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PubMed2004

Affinity purification of the tobacco plastid RNA polymerase and in vitro reconstitution of the holoenzyme.

We affinity-purified the tobacco plastid-encoded plastid RNA polymerase (PEP) complex by the alpha subunit containing a C-terminal 12 x histidine tag using heparin and Ni(2+) chromatography. The composition of the complex was determined by mass spectrometry after separating the proteins of the >900 kDa complex in blue native and SDS polyacrylamide gels. The purified PEP contained the core alpha, beta, beta', beta" subunits and five major associated proteins of unknown function, but lacked sigma factors required for promoter recognition. The holoenzyme efficiently recognized a plastid psbA promoter when it was reconstituted from the purified PEP and recombinant plastid sigma factors. Reconstitution of a plastid holoenzyme with individual sigma factors will facilitate identification of sigma factor-specific promoter elements.

Base Sequence↗

PubMed1992

Alteration of the curved helical structure located in the upstream region of the beta-lactamase promoter of plasmid pUC19 and its effect on transcription.

The region preceding the beta-lactamase promoter of Escherichia coli plasmid pUC19 has a curved DNA (bent DNA) structure. The center of the curvature was revealed to exist around nucleotide position 2580 of the plasmid, which is just beside RNA polymerase binding region. It was indicated that the identified region is curved even at 60 degrees C. The gross geometry of the curvature was altered by inserting synthetic double-stranded oligonucleotides between positions 2585 and 2586. Effect of the alteration on strength of the promoter was not detected in vitro. However, in vivo analyses showed that the promoter strength is apparently dependent, in part, on the gross geometry of the curvature. Insertions of 4 and 16 bp, both of which altered the gross geometry of the curvature greatly, caused considerable reductions of in vivo level of beta-lactamase mRNA. In vivo, overall three-dimensional structure of the region covering the promoter and the curvature seems to play some significant role in transcription of the gene.

Base Sequence↗

PubMed1985

Analysis of a sequence region of 5S RNA from E. coli cross-linked in situ to the ribosomal protein L25.

70S ribosomes from E. coli were chemically cross-linked under conditions of in vitro protein biosynthesis. The ribosomal RNAs were extracted from reacted ribosomes and separated on sucrose gradients. The 5S RNA was shown to contain the ribosomal protein L25 covalently bound. After total RNase T1 hydrolysis of the covalent RNA-protein complex several high molecular weight RNA fragments were obtained and identified by sequencing. One fragment, sequence region U103 to U120, was shown to be directly linked to the protein first by protein specific staining of the particular fragment and second by phosphor cellulose chromatography of the covalent RNA-protein complex. The other two fragments, U89 to G106 and A34 to G51, could not be shown to be directly linked to L25 but were only formed under cross-linking conditions. While the fragment U89 to G106 may be protected from RNase T1 digestion because of a strong interaction with the covalent RNA-protein complex, the formation of the fragment A34 to G51 is very likely the result of a double monovalent modification of two neighbouring guanosines in the 5S RNA. The RNA sequences U103 to U120 established to be in direct contact to the protein L25 within the ribosome falls into the sequence region previously proposed as L25 binding site from studies with isolated 5S RNA-protein complexes.

Electrophoresis, Polyacrylamide Gel↗

PubMed2001

Restructuring of an RNA polymerase holoenzyme elongation complex by lambdoid phage Q proteins.

The structure of an intermediate in the initiation to elongation transition of Escherichia coli RNA polymerase has been visualized through region-specific DNA cleavage by the hydroxyl radical reagent FeBABE. FeBABE was tethered to specific sites of the final sigma(70) subunit and incorporated into two specialized paused elongation complexes that obligatorily retain the final sigma(70) initiation subunit and are targets for modification by lambdoid phage late gene antiterminators. The FeBABE cleavage pattern reveals structures similar to open complex, except for notable changes to region 3 of final sigma(70) that might reflect the presence of stably bound transcript. Binding of the antiterminator protein Q displaces the reactivity of FeBABE conjugated to region 4 of final sigma(70), suggesting that final sigma(70) subunit rearrangement is a step in conversion of RNAP to the antiterminating form.

Bacteriophage lambda↗

PubMed1992

Over-production of 5-enolpyruvylshikimate-3-phosphate synthase in Escherichia coli: use of the T7 promoter.

5-Enolpyruvylshikimate-3-phosphate (EPSP) synthase, the product of the Escherichia coli aroA gene, has been overproduced in E. coli BL21(lambda DE3) under the control of the T7 gene 10 promoter and ribosome binding site, to a level of approximately 50% of total cell protein. EPSP synthase is the primary target of the post-emergence herbicide, glyphosate, commonly known as Roundup. A simple two step purification is described, which results in 99% pure homogeneous protein (as determined by PAGE). The integrity of the protein has been compared with previously characterized protein from E. coli AB2829(pKD501) by determination of its kinetic parameters, N-terminal protein and DNA sequences, amino acid analysis and 13C-NMR spectroscopy. This new overproducing strain readily provides the gram quantities of highly pure protein required for NMR studies of the active site and the development of novel time-resolved solid-state NMR techniques currently underway in this laboratory.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗

PubMed1999

Natural selection of the Pol gene of bovine immunodeficiency virus.

Genetic variability is a salient feature of lentiviruses, contributing to the pathogenesis of these viruses by enabling them to persist in the host and to resist anti-retroviral treatment. Bovine immunodeficiency virus (BIV), a lentivirus of unknown pathology, infects cattle in the United States and worldwide. Genetic diversity of BIV that is associated with naturally infected cattle is not well studied. We examined the genetic diversity and natural selection of a segment of the BIV pol gene amplified from the leukocyte DNA of naturally infected cattle. A portion of the reverse transcriptase domain (183 bp) of the pol region was targeted for amplification by PCR. PCR products were sequenced directly and aligned. When compared to the sequences of BIV R29-127, a molecular clone of the original BIV R29 isolate, all isolates were greater than 91% identical in nucleotide sequences and 77% identical in amino acid sequences. Pol genotypes were polymorphic at 14% of the nucleotide sites. The ratio of nonsynonymous to synonymous nucleotide substitutions (relative to the number of respective sites, Ka/Ks) was 0.16, indicating that this region of the BIV genome, like that of HIV-1, is subject to purifying selection. Based on the McDonald-Kreitman analysis, this region also was under positive Darwinian selection as HIV-1 and BIV diverged from a common progenitor. Phylogenetic analysis revealed that genotypes were geographically distinct, possibly indicating a common source of infection for animals within a herd.

Amino Acid Sequence↗

PubMed1993

A novel open reading frame in tobacco mosaic virus genome coding for a putative small, positively charged protein.

From sequence comparisons between the tobramovirus genomes an open reading frame (ORF-X) potentially encoding a small, positively charged protein (33- to 45-amino-acids long) was found to overlap the immediate 3' and 5' sides of the transport protein gene and coat protein gene, respectively. In vitro translation of the monocistronic artificial transcripts generated with T7 RNA polymerase yielded a protein of M(r) 4000 (p4) and an unexpected trypsin-sensitive complex of M(r) 54,000 that was resistant to reduction with 2-mercaptoethanol but could be dissociated by 8 M urea. Assembly of this complex was inhibited completely by site-directed mutagenesis within a conserved, positively charged 5-amino-acid long segment of the ORF-X protein. After centrifugation in low salt buffer the 54-kDa complex remained mostly associated with ribosomes. Apparently this complex represents a specific aggregate of the p4 product of ORF-X with a protein of approximate M(r) 50,000 that is a component of the translation apparatus.

Amino Acid Sequence↗

PubMed1991

Purification and partial characterization of equine infectious anemia virus reverse transcriptase.

Previously we raised a rabbit monospecific antibody (C2003) against a synthetic peptide derived from a sequence within the C-terminal portion of the reverse transcriptase (RT) of the human immunodeficiency virus type 1 (HIV-1). This sequence is found to be conserved in the predicted amino acid sequence of a related lentivirus, the equine infectious anemia virus (EIAV). It was previously determined that the C2003 antibody could cross-react with native EIAV RT and directly inhibit the DNA polymerase activity of the enzyme. We have now fractionated EIAV RT by immunoaffinity chromatography with immobilized C2003 antibody. The procedure yielded an equimolar mixture of two proteins of 66 and 51 kDa associated with both DNA polymerase and RNase H activities. When the EIAV RT proteins were examined by in situ activity gel assays, polymerase activity was found to be principally associated with the 66-kDa component. The fidelity of DNA synthesis by EIAV RT was found to be equivalent to that of HIV-1 RT and lower than that of AMV RT. These observations indicate that the RTs of EIAV and HIV-1 share similar structural and functional properties.

Amino Acid Sequence↗

PubMed1996

Structural modules of the large subunits of RNA polymerase. Introducing archaebacterial and chloroplast split sites in the beta and beta' subunits of Escherichia coli RNA polymerase.

The beta and beta' subunits of Escherichia coli DNA-dependent RNA polymerase are highly conserved throughout eubacterial and eukaryotic kingdoms. However, in some archaebacteria and chloroplasts, the corresponding sequences are "split" into smaller polypeptides that are encoded by separate genes. To test if such split sites can be accommodated into E. coli RNA polymerase, subunit fragments encoded by the segments of E. coli rpoB and rpoC genes corresponding to archaebacterial and chloroplast split subunits were individually overexpressed. The purified fragments, when mixed in vitro with complementing intact RNA polymerase subunits, yielded an active enzyme capable of catalyzing the phosphodiester bond formation. Thus, the large subunits of eubacteria and eukaryotes are composed of independent structural modules corresponding to the smaller subunits of archaebacteria and chloroplasts.

Amino Acid Sequence↗

PubMed1993

Phosphorylation and dephosphorylation affect functional characteristics of chloroplast and etioplast transcription systems from mustard (Sinapis alba L.).

Chloroplast and etioplast RNA polymerase preparations each consist of a multi-subunit core and a set of three sigma-like transcription factors, SLF67, SLF52 and SLF29. Despite this structural similarity, the enzymes from either plastid type are functionally distinct, as is reflected by their different promoter usage and the tight core-SLF association in the etioplast but not the chloroplast holoenzyme. We tested whether these differences are related to phosphorylation. Treatment of the chloroplast enzyme with protein kinase converted it to an etioplast-type form and vice versa, treatment of the etioplast enzyme with phosphatase generated chloroplast-type properties. Although both the core enzyme and the SLF polypeptides were phosphorylation targets, only the SLFs seem to confer plastid-type-specific DNA binding characteristics. Methylation interference and DNase I footprint patterns in the psbA promoter region were found to correlate with the phosphorylation state of the chloroplast and etioplast enzymes.

Bacterial Proteins↗

PubMed2003

Spx-dependent global transcriptional control is induced by thiol-specific oxidative stress in Bacillus subtilis.

The Spx protein of Bacillus subtilis represses activator-stimulated transcription by interacting with the C-terminal domain of RNA polymerase (RNAP) alpha subunit. Its concentration increases in cells lacking the ATP-dependent protease, ClpXP, resulting in severe effects on growth and developmental processes. Microarray analysis was undertaken to identify genes that are induced or repressed when Spx interacts with RNAP. The induced genes included those encoding products known to function in maintaining thiol homeostasis. Two genes, thioredoxin (trxA) and thioredoxin reductase (trxB), are transcriptionally induced under conditions of thiol-specific oxidative (disulfide) stress by a mechanism involving Spx-RNAP interaction. Disulfide stress also results in an increase in Spx-dependent transcriptional repression. The increase in Spx activity in cells encountering disulfide stress is due in part to a posttranscriptional mechanism of spx control resulting in an increase in Spx concentration. An spx null mutant and a strain bearing an allele of rpoA that prevents Spx-RNAP interaction show hypersensitivity to disulfide stress. From these results, it is proposed that Spx is an activator that mobilizes the operations necessary to reverse the effects of oxidative damage, but it also serves as a negative regulator that causes the postponement of developmental programs and energy-consuming growth-related functions while the cell copes with the period of stress.

Alleles↗

PubMed2004

In vitro transcription analysis by reconstituted cyanobacterial RNA polymerase: roles of group 1 and 2 sigma factors and a core subunit, RpoC2.

The RNA polymerase (RNAP) core enzyme of cyanobacterium Synechocystis sp. strain PCC 6803 was reconstituted with overproduced recombinant subunits and purified with C-terminal histidine-tagged RpoA. The core enzyme with purified a sigma factor, SigA/SigD or SigB, allowed specific in vitro transcription from the light-inducible psbA2 or the dark-/heat-inducible lrtA/hspA promoters, respectively. Further analysis using a mutant psbA2 promoter revealed that the -35 hexamer of the promoter was essential for SigA but not SigD. Similar but distinct patterns of psbA2 transcription were found for two types of RNAP, cyanobacterial (alpha2betabeta'gamma) and E. coli (alpha2betabeta') core enzymes. Specific binding of PCC 6803 RpoC2 (beta') to E. coli core enzyme and its contribution to efficient psbA2 transcription by RNAP-SigA/D suggest that this subunit could confer an important role on the cyanobactrial RNAP. Differences in affinity and specificity among cyanobacterial sigma factors for the core enzyme and promoters were discussed.

Bacterial Proteins↗

PubMed1997

Rifampicin resistance in Neisseria meningitidis: evidence from a study of sibling strains, description of new mutations and notes on population genetics.

To provide direct evidence for the mechanism leading to resistance to rifampicin, two Neisseria meningitidis strains from one clonal lineage (so-called sibling strains) were studied; one of these strains was resistant, the other sensitive to rifampicin. The polymerase chain reaction (PCR) was used to amplify fragments of the known rifampicin resistance region on the rpoB gene coding for the beta subunit of DNA-directed RNA polymerase and the amplimers were then sequenced. In addition to the DNA from the sibling strains, DNA from further strains was analysed, including two Spanish, rifampicin-resistant strains, eight further N. meningitidis strains, strains of four further Neisseria spp. and one reference strain. The results demonstrated how quickly and easily N. meningitidis can acquire resistance to rifampicin, and also suggest a clonal population structure within the collection of strains studied. This finding is discussed with respect to recent studies that indicate a more panmictic population structure within particular serogroups of N. meningitidis.

Antibiotics, Antitubercular↗

PubMed2004

Cloning and overproduction of the rpoZ gene encoding an RNA polymerase omega subunit from a deep-sea piezophilic Shewanella violacea strain DSS12.

We have cloned the rpoZ gene, encoding RNA polymerase omega protein, by PCR approach from the deep-sea piezophilic and psychrophilic bacterium, Shewanella violacea strain DSS12. The cloned gene, 285bp in length, was found to encode a protein consisting of 94 amino acid residues with a molecular mass of 10,327 Da. Significant homology was evident comparing the RpoZ protein of S. violacea with that of Shewanella oneidensis (69% identity), Vibrio cholerae (65% identity), Escherichia coli K-12 (64% identity) and Haemophilus influenzae (61% identity). From the Northern blot analysis, S. violacea rpoZ gene was expressed constitutively under pressure conditions of 0.1, 30 and 50MPa. We constructed expression plasmid to overproduce the RpoZ protein and transformed into E. coli JM109 as a host of overproduction. Upon induction, the recombinant protein encoded by plasmid pQrpoZ was overexpressed and purified using Ni2+ affinity column.

Amino Acid Sequence↗

PubMed1993

Methodologies for specific intron and exon RNA localization in cultured cells by haptenized and fluorochromized probes.

We have determined optimal conditions for the detection of mRNA sequences in cultured cells by nonradioactive in situ hybridization. For this purpose a number of different cell lines have been used: rat 9G cells for the detection of human cytomegalovirus immediate early mRNA, and HeLa as well as 5637 carcinoma cells for the detection of housekeeping gene mRNAs. Extensive optimization of fixation and pretreatment conditions revealed that most intense hybridization signals are obtained when cells are grown on glass microscope slides, fixed with a mixture of formaldehyde and acetic acid, pretreated with pepsin and denatured prior to hybridization. In addition, we also studied the potential of fluorochromized probes for the direct detection of multiple RNA sequences. The optimized in situ hybridization procedure revealed that immediate early mRNA transcripts are, in addition to a cytoplasmic localization, localized within nuclei of rat 9G cells. Double hybridization experiments showed that intron and exon sequences colocalize within the main nuclear signal. In addition, the presence of small, intron-specific, fluorescent spots scattered around the main nuclear signals indicates that intron sequences which are spliced out can be visualized. Additional information about the functioning of cells could be obtained by the detection of mRNA simultaneously with bromodeoxyuridine, incorporated during S-phase, or its cognate protein. The sensitivity of these methods is such that mRNAs of abundantly expressed housekeeping genes can be detected in a variety of cell lines with high signal to noise ratios.

Animals↗

PubMed1995

Structural map of the alpha subunit of Escherichia coli RNA polymerase: structural domains identified by proteolytic cleavage.

The alpha subunit of Escherichia coli RNA polymerase plays essential roles in protein-protein contacts, not only for RNA polymerase assembly, but also for transcription activation by class I factors. To reveal the structure-function relationship of the alpha subunit, we attempted to elucidate the organization of the structural domains by analysis of the pattern of limited proteolysis with two endoproteases, V8 protease and trypsin. The results indicate that one region, Arg235 to Glu244, is highly accessible to endoproteases. We propose that the alpha subunit consists of two major structural domains, the amino-terminal domain upstream from Arg235 and the carboxy-terminal domain downstream from Glu245, each being connected by an inter-domain linker formed by the spacer between these two amino acid residues. The structural organization is in good agreement with its functional map, i.e., the amino-terminal subunit assembly determinants and the carboxy-terminal transcription activation determinants, including the contact sites with class I transcription factors and DNA UP (enhancer) elements. The secondary proteolytic cleavage sites were also determined, in order to analyse intra-domain structures.

Amino Acid Sequence↗

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↗