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At least 433 records · Page 24Linked to original sources
PubMed1996

Mouse RNA polymerase I 16-kDa subunit able to associate with 40-kDa subunit is a homolog of yeast AC19 subunit of RNA polymerases I and III.

We have previously isolated a mouse RPA40 (mRPA40) cDNA encoding the 40-kDa subunit of mouse RNA polymerase I and demonstrated that mRPA40 is a mouse homolog of the yeast subunit AC40, which is a subunit of RNA polymerases I and III, having a limited homology to bacterial RNA polymerase subunit alpha (Song, C. Z., Hanada, K., Yano, K., Maeda, Y., Yamamoto, K., and Muramatsu, M. (1994) J. Biol. Chem. 269, 26976-26981). In an extension of the study we have now cloned mouse RPA16 (mRPA16) cDNA encoding the 16-kDa subunit of mouse RNA polymerase I by a yeast two-hybrid system using mRPA40 as a bait. The deduced amino acid sequence shows 45% identity to the yeast subunit AC19 of RNA polymerases I and III, known to associate with AC40, and a local similarity to bacterial alpha subunit. We have shown that mRPA40 mutants failed to interact with mRPA16 and that neither mRPA16 nor mRPA40 can interact by itself in the yeast two-hybrid system. These results suggest that higher eukaryotic RNA polymerase I conserves two distinct alpha-related subunits that function to associate with each other in an early stage of RNA polymerase I assembly.

Amino Acid Sequence↗
PubMed2024

Double-stranded RNA sequencing reveals distinct riboviruses associated with thermoacidophilic bacteria from hot springs in Japan.

Metatranscriptome sequencing expanded the known diversity of the bacterial RNA virome, suggesting that additional riboviruses infecting bacterial hosts remain to be discovered. Here we employed double-stranded RNA sequencing to recover complete genome sequences of two ribovirus groups from acidic hot springs in Japan. One group, denoted hot spring riboviruses (HsRV), consists of viruses with distinct RNA-directed RNA polymerases (RdRPs) that seem to be intermediates between typical ribovirus RdRPs and viral reverse transcriptases. This group forms a distinct phylum, Artimaviricota, or even kingdom within the realm Riboviria. We identified viruses encoding HsRV-like RdRPs in marine water, river sediments and salt marshes, indicating that this group is widespread beyond extreme ecosystems. The second group, denoted hot spring partiti-like viruses (HsPV), forms a distinct branch within the family Partitiviridae. The genome architectures of HsRV and HsPV and their identification in bacteria-dominated habitats suggest that these viruses infect thermoacidophilic bacteria.

Hot Springs↗
PubMed2004

Normal-mode analysis suggests protein flexibility modulation throughout RNA polymerase's functional cycle.

To explore the domain-scale flexibility of bacterial RNA polymerase (RNAP) throughout its functional cycle, block normal-mode analyses (BNM) were performed on several important functional states, including the holoenzyme, the core complex, a model of RNAP bound to primarily duplex DNA, and a model of the ternary elongation complex. The calculations utilized a molecular mechanics (MM) force field with physical interactions; this is made possible by the use of BNM and the implementation of a sparse-matrix diagonalization routine. The use of homology models necessitated the MM force field rather than the simpler elastic network model (ENM). From the MM/BNM, we have systematically and semiquantitatively calculated the atomic fluctuations in the four functional states without bias due to crystal packing or other artifactual forces. We have observed that both alpha subunits and the omega subunit are rigid, in line with their roles as structural motifs that are not mechanistically involved in RNAP's functional cycle. It has been observed that the beta subunit has two highly mobile domains; these are commonly known as the beta1 and beta2 domains. Our calculations suggest that the flexibility of these domains is modulated throughout the functional cycle and that they move entirely independently of each other unless DNA is bound. From an energetic perspective, we have shown the beta2 domain can flex into and out of the cleft, forming interactions with DNA in the TEC as has been previously proposed. Our calculations also confirm that the beta' subunit's likely flexibility into and out of the DNA binding cleft is energetically allowed. These two observations validate that both of the RNAP crab claw's pincers are mobile, as both beta and beta' have substantial flexibility.

Bacterial Proteins↗
PubMed2004

Reorganisation of an RNA polymerase-promoter DNA complex for DNA melting.

Sigma factors, the key regulatory components of the bacterial RNA polymerase (RNAP), direct promoter DNA binding and DNA melting. The sigma(54)-RNAP forms promoter complexes in which DNA melting is only triggered by an activator and ATP hydrolysis-driven reorganisation of an initial sigma(54)-RNAP-promoter complex. We report that an initial bacterial RNAP-DNA complex can be reorganised by an activator to form an intermediate transcription initiation complex where full DNA melting has not yet occurred. Using sigma(54) as a chemical nuclease we now show that the reorganisation of the initial sigma(54)-RNAP-promoter complex occurs upon interaction with the activator at the transition point of ATP hydrolysis. We demonstrate that this reorganisation event is an early step in the transcription initiation pathway that occurs independently of RNAP parts normally associated with stable DNA melting and open complex formation. Using photoreactive DNA probes, we provide evidence that within this reorganised sigma(54)-RNAP-promoter complex, DNA contacts across the 'to be melted' sequences are made by the sigma(54) subunit. Strikingly, the activator protein, but not core RNAP subunits, is close to these DNA sequences.

Adenosine Diphosphate↗
PubMed1985

Mutations in the sigma subunit of E. coli RNA polymerase which affect positive control of transcription.

The sigma subunits of bacterial RNA polymerases are required for the selective initiation of transcription. We have isolated and characterized mutations in rpoD, the gene which encodes the major form of sigma in E. coli, which affect the selectivity of transcription. These mutations increase the expression of araBAD up to 12-fold in the absence of CAP-cAMP. Expression of lac is unaffected, while expression of malT-activated operons is decreased. We determined the DNA sequence of 17 independently isolated mutations, and found that they consist of three different changes in a single CGC arginine codon at position 596 in the sigma polypeptide.

Arabinose↗
PubMed2005

Stable DNA opening within open promoter complexes is mediated by the RNA polymerase beta'-jaw domain.

DNA opening for transcription-competent open promoter complex (OC) formation by the bacterial RNA polymerase (RNAP) relies upon a complex network of interactions between the structurally conserved and flexible modules of the catalytic beta and beta'-subunits, RNAP-associated sigma-subunit, and the DNA. Here, we show that one such module, the beta'-jaw, functions to stabilize the OC. In OCs formed by the major sigma70-RNAP, the stabilizing role of the beta'-jaw is not restricted to any particular melted DNA segment. In contrast, in OCs formed by the major variant sigma54-RNAP, the beta'-jaw and a conserved sigma54 regulatory domain co-operate to stabilize the melted DNA segment immediately upstream of the transcription start site. Clearly, regulated communication between the mobile modules of the RNAP and the functional domain(s) of the sigma subunit is required for stable DNA opening.

Bacteriophage T7↗
PubMed2002

Efficient isolation of total RNA from antibiotic-producing bacterium Amycolatopsis mediterranei.

RNA extraction from antibiotic-producing actinomycetes can be a difficult and time-consuming process due to their special peptidoglycans cell wall composition and the short life of RNA. Hence, the rapidity of cellular lysis and complete inhibition of RNase are of particular importance for isolating intact RNA of high quality. The genus of Amycolatopsis mediterranei produces many clinically important antibiotics, such as rifamycin and vancomycin; however, the available methods for bacterial RNA isolation did not work very well with this genus. In this report, we described a new method for RNA isolation using the combination of LiCl, urea and guanidinium thiocyanate to disrupt the cell wall of Amycolatopsis. Compared with earlier published RNA isolation methods, the method gave higher yields of pure and intact RNA. About 1 microg total RNA free of DNA contamination can be obtained from 1 mg wet weight of A. mediterranei. The integrity of the RNA was demonstrated by formaldehyde agarose gel electrophoresis and Northern blot analyses.

Actinomycetales↗
PubMed1999

Direct detection of bacteria in cellular blood products using bacterial ribosomal RNA-directed probes coupled to electrochemiluminescence.

Various techniques for detecting bacteria in blood products exist; however, none has been widely accepted. Our method permits direct bacterial detection in both platelet concentrates (PC) and packed red blood cells (RBC). This novel procedure targets bacterial ribosomal RNA (rRNA) but does not utilize culture or nucleic acid amplification. The assay comprises five steps: (1) release of bacterial rRNA by cell lysis with a combination of detergents and high heat; (2) hybridization of bacterial rRNA using a biotin- and a ruthenium (ORIGEN)-labelled oligonucleotide probe pair; (3) capture of labelled rRNA with streptavidin-coated magnetic beads; (4) concentration of labelled rRNA/bead complexes out of solution and onto an electrode surface with a magnet; (5) detection of ruthenium-labelled bacterial rRNA by application of voltage and consequent generation of the electrochemiluminescent (ECL) signal. Results using PC and RBC samples, spiked with clinically relevant gram-negative and -positive bacterial species, consistently demonstrated a linear relationship between ECL signal (equates to rRNA level) and colony forming units (CFU) mL(-1). Signals were generated in the range of 1400-80000 and 3500-500000 ECL units for unwashed and washed samples, respectively. This is equivalent to 10(5)-10(8)CFU mL(-1). These data demonstrate that therapeutic blood products significantly contaminated with bacteria may be identified prior to issue.

Bacteria↗
PubMed1997

Multiple pathways to bypass the enhancer requirement of sigma 54 RNA polymerase: roles for DNA and protein determinants.

Sigma 54 is a required factor for bacterial RNA polymerase to respond to enhancers and directs a mechanism that is a hybrid between bacterial and eukaryotic transcription. Three pathways were found that bypass the enhancer requirement in vitro. These rely on either deletion of the sigma 54 N terminus or destruction of the DNA consensus -12 promoter recognition element or altering solution conditions to favor transient DNA melting. Each of these allows unstable heparin-sensitive pre-initiation complexes to form that can be driven to transcribe in the absence of both enhancer protein and ATP beta-gamma hydrolysis. These disparate pathways are proposed to have a common basis in that multiple N-terminal contacts may mediate the interactions between the polymerase and the DNA region where melting originates. The results raise possibilities for common features of open complex formation by different RNA polymerases.

Amino Acid Sequence↗
PubMed1999

The acid-inducible asr gene in Escherichia coli: transcriptional control by the phoBR operon.

Escherichia coli responds to external acidification (pH 4.0 to 5.0) by synthesizing a newly identified, approximately 450-nucleotide RNA component. At maximal levels of induction it is one of the most abundant small RNAs in the cell and is relatively stable bacterial RNA. The acid-inducible RNA was purified, and the gene encoding it, designated asr (for acid shock RNA), mapped at 35.98 min on the E. coli chromosome. Analysis of the asr DNA sequence revealed an open reading frame coding for a 111-amino-acid polypeptide with a deduced molecular mass of approximately 11.6 kDa. According to computer-assisted analysis, the predicted polypeptide contains a typical signal sequence of 30 amino acids and might represent either a periplasmic or an outer membrane protein. The asr gene cloned downstream from a T7 promoter was translated in vivo after transcription using a T7 RNA polymerase transcription system. Expression of a plasmid-encoded asr::lacZ fusion under a native asr promoter was reduced approximately 15-fold in a complex medium, such as Luria-Bertani medium, versus the minimal medium. Transcription of the chromosomal asr was abolished in the presence of a phoB-phoR (a two-component regulatory system, controlling the pho regulon inducible by phosphate starvation) deletion mutant. Acid-mediated induction of the asr gene in the Delta(phoB-phoR) mutant strain was restored by introduction of the plasmid with cloned phoB-phoR genes. Primer extension analysis of the asr transcript revealed a region similar to the Pho box (the consensus sequence found in promoters transcriptionally activated by the PhoB protein) upstream from the determined transcription start. The asr promoter DNA region was demonstrated to bind PhoB protein in vitro. We discuss our results in terms of how bacteria might employ the phoB-phoR regulatory system to sense an external acidity and regulate transcription of the asr gene.

Acids↗
PubMed2000

Interaction of sigma 70 with Escherichia coli RNA polymerase core enzyme studied by surface plasmon resonance.

The interaction between the core form of bacterial RNA polymerases and sigma factors is essential for specific promoter recognition, and for coordinating the expression of different sets of genes in response to varying cellular needs. The interaction between Escherichia coli core RNA polymerase and sigma 70 has been investigated by surface plasmon resonance. The His-tagged form of sigma 70 factor was immobilised on a Ni2+-NTA chip for monitoring its interaction with core polymerase. The binding constant for the interaction was found to be 1.9x10(-7) M, and the dissociation rate constant for release of sigma from core, in the absence of DNA or transcription, was 4x10(-3) s(-1), corresponding to a half-life of about 200 s.

Bacterial Proteins↗
PubMed1973

Analysis of isoaccepting transfer ribonucleic acid species of Bacillus subtilis: changes in chromatography of transfer ribonucleic acids associated with stage of development.

Changes in chromatographic profiles of tyrosyl-, leucyl-, tryptophanyl-, and lysyl-transfer ribonucleic acids (tRNAs) are presented as a function of the growth stage in Bacillus subtilis. All of the tRNA groups investigated expressed different temporal patterns of change in isoaccepting species. Tyrosyl-tRNAs were the earliest to change and were followed by changes in leucyl- and then tryptophanyl-tRNAs. Lysyl-tRNAs were unique in having two times of change: one early and one very late. As an aid in understanding the temporal aspect of tRNA alterations during sporulation, the chromatographic profiles of aminoacyl tRNAs from an early blocked asporogenous mutant were studied. The asporogenous mutant used was blocked at the axial filament stage, stage 0 of sporulation. Nevertheless, those tRNAs which showed differences between the spore and cells in exponential growth exhibited similar changes in the asporogenous mutant after 24 h of growth. The data suggest that several tRNA changes occur during development in B. subtilis but that the events leading to these changes are either independent of, or occur before, stage 0 of sporulation, except in the case of lysyl-tRNA.

Amino Acyl-tRNA Synthetases↗
PubMed2002

Identification of characteristic oligonucleotides in the bacterial 16S ribosomal RNA sequence dataset.

MOTIVATION: The phylogenetic structure of the bacterial world has been intensively studied by comparing sequences of 16S ribosomal RNA (16S rRNA). This database of sequences is now widely used to design probes for the detection of specific bacteria or groups of bacteria one at a time. The success of such methods reflects the fact that there are local sequence segments that are highly characteristic of particular organisms or groups of organisms. It is not clear, however, the extent to which such signature sequences exist in the 16S rRNA dataset. A better understanding of the numbers and distribution of highly informative oligonucleotide sequences may facilitate the design of hybridization arrays that can characterize the phylogenetic position of an unknown organism or serve as the basis for the development of novel approaches for use in bacterial identification. RESULTS: A computer-based algorithm that characterizes the extent to which any individual oligonucleotide sequence in 16S rRNA is characteristic of any particular bacterial grouping was developed. A measure of signature quality, Q(s), was formulated and subsequently calculated for every individual oligonucleotide sequence in the size range of 5-11 nucleotides and for 15mers with reference to each cluster and subcluster in a 929 organism representative phylogenetic tree. Subsequently, the perfect signature sequences were compared to the full set of 7322 sequences to see how common false positives were. The work completed here establishes beyond any doubt that highly characteristic oligonucleotides exist in the bacterial 16S rRNA sequence dataset in large numbers. Over 16,000 15mers were identified that might be useful as signatures. Signature oligonucleotides are available for over 80% of the nodes in the representative tree.

Algorithms↗
PubMed1993

Promoter opening (melting) and transcription initiation by RNA polymerase I requires neither nucleotide beta,gamma hydrolysis nor protein phosphorylation.

With some bacterial RNA polymerases and in eukaryotic RNA polymerase II, DNA melting during initiation requires the coupling of energy derived from beta,gamma hydrolysis of ATP. A detailed analysis of this possible requirement for eukaryotic RNA polymerase I reveals no such requirement. However, in some cases, beta,gamma non-hydrolyzable derivatives (beta,gamma imido or methylene) of nucleotide substrates have been found to significantly inhibit transcription initiation because of their inefficient use as the first nucleotide of the transcript. In addition, the results presented here show that protein kinase activity is not required as an integral part of transcription initiation by RNA polymerase I. Prior phosphorylation of proteins participating in the process is not ruled out.

Acanthamoeba↗
PubMed2002

Role of the RNA polymerase sigma subunit in transcription initiation.

In bacteria, sigma subunits direct the catalytically competent RNA polymerase core enzyme to promoters. Recent advances in our understanding of bacterial RNA polymerase reveal that sigma subunits are intimately involved in all aspects of transcription initiation including promoter location, promoter melting, initiation of RNA synthesis, abortive initiation and promoter escape.

Amino Acid Sequence↗
PubMed1976

The effect of dibromodulcitol on the template activity of DNA chromatin and nuclei from Yoshida sarcoma cells.

It has been shown earlier that after in vivo administration, dibromodulcitol (DBD) reacts with DNA and to a greater extent with chromosomal proteins of Yoshida sarcoma cells. The present experiments were designed to show if the binding of DBD to the chromatin elements of Yoshida sarcoma cells causes any changes in RNA synthesis using either DNA or chromatin as template in bacterial RNA polymerase system. During 4 to 24 h following in vivo administration, DBD reduces the template activity of dna without detectable single-strand breaks in the template DNA in alkaline sucrose gradients. Using chromatin as template the same dose of DBD produces no or very slight inhibition of RNA synthesis. Measuring the DNA-dependent RNA synthesis in nuclei isolated from Yoshida cells of treated rats, the dose of DBD which markedly inhibited the template activity of DNA, resulted in a significant stimulation of the nuclear RNA synthesis. The increased RNA synthesis was not due to an inhibition of ribonuclease activity. The observed alterations of the transcriptive properties of chromatin and nuclei produced by DBD are interpreted as being due to a modification of the whole nucleoprotein structure caused by the interaction of DBD with both DNA and chromosomal proteins.

Animals↗