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At least 919 records · Page 51Linked to original sources
PubMed1988

Isolation of a Bacillus thuringiensis RNA polymerase capable of transcribing crystal protein genes.

We report the isolation of an RNA polymerase from sporulating cells of Bacillus thuringiensis subsp. kurstaki HD-1-Dipel that directs transcription from the promoter region of an insecticidal crystal protein gene. The core components of this RNA polymerase are associated with a polypeptide that has an apparent mass of 35 kDa. Neither RNA polymerase holoenzyme isolated from vegetative B. thuringiensis, nor the core derived from this enzyme, is capable of transcribing from the crystal protein gene promoter region; the addition of gel-purified 35-kDa polypeptide to the core reconstitutes the specific transcribing capability. The reconstituted enzyme does not direct transcription from the promoters for the ctc or spoVG genes of Bacillus subtilis; however, this form of RNA polymerase does direct transcription from a promoter for the 27-kDa crystal protein of B. thuringiensis subsp. israelensis and from a promoter for a 29-kDa polypeptide present in cuboidal crystals of B. thuringiensis subsp. kurstaki HD-1. We propose a tentative consensus sequence based on the alignment of the three B. thuringiensis promoters. This consensus sequence is different from consensus sequences reported for promoters recognized by enzymes containing other sigma subunits, suggesting that the 35-kDa polypeptide is an unusual sigma subunit.

Bacillus thuringiensis↗
PubMed1998

Conserved core structure in the internal transcribed spacer 1 of the Schizosaccharomyces pombe precursor ribosomal RNA.

The structure of the internal transcribed spacer 1 (ITS1) in Schizosaccharomyces pombe was examined with respect to phylogenetically conserved features in yeasts as well as the binding of transacting factors that potentially play a role in ribosomal maturation. Computer analyses and probes for nuclease protection indicate a compact, more highly organized structure than previously proposed in Saccharomyces cerevisiae, with distinct structural features which can be recognized in S. cerevisiae. These include a central extended hairpin structure as well as smaller hairpins immediately adjacent to the maturing termini. Comparisons with ITS sequences in more diverse organisms indicate that the same features also can be recognized. This is especially clear in organisms which contain very short sequences in which the putative structures are much less ambiguous. Again nuclease protection analyses in one of these, Verticillium albo-atrum, confirm a central hairpin with additional hairpins linked to the maturing termini. Protein binding and gel retardation studies with the S. pombe ITS1 further indicate that, as observed in the 3' external transcripted spacer (ETS) region, the extended hairpin is not only the site of intermediate RNA cleavage during rRNA processing, but also a site for specific interactions with one or more soluble factors. Taken together with other analyses on transcribed spacer regions, the present data provide evidence that the spacer regions act not only to organize the maturing terminal sequences but also may serve to organize specific soluble factors, possibly acting in a manner which is analogous with that of the free small nucleolar ribonucleo protein particles (snoRNPs).

Animals↗
PubMed2002

The molecular biology of West Nile Virus: a new invader of the western hemisphere.

West Nile virus (WNV) is a mosquito-borne flavivirus that primarily infects birds but occasionally also infects humans and horses. In recent years, the frequency of WNV outbreaks in humans has increased, and these outbreaks have been associated with a higher incidence of severe disease. In 1999, the geographical distribution of WNV expanded to the Western hemisphere. WNV has a positive strand RNA genome of about 11 kb that encodes a single polyprotein. WNV replicates in the cytoplasm of infected cells. Although there are still many questions to be answered, a large body of data on the molecular biology of WNV and other flaviviruses has already been obtained. Aspects of virion structure, the viral replication cycle, viral protein function, genome structure, conserved viral elements, host factors, virus-host interactions, and vaccines are discussed in this review.

Animals↗
PubMed1995

Molecular characterisation of small round structured viruses associated with gastroenteritis in South Africa.

The application of the reverse transcriptase polymerase chain reaction (RT-PCR) has enabled several morphologically and physically similar small round structured viruses (SRSVs), including the prototype Norwalk virus (NV), to be classified within the Caliciviridae. This technique, using primers directed to the RNA-dependent RNA polymerase region within the ORF1 of NV, was used to characterise SRSVs associated with epidemic gastroenteritis in adults and sporadic paediatric gastroenteritis in South Africa. Genomic variation was investigated by sequence analysis of the amplified 209bp cDNA region from six isolates and comparison with other characterised SRSVs including NV. Antigenic variation was investigated by the use of the recombinant enzyme immunoassay described recently for the detection of Snow Mountain agent-like antigen in stool specimens. Two distinct antigenic groups were evident with NV-like viruses associated with adult gastroenteritis, and Mexico viruslike viruses associated with paediatric gastroenteritis. Viral isolates from two of the outbreaks of adult gastroenteritis showed a high degree of nucleotide sequence identity with NV, i.e., 84% and 98%, respectively, whereas the paediatric isolates showed 92-95% sequence similarity with the Snow Mountain-like virus, MxV. These data show concordance between antigenic and genomic analyses.

Adult↗
PubMed2002

NusA-stimulated RNA polymerase pausing and termination participates in the Bacillus subtilis trp operon attenuation mechanism invitro.

The trp RNA-binding attenuation protein (TRAP) regulates expression of the Bacillus subtilis trpEDCFBA operon by transcription attenuation and translation control mechanisms. Both mechanisms require the binding of tryptophan-activated TRAP to the 11 (G/U)AG-repeat segment in the trp leader transcript. To promote termination, TRAP must bind to the nascent RNA before the antiterminator structure forms. Because only 20 nucleotides separate the TRAP-binding site from the 3' end of the antiterminator, TRAP has a short time frame to control this regulatory decision. Synchronization of factor binding and/or RNA folding with the RNA polymerase position is a major challenge in all attenuation mechanisms. Because RNA polymerase pausing allows this synchronization in many attenuation mechanisms, we performed experiments in vitro to determine whether pausing participates in the B. subtilis trp attenuation mechanism. We identified two NusA-stimulated pause sites in the trp leader region. Formation of pause hairpins participates in pausing at both positions. The first pause occurred at the nucleotide just preceding the critical overlap between the alternative antiterminator and terminator structures. TRAP binding to transcripts containing preexisting pause complexes releases RNA polymerase, suggesting that pausing provides additional time for TRAP to bind and promote termination. The second pause is downstream from the trp leader termination point, raising the possibility that this pause event participates in the trpE translation control mechanism. NusA also increases the efficiency of termination in the trp leader region and shifts termination one nucleotide upstream. Finally, NusA-stimulated termination is cooperative, suggesting that binding of multiple NusA molecules influences termination.

Bacillus subtilis↗
PubMed1991

RNA polymerases from Pseudomonas aeruginosa and Pseudomonas syringae respond to Escherichia coli activator proteins.

The activities of RNA polymerases (RNAPs) from Pseudomonas aeruginosa and Pseudomonas syringae were compared with that of Escherichia coli RNAP. All three enzymes are able to initiate transcription at the trpBA promoter of P. aeruginosa and at the coliphage lambda promoters, pRM and pRE, in response to heterospecific activators (TrpI protein, repressor, and cII protein, respectively). However, both Pseudomonas polymerases have less stringent requirements for promoter recognition in the absence of activators than does E. coli RNAP.

Bacterial Proteins↗
PubMed1986

Terminators of transcription with RNA polymerase from Escherichia coli: what they look like and how to find them.

We present here a compilation of prokaryotic transcription terminator sequences (ref. 1-152). The compilation includes 49 independent terminators, 52 speculated independent terminators, 27 sites shown to function in vivo, and some 20 proven or speculated rho-dependent terminators. In addition to the well-known features of independent terminators (dyad symmetry and T-run), two consensus are found: CGGG(C/G) upstream and TCTG downstream of the termination point. A subset of the collection of sequence has been used to construct a computer algorithm to locate independent terminators by sequence analysis.

Algorithms↗
PubMed2001

Genetic classification of "Sapporo-like viruses".

"Sapporo-like viruses" (SLVs) and "Norwalk-like viruses" (NLVs) are an important cause of acute gastroenteritis in humans. While NLVs have been genetically classified into three major genetic groups consisting of 17 genetic subgroups, a classification of SLVs into comparable genetic groups remains to be determined. In an attempt to classify both SLVs and NLVs uniformly, the sequences of 2 SLV strains newly detected from French infants were analysed together with the published sequences of 9 SLV and 19 NLV strains. Distance and phylogenetic analyses were conducted on the sequences of the capsid gene, RNA polymerase gene, 3' open reading frame (3'ORF), ORF overlapping the capsid gene, and 3' untranslated region (3'UTR). The histogram showing frequency distribution of pairwise distances and the topology of the phylogenetic tree demonstrated that SLVs and NLVs could be classified uniformly on the basis of the entire capsid sequences and that the 11 SLV strains could be genetically classified into 3 major genetic groups, genogroups I, II and III, comprised of 5 genetic subgroups. The differentiation of the 11 SLV strains into these genetic groups was also maintained in the 4 remaining genome regions, while the sequences at the junction between the RNA polymerase and capsid genes were shown to be genogroup-specific.

3' Untranslated Regions↗
PubMed1982

Function of transcription termination factor rho in a model transcription system using synthetic deoxyribonucleic acid as template.

The function of a transcription termination factor, rho, has been studied by using several synthetic DNAs with simple repetitive base sequences as templates for transcription. rho actually exhibits various effects on transcription depending on the base sequence of the template: (1) rho terminates poly(A) synthesis with poly(dA) x poly(dT), poly(dT), or oligo(dT), leading to release of RNA from RNA polymerase. rho also inhibits the synthesis of other homoribopolymers such as poly(U) directed by poly(dA) x poly(dT) and poly(C) and poly(I) directed by poly(dG) x poly(dC), presumably by a similar mechanism. (2) rho inhibits the synthesis of another homoribopolymer, poly(G), directed by poly(dG) x poly(dC) at the step of initiation rather than propagation of transcription. (3) rho stimulates rather than inhibits the synthesis of poly(A-C) and poly(G-U) directed by poly[d(A-C)] x poly[d(G-T)], presumably by enhancing the dissociation of transcription complexes. (4) rho has no influence on the synthesis of poly(A-U) and poly(G-C) directed by poly[d(A-T)] and poly[d(G-C)], respectively. In the first case, but not otherwise, the effect of rho is coupled with its RNA-dependent nucleosidetriphosphate phosphohydrolase activity, as is rho-mediated transcription termination on natural templates. The implication of these results is discussed in reference to the current view that rho acts on transcription complexes that have ceased elongation and causes release of RNA in an energy-requiring reaction.

Base Sequence↗
PubMed1984

Both inverted repeat sequences located at the ends of IS1 provide promoter functions.

Escherichia coli RNA polymerase was found to bind specifically to restriction fragments containing either end of IS1. DNase I footprint analyses indicate that RNA polymerase protects approximately 70 base-pairs at each end of IS1, including the left or right terminal inverted repeat sequences in IS1 (termed insL or insR, respectively) as well as some non-IS1 sequence directly adjacent to each end of IS1. Analysis of transcripts from the left terminal region of IS1 shows that the insL sequence contains a promoter (named insPL), and that RNA synthesis initiates apparently at one in a stretch of five adenylate residues within insL and continues toward the interior region of IS1. Interestingly, most of the resulting transcripts contain polyuridylate residues (more than 5 U residues) at their 5'-ends. Analysis of transcripts from the right terminal region of IS1 indicates that the insR sequence also contains a promoter (named insPR). RNA synthesis initiates specifically at an adenylate residue within insR and continues toward the interior region of IS1, i.e. in the opposite direction to RNA synthesis initiating at insPL, which is present at the other end of IS1. We propose that insPL is used to make the messenger RNA for the IS1-encoded genes insA and insB, while insPR might be used to synthesize an anti-mRNA and thereby negatively regulate insPL.

Base Sequence↗
PubMed1988

PL of coliphage lambda: an alternative solution for an efficient promoter.

Promoter PL of coliphage lambda is highly active in vivo although it is recognized 15-30 times less efficiently by RNA polymerase when compared with promoters of similar strength. Moreover, it differs significantly from the consensus sequence for Escherichia coli promoters. Sequence variants of PL which are more homologous to consensus promoters bind RNA polymerase with increased efficiency. They are nevertheless significantly reduced in their in vivo strength. High activity can be restored by a downstream sequence of a typical consensus-like promoter. Evidently, such elements are required for the efficient release of a stably bound RNA polymerase into a transcriptional elongation complex. We propose that the functional programme encoded in a promoter sequence can be optimized in alternative ways.

Bacteriophage lambda↗
PubMed1997

Changing the mechanism of transcriptional activation by phage lambda repressor.

The first steps of transcription initiation include binding of RNA polymerase to a promoter to form an inactive, unstable, closed complex (described by an equilibrium constant, K(B)) and isomerization of the closed complex to an active, stable, open complex (described by a forward rate constant, k(f)). lambda cI protein activates the PRM promoter by specifically increasing k(f). A positive control mutant, cI-pc2, is defective for activation because it fails to raise k(f). An Arg to His change in the sigma70 subunit of RNA polymerase was previously obtained as an allele-specific suppressor of cI-pc2. To elucidate how the mutant polymerase restores the activation function of the mutant activator, abortive initiation assays were performed, using purified cI proteins and RNA polymerase holoenzymes. The change in sigma does not significantly alter K(B) or k(f) in the absence of cI protein. As expected, cI-pc2 activates the mutant polymerase in the same way that wild-type cI activates the wild-type polymerase, by increasing k(f). An unexpected and novel finding is that the wild-type activator stimulates the mutant polymerase, but not wild-type polymerase, by increasing K(B).

Bacteriophage lambda↗
PubMed1989

Structure and stability of mRNA synthesized by vaccinia virus-encoded bacteriophage T7 RNA polymerase in mammalian cells. Importance of the 5' untranslated leader.

We have analyzed the structure and stability of RNA synthesized by bacteriophage T7 RNA polymerase in mammalian cells. The T7 polymerase, expressed by a recombinant vaccinia virus, transcribed the Escherichia coli lacZ gene flanked by T7 promoter and terminator signals. The lacZ gene cassette was introduced into infected cells within either a transfected plasmid or a second recombinant vaccinia virus. The T7-lacZ transcripts, which had a half-life of approximately 75 minutes, represented approximately 30% of total cytoplasmic RNA after a 24 hour period. The latter estimation indicated a disparity between the levels of lacZ RNA and beta-galactosidase synthesis. Analysis of the T7 transcripts indicated that they were initiated correctly but that only 5 to 10% contained terminal cap structures, providing an explanation for the low translatability of the RNA. Since the 5' end of the T7 transcripts can form a stem-loop structure that might interfere with capping by vaccinia virus RNA guanylyltransferase, as well as ribosome binding and scanning, a similar vector lacking such sequences was constructed. In vitro experiments demonstrated that T7 RNA polymerase transcribed both templates with similar efficiency and that the RNA lacking the potential to form the stem-loop was capped more rapidly by the purified vaccinia virus enzyme. Nevertheless, when the stem-loop was removed, beta-galactosidase was not expressed in infected cells; moreover, no T7 transcripts could be detected, suggesting that the RNA was not made or more likely was degraded during or shortly after synthesis. There is previous evidence that vaccinia virus RNA guanylyltransferase is associated with the viral transcription complex, thereby allowing RNA synthesis and capping to occur concurrently. We suggest that a lack of coupling between the vaccinia viral RNA guanylyltransferase and bacteriophage T7 RNA polymerase delays capping of T7 transcripts and that, under these conditions, the 5'-terminal double-stranded stem is required to stabilize the nascent RNA against degradation. Although deletion of the 3' palindromic sequence specifying T7 transcriptional termination from the expression cassette resulted in RNA of more heterogeneous lengths, neither the apparent turnover rate nor translation of the RNAs was diminished appreciably.

Animals↗
PubMed1994

Synthesis of biologically active influenza virus core proteins using a vaccinia virus-T7 RNA polymerase expression system.

An in vivo system in which expression of a synthetic influenza virus-like chloramphenicol acetyltransferase (CAT) RNA is driven by influenza virus proteins synthesized from cloned cDNAs has been developed. Expression of the four influenza virus core proteins (nucleoprotein, PA, PB1 and PB2) was performed by transfection of four pGEM recombinant plasmids, each containing one of the four viral genes, into cell cultures previously infected with a vaccinia virus recombinant encoding the T7 RNA polymerase (vTF7-3). When a naked negative-sense influenza virus-like CAT RNA was transfected into cells expressing the four influenza virus proteins, CAT activity was detected in the cell extracts, demonstrating that the expressed proteins had RNA-synthesizing activity. In this system, CAT RNA templates containing additional nucleotides at the 3' end were also expressed, resulting in CAT activity. This showed that the influenza virus polymerase can recognize its promoter when located internally on an RNA template. In influenza virus-infected cells however, CAT activity was detected only when the CAT RNA contained the viral promoter at the exact 3' end and was transfected as in vitro assembled ribonucleoprotein. These results are discussed in terms of the different requirements of the two helper systems for expression of an exogenously added RNA.

Animals↗
PubMed2004

Real time monitoring of the interaction of T7 RNA polymerase with azobenzene-tethered T7 promoter by biosensor.

We have already reported that transcription reaction by phage T7 or SP6 RNA polymerase (RNAP) can be reversibly photo-regulated with azobenzene-tethered promoter. Transcription reaction proceeded faster by UV irradiation than visible light irradiation. In the present study, binding of T7 RNAP to its azobenzene-tethered promoter was directly monitored by use of affinity biosensor. When azobenzene-tethered T7 promoter was immobilized on the sensor surface, response based on the binding of RNAP increased by UV light irradiation rather than by visible light irradiation. Thus, photo-regulation of transcription reaction by azobenzene-tethered promoter was attributed to the change of binding property of RNAP to the promoter by trans-cis isomerization of azobenzene.

Azo Compounds↗
PubMed1994

A baculovirus gene involved in late gene expression predicts a large polypeptide with a conserved motif of RNA polymerases.

We have identified and sequenced a novel baculovirus gene, late expression factor eight gene (lef-8), of Autographa californica nuclear polyhedrosis virus that is necessary for efficient expression from late and very late virus gene promoters in a transient expression assay. The predicted gene product, LEF-8, has a molecular mass of 102 kDa and contains a conserved sequence motif, GXKX4HGQ/NKG, found in DNA-directed RNA polymerases throughout the animal, plant, and microbial kingdoms.

Amino Acid Sequence↗