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PubMed2006

The sigma factors of Mycobacterium tuberculosis.

Mycobacterium tuberculosis is a remarkable pathogen capable of adapting and surviving in various harsh conditions. Correct gene expression regulation is essential for the success of this process. The reversible association of different sigma factors is a common mechanism for reprogramming bacterial RNA polymerase and modulating the transcription of numerous genes. Thirteen putative sigma factors are encoded in the M. tuberculosis genome, several being important for virulence. Here, we analyse the latest information available on mycobacterial sigma factors and discuss their roles in the physiology and virulence of M. tuberculosis.

DNA-Directed RNA Polymerases↗

PubMed1964

RIBONUCLEIC ACIDS OF BACILLUS SUBTILIS SPORES AND SPORULATING CELLS.

Doi, Roy H. (Syracuse University, Syracuse N.Y.), and Richard T. Igarashi. Ribonucleic acids of Bacillus subtilus spores and sporulating cells. J. Bacteriol. 87:323-328. 1964.-The ribonucleic acids (RNA) of Bacillus subtilis spores were analyzed for their size and base composition. Soluble and ribosomal RNA identical to those found in vegetative cells were present in spores. A base ratio difference was observed between the 16s and 23s ribosomal RNA. The 16s RNA had a higher cytidylate and lower adenylate base composition. The ratio of soluble of ribosomal RNA in spores was 30%, in contrast to 15% in vegetative cells, and may be related to their difference in biosynthetic activity. Although active synthesis of informational RNA occurred prior to endospore formation, no detectable level of informational RNA was found in spores. The informational RNA was shown to have the same base composition as that of B. subtilis deoxyribonucleic acid. The compositional difference between spore and vegetative cell RNA appeared to be strictly quantitative.

Bacillus↗

PubMed2003

Transcription termination control of the S box system: direct measurement of S-adenosylmethionine by the leader RNA.

Modulation of the structure of a leader RNA to control formation of an intrinsic termination signal is a common mechanism for regulation of gene expression in bacteria. Expression of the S box genes in Gram-positive organisms is induced in response to limitation for methionine. We previously postulated that methionine availability is monitored by binding of a regulatory factor to the leader RNA and suggested that methionine or S-adenosylmethionine (SAM) could serve as the metabolic signal. In this study, we show that efficient termination of the S box leader region by bacterial RNA polymerase depends on SAM but not on methionine or other related compounds. We also show that SAM directly binds to and induces a conformational change in the leader RNA. Both binding of SAM and SAM-directed transcription termination were blocked by leader mutations that cause constitutive expression in vivo. Overproduction of SAM synthetase in Bacillus subtilis resulted in delay in induction of S box gene expression in response to methionine starvation, consistent with the hypothesis that SAM is the molecular effector in vivo. These results indicate that SAM concentration is sensed directly by the nascent transcript in the absence of a trans-acting factor.

5' Untranslated Regions↗

PubMed2004

Nucleotide-dependent interactions between a fork junction-RNA polymerase complex and an AAA+ transcriptional activator protein.

Enhancer-dependent transcriptional activators that act upon the sigma54 bacterial RNA polymerase holoenzyme belong to the extensive AAA+ superfamily of mechanochemical ATPases. Formation and collapse of the transition state for ATP hydrolysis engenders direct interactions between AAA+ activators and the sigma54 factor, required for RNA polymerase isomerization. A DNA fork junction structure present within closed complexes serves as a nucleation point for the DNA melting seen in open promoter complexes and restricts spontaneous activator-independent RNA polymerase isomerization. We now provide physical evidence showing that the ADP.AlF(x) bound form of the AAA+ domain of the transcriptional activator protein PspF changes interactions between sigma54-RNA polymerase and a DNA fork junction structure present in the closed promoter complex. The results suggest that one functional state of the nucleotide-bound activator serves to alter DNA binding by sigma54 and sigma54-RNA polymerase and appears to drive events that precede DNA opening. Clear evidence for a DNA-interacting activity in the AAA+ domain of PspF was obtained, suggesting that PspF may make a direct contact to the DNA component of a basal promoter complex to promote changes in sigma54-RNA polymerase-DNA interactions that favour open complex formation. We also provide evidence for two distinct closed promoter complexes with differing stabilities.

Adenosine Diphosphate↗

PubMed1977

Protection of mice against encephalomyocarditis virus infection by preparations of transfer RNA.

Preparations of bacterial transfer RNA (tRNA), give dose-dependent protection of mice against encephalomyocarditis (EMC) virus infection at up to I mg tRNA per mouse with maximum response when the tRNA is administered around 6 h before infection. Protection occurs with intraperitoneally and intravenously administered tRNA against infections by both these routes. In some experiments significant protection occurs by single treatments of tRNA up to 24 h after infection with virus doses of I X LD100. Some tRNA preparations of eukaryotic origin do not give significant protection. Protection is not a feature of all species of bacterial tRNA; partially purified valine, tyrosine and phenylalanine tRNAs from Escherichia coli are not protective. tRNA treatment does not induce circulating interferon nor does it 'hypo-reactivate' the protective effect of poly (I).poly (C) treatment of mice. Humoral and cell mediated immune responses do not seem to be involved in tRNA mediated protection since first, cytosine arabinoside treatment does not affect protection by tRNA; second, serum from mice treated with tRNA and an EMC vaccine does not protect other mice against infection, and third, mice that survive normally lethal infections as a result of tRNA treatment are generally just as susceptible to re-infection as previously untreated, uninfected mice. Silica treatment abolishes protection of mice by tRNA implying that macrophages are necessary. However, tRNA does not seem to act by clearance of virus particles since vaccination of mice by inactivated EMC virus is not affected by tRNA treatment. These results are considered in relation to the presence of a tRNA-like structure in EMC virus RNA and protection of mice by other single stranded polynucleotides.

Animals↗

PubMedDate not supplied

Analysis of the tertiary structure of bacterial RNase P RNA.

The ubiquitous occurrence of ribonuclease P (RNase P) as a ribonucleoprotein and the catalytic properties of bacterial RNase P RNAs indicate that RNA fulfills an ancient and important role in the function of this enzyme. This review focuses on efforts to determine the structure of the bacterial RNase P RNA ribozyme. Phylogenetic comparative analysis of a library of bacterial RNase P RNA sequences has resulted in a well-developed secondary structure model and allowed identification of some elements of tertiary structure. The native structure has been redesigned by circular permutation to facilitate intra- and inter-molecular crosslinking experiments in order to gain further structural information. The crosslinking constraints, together with the constraints provided by comparative analyses, have been incorporated into a first-order model of the structure of of the ribozyme-substrate complex. The developing structural perspective allows the design of self-cleaving pre-tRNA-RNase P RNA conjugates which are useful tools for additional structure-probing experiments.

Bacteria↗

PubMed1978

Existence of differences in repressor properties between serine-rich histones (H5, F2c) from immature and mature pigeon erythroid cells.

The abilities of H1 and H5 histones from immature and mature pigeon erythroid cells and subfractions of H5 histones with different content of alkali-labile phosphorus to restrict RNA synthesis were compared in an in vitro transcription system with bacterial RNA polymerase. It has been found that: a) H1 histones from both sources exhibit similar efficiency. b) H5 histones from immature cells restrict transcription to a lesser extent than the same histone from the mature erythrocyte. c) The subfraction of H5 histone from immature cells with the higher content of alkali-labile phosphorus restrict transcription less than the subfraction with the lower degree of phosphorylation. The results suggest that H5 histone, which first appears in the erythroblasts in a highly phosphorylated form, does not display its potential 'repressor' properties. During erythrocyte maturation H5 histone is dephosphorylated and this causes the massive inactivation of erythrocyte genome.

Animals↗

PubMed2006

Deletion of the rpoZ gene, encoding the omega subunit of RNA polymerase, results in pleiotropic surface-related phenotypes in Mycobacterium smegmatis.

The omega subunit, the smallest subunit of bacterial RNA polymerase, is known to be involved in maintaining the conformation of the beta' subunit and aiding its recruitment to the rest of the core enzyme assembly in Escherichia coli. It has recently been shown in Mycobacterium smegmatis, by creating a deletion mutation of the rpoZ gene encoding omega, that the physiological role of the omega subunit also includes providing physical protection to beta'. Interestingly, the mutant had altered colony morphology. This paper demonstrates that the mutant mycobacterium has pleiotropic phenotypes including reduced sliding motility and defective biofilm formation. Analysis of the spatial arrangement of biofilms by electron microscopy suggests that the altered phenotype of the mutant arises from a deficiency in generation of extracellular matrix. Complementation of the mutant strain with a copy of the wild-type rpoZ gene integrated in the bacterial chromosome restored both sliding motility and biofilm formation to the wild-type state, unequivocally proving the role of omega in the characteristics observed for the mutant bacterium. Analysis of the cell wall composition demonstrated that the mutant bacterium had an identical glycopeptidolipid profile to the wild-type, but failed to synthesize the short-chain mycolic acids characteristic of biofilm growth in M. smegmatis.

Bacterial Proteins↗

PubMed2005

Allosteric modulation of the RNA polymerase catalytic reaction is an essential component of transcription control by rifamycins.

Rifamycins, the clinically important antibiotics, target bacterial RNA polymerase (RNAP). A proposed mechanism in which rifamycins sterically block the extension of nascent RNA beyond three nucleotides does not alone explain why certain RNAP mutations confer resistance to some but not other rifamycins. Here we show that unlike rifampicin and rifapentin, and contradictory to the steric model, rifabutin inhibits formation of the first and second phosphodiester bonds. We report 2.5 A resolution structures of rifabutin and rifapentin complexed with the Thermus thermophilus RNAP holoenzyme. The structures reveal functionally important distinct interactions of antibiotics with the initiation sigma factor. Strikingly, both complexes lack the catalytic Mg2+ ion observed in the apo-holoenzyme, whereas an increase in Mg2+ concentration confers resistance to rifamycins. We propose that a rifamycin-induced signal is transmitted over approximately 19 A to the RNAP active site to slow down catalysis. Based on structural predictions, we designed enzyme substitutions that apparently interrupt this allosteric signal.

Allosteric Regulation↗

PubMed1979

Comparison of the effect of linear gramicidin analogues on bacterial sporulation, membrane permeability, and ribonucleic acid polymerase.

Various analogues of linear gramicidin were tested for their biological activity in restoring the normal spore phenotype of gramicidin-negative mutants of Bacillus brevis and for their ability to increase cation conductivity of black lipid membranes and to inhibit bacterial RNA polymerase. Whereas many biologically active gramicidin analogues had no effect on membrane permeability, all biologically active peptides were able to inhibit ribonucleic acid (RNA) polymerase. These observations make it unlikely that membranes are the site of action of gramicidin during bacterial sporulation, but they are consistent with the notion that gramicidin functions to control RNA synthesis during the transition from vegetative growth to sporulation (Sarkar & Paulus, 1972). The relationship between peptide structure and the ability to restore normal sporulation and inhibit RNA polymerase showed that the eight amino-terminal residues have little influence on the function of gramicidin, whereas the highly nonpolar repeating sequence D-leucyl-L-tryptophan is essential for biological activity and may represent the site of interaction with RNA polymerase.

Bacillus↗

PubMed2002

Multiple roles of the RNA polymerase beta subunit flap domain in sigma 54-dependent transcription.

Recent determinations of the structures of the bacterial RNA polymerase (RNAP) and promoter complex thereof establish that RNAP functions as a complex molecular machine that contains distinct structural modules that undergo major conformational changes during transcription. However, the contribution of the RNAP structural modules to transcription remains poorly understood. The bacterial core RNAP (alpha(2)beta beta'omega; E) associates with a sigma (sigma) subunit to form the holoenzyme (E sigma). A mutation removing the beta subunit flap domain renders the Escherichia coli sigma(70) RNAP holoenzyme unable to recognize promoters. sigma(54) is the major variant sigma subunit that utilizes enhancer-dependent promoters. Here, we determined the effects of beta flap removal on sigma(54)-dependent transcription. Our analysis shows that the role of the beta flap in sigma(54)-dependent and sigma(70)-dependent transcription is different. Removal of the beta flap does not prevent the recognition of sigma(54)-dependent promoters, but causes multiple defects in sigma(54)-dependent transcription. Most importantly, the beta flap appears to orchestrate the proper formation of the E sigma(54) regulatory center at the start site proximal promoter element where activator binds and DNA melting originates.

Amino Acid Sequence↗

PubMed1987

Evolution in bacteria: evidence for a universal substitution rate in cellular genomes.

This paper constructs a temporal scale for bacterial evolution by tying ecological events that took place at known times in the geological past to specific branch points in the genealogical tree relating the 16S ribosomal RNAs of eubacteria, mitochondria, and chloroplasts. One thus obtains a relationship between time and bacterial RNA divergence which can be used to estimate times of divergence between other branches in the bacterial tree. According to this approach, Salmonella typhimurium and Escherichia coli diverged between 120 and 160 million years (Myr) ago, a date which fits with evidence that the chief habitats occupied now by these two enteric species became available that long ago. The median extent of divergence between S. typhimurium and E. coli at synonymous sites for 21 kilobases of protein-coding DNA is 100%. This implies a silent substitution rate of 0.7-0.8%/Myr--a rate remarkably similar to that observed in the nuclear genes of mammals, invertebrates, and flowering plants. Similarities in the substitution rates of eucaryotes and procaryotes are not limited to silent substitutions in protein-coding regions. The average substitution rate for 16S rRNA in eubacteria is about 1%/50 Myr, similar to the average rate for 18S rRNA in vertebrates and flowering plants. Likewise, we estimate a mean rate of roughly 1%/25 Myr for 5S rRNA in both eubacteria and eucaryotes. For a few protein-coding genes of these enteric bacteria, the extent of silent substitution since the divergence of S. typhimurium and E. coli is much lower than 100%, owing to extreme bias in the usage of synonymous codons. Furthermore, in these bacteria, rates of amino acid replacement were about 20 times lower, on average, than the silent rate. By contrast, for the mammalian genes studied to date, the average replacement rate is only four to five times lower than the rate of silent substitution.

Bacteria↗

PubMed2004

New enzymes from environmental cassette arrays: functional attributes of a phosphotransferase and an RNA-methyltransferase.

By targeting gene cassettes by polymerase chain reaction (PCR) directly from environmentally derived DNA, we are able to amplify entire open reading frames (ORFs) independently of prior sequence knowledge. Approximately 10% of the mobile genes recovered by these means can be attributed to known protein families. Here we describe the characterization of two ORFs which show moderate homology to known proteins: (1) an aminoglycoside phosphotransferase displaying 25% sequence identity with APH(7") from Streptomyces hygroscopicus, and (2) an RNA methyltransferase sharing 25%-28% identity with a group of recently defined bacterial RNA methyltransferases distinct from the SpoU enzyme family. Our novel genes were expressed as recombinant products and assayed for appropriate enzyme activity. The aminoglycoside phosphotransferase displayed ATPase activity, consistent with the presence of characteristic Mg(2+)-binding residues. Unlike related APH(4) or APH(7") enzymes, however, this activity was not enhanced by hygromycin B or kanamycin, suggesting the normal substrate to be a different aminoglycoside. The RNA methyltransferase contains sequence motifs of the RNA methyltransferase superfamily, and our recombinant version showed methyltransferase activity with RNA. Our data confirm that gene cassettes present in the environment encode folded enzymes with novel sequence variation and demonstrable catalytic activity. Our PCR approach (cassette PCR) may be used to identify a diverse range of ORFs from any environmental sample, as well as to directly access the gene pool found in mobile gene cassettes commonly associated with integrons. This gene pool can be accessed from both cultured and uncultured microbial samples as a source of new enzymes and proteins.

Amino Acid Sequence↗

PubMed1989

Interaction of RNA polymerase II with structurally altered nucleosomal particles. Transcription is facilitated by loss of one H2A.H2B dimer.

The loss of one H2A.H2B dimer from the nucleosomal core increases its affinity for RNA polymerase II and its efficiency as a transcription template, allowing transcription of the entire DNA present in the particle. In contrast, the nucleosomal core lacking the amino-terminal ends of histones, which has an affinity for polymerase equal to that of the H2A.H2B-deficient core, shows transcription properties similar to those of the whole nucleosomal core, with synthesis of short RNA chains (40 nucleotides or less). Similar results were obtained with a bacterial RNA polymerase. The improved efficiency of the H2A.H2B-deficient cores as transcription templates does not appear to be produced by nonspecific loss of protein or structural relaxation of the particle. These results suggest that a particle lacking one H2A.H2B dimer might be a necessary intermediate during in vivo transcription.

Animals↗

PubMed1975

Biochemical and immunological properties of ribonucleic acid-rich extracts from Francisella tularensis.

Ribonucleic acid (RNA)-rich extracts derived from the attenuated strain of Francisella tularensis (strain LVS) protected Swiss mice against lethal challenge with F. tularensis strain 425 but not against strain SCHU S4. No killed preparation, including an RNA-rich extract from SCHU S4 itself, offered protection against strain SCHU S4 in contrast to the high level of protection offered against this strain by vaccination with live strain LVS. The protective activity observed against strain 425 was sensitive to ribonuclease but not to Pronase. Protective activity is not a general property of bacterial RNA, since RNA-rich extracts from Staphylococcus aureus offered no protection against tularemia, although disc gel electrophoresis showed similar kinds and amounts of RNA in preparations form F. tularensis and S. aureus. Furthermore, inability to localize activity to a specific region in sucrose gradients suggests a structural rather than an informational role for the RNA in such extracts. RNA-rich extracts from F. tularensis but not from S. aureus were efficient inducers of F. tularensis opsonins in mouse serum, suggesting one mechanism by which such extracts confer protection.

Animals↗

PubMed2003

RNA polymerase holoenzyme: structure, function and biological implications.

The past three years have marked the breakthrough in our understanding of the structural and functional organization of RNA polymerase. The latest major advance was the high-resolution structures of bacterial RNA polymerase holoenzyme and the holoenzyme in complex with promoter DNA. Together with an array of genetic, biochemical and biophysical data accumulated to date, the structures provide a comprehensive view of dynamic interactions between the major components of transcription machinery during the early stages of the transcription cycle. They include the binding of sigma factor to the core enzyme, and the recognition of promoter sequences and DNA melting by holoenzyme, transcription initiation and promoter clearance.

Bacteria↗

PubMed1987

In organello transcription in maize mitochondria and its sensitivity to inhibitors of RNA synthesis.

Purified mitochondrial preparations from etiolated maize shoots support the incorporation of radioactivity from labeled UTP into RNA. The incorporation is linear with time for up to 2 hours, shows Michaelis-Menton kinetics with respect to the concentration of the labeled substrate, UTP, and has salt and pH optima which are different than those previously reported for RNA synthesis by isolated chloroplasts. When a crude mitochondrial preparation is subjected to isopycnic sucrose gradient centrifugation, the bulk of the RNA synthetic activity co-sediments with mitochondrial marker enzymes and with the mitochondrial 26S and 18S rRNAs. Maize mitochondrial RNA synthesis is prevented by actinomycin D and ethidium bromide but unaffected by alpha-amanitin. It is strongly inhibited by rifampicin at concentrations which have no effect on nuclear and chloroplast RNA synthesis, but only moderately inhibited by rifampicin at concentrations which completely inhibit bacterial RNA synthesis. The optimization, cell fractionation, and inhibitor data all suggest that contaminating organelles and bacteria do not contribute appreciably to the RNA synthesis in purified mitochondrial preparations.

Journal Article↗

PubMed1980

Effect on transcription of low-molecular-weight RNA from calf thymus chromatin.

1. Two low-molecular-weight nuclear RNA fractions were isolated from the loosely-bound non-histone proteins of calf thymus chromatin: RNA I (of 4 - 5.5S, heterogeneous on polyacrylamide-gel electrohoresis), and RNA IV (of about 3S). 2. Under the reinitiation conditions, RNA I stimulates about 2.5-fold the template properties of homologous chromatin with bacterial RNA polymerase, while RNA IV inhibits these properties by about 50%. 3. When reinitiation is blocked, RNA I stimulates both the initiation and elongation steps (by 20 - 30% and 60%, respectively). 4. The two RNA fractions equally inhibit transcription (by 30 - 35%) with free homologous DNA as a template.

Animals↗