators, and that PhoB and CRP require distinct subsites.

Base Sequence↗
PubMed2006

RNA-mediated destabilization of the sigma(70) region 4/beta flap interaction facilitates engagement of RNA polymerase by the Q antiterminator.

The bacterial RNA polymerase (RNAP) holoenzyme consists of a catalytic core enzyme (alpha(2)betabeta'omega) complexed with a sigma factor that is required for promoter-specific transcription initiation. During early elongation, the stability of interactions between sigma(70) (the primary sigma factor in Escherichia coli) and core decreases due to an ordered displacement of segments of sigma(70) from core triggered by growth of the nascent RNA. Here we demonstrate that the nascent RNA-mediated destabilization of an interaction between sigma(70) region 4 and the flap domain of the beta subunit is required for the bacteriophage lambda Q antiterminator protein to contact holoenzyme during early elongation. We demonstrate further that the requirement for nascent RNA in the process by which Q engages RNAP can be bypassed if sigma(70) region 4 is removed. Our findings illustrate how a regulator can exploit the nascent RNA-mediated reconfiguration of the holoenzyme to gain access to the enzyme during early elongation.

Base Sequence↗
PubMed1992

Transcriptional commitment of mitochondrial RNA polymerase from Saccharomyces cerevisiae.

The transcriptional commitment of mitochondrial RNA (mtRNA) polymerase and the conditions required for the formation of a stable ternary complex have been determined by in vitro transcription study. Four different transcription complexes were made in vitro by incubating purified mtRNA polymerase, cloned synthetic mitochondrial promoters and selective ribonucleotides. The responses of these complexes to heparin, an inhibitor of unbound mtRNA polymerase, have been examined to determine their involvement in transcription. This study leads to the following observations. (1) Under normal reaction conditions, 40 nM-heparin completely inhibited mitochondrial transcription. (2) A preinitiation mitochondrial DNA-RNA polymerase complex (complex 0) showed partial resistance to heparin (approximately 25% resistant to 40 nm-heparin) when heparin and ribonucleoside triphosphates (rNTPs) were added together to the preformed complex. This complex was rapidly inactivated when preincubated with heparin before the addition of rNTPs. (3) The early initiation (complexes 2 and 4) containing DNA template, RNA polymerase and a short RNA product showed more resistance (approx. 40 to 50%) to 40 nM-heparin but destabilized upon further incubation with heparin before addition of the rest of the rNTPs. (4) After generation of ten or more phosphodiester bonds (complex 11), the early transcription complex is converted into a stable initiation complex, leading to the polymerase consignment to elongation. On the basis of stability and heparin sensitivity, three initial steps of mitochondrial transcription have been defined: polymerase-promoter interaction, initiation, and the transition from initiation to elongation. The formation of preinitiation complex is the rate-limiting step t 1/2 approx. 50 s), whereas the initiation and elongation reactions are very fast processes (t 1/2 greater than 5 s) in mitochondrial transcription.

Base Sequence↗
PubMed2006

The role of the largest RNA polymerase subunit lid element in preventing the formation of extended RNA-DNA hybrid.

Analysis of multi-subunit RNA polymerase (RNAP) structures revealed several distinct elements that may perform partial functions of the enzyme. One such element, the "lid", is formed by an evolutionarily conserved segment of the RNAP largest subunit (beta' in bacterial RNAP). The beta' lid contacts the nascent RNA at the upstream edge of the RNA-DNA hybrid, where the RNA gets separated from the DNA template-strand and double-stranded upstream DNA is formed. To test the beta' lid functions, we generated bacterial RNAP lacking the lid and studied the mutant enzyme's properties in vitro. Our results demonstrate that removal of the lid has minimal consequences on transcription elongation from double-stranded DNA. On single-stranded DNA, the mutant RNAP generates full-sized transcripts that remain annealed to the DNA throughout their length. In contrast, the wild-type enzyme produces short, 18-22 nucleotide transcripts that remain part of the transcription complex but cannot be further elongated. The cessation of transcription is apparently triggered by a clash between the lid and the nascent RNA 5' end. The results show that the lid's function is redundant in the presence of the non-template DNA strand, which alone can control the proper geometry of nucleic acids at the upstream edge of the transcription complex. Structural considerations suggest that in the absence of the non-template strand and the lid, a new channel opens within the RNAP molecule that allows continuous DNA-RNA hybrid to exit RNAP.

Amino Acid Sequence↗
PubMed1994

Molecular epidemiology of enterovirus outbreaks in Canada during 1991-1992: identification of echovirus 30 and coxsackievirus B1 strains by amplicon sequencing.

The relatedness of enteroviral isolates associated with two recent outbreaks in Canada was assessed using direct sequencing of amplicons derived from a large portion of the 5' nontranslated region (NTR) of the viral genome. The amplicons of 60 echovirus 30 isolates originating from seven different provinces in 1991 were found to share 99% or greater sequence identity. Recent coxsackievirus B1 isolates characterised in the same manner were identical to each other. When the 5' NTR sequence of these isolates was compared to prototype strains a difference of 11-15% in nucleotide composition was observed. These results indicate that the variability of nucleotide sequence found in 5' NTRs can be utilized to identify rapidly enteroviral strains associated with particular outbreaks and distinguish them from other strains and serotypes.

Animals↗