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PubMed1988

Engineered recombinant messenger RNA can be replicated and expressed inside bacterial cells by an RNA bacteriophage replicase.

In this paper we describe how an RNA replicase can be "tricked" into recognizing, binding to, and replicating host-cell message RNAs. In our system, the gene encoding Q beta replicase is constitutively expressed from a plasmid vector present in an Escherichia coli host, while a second plasmid directs the transcription of a replication-competent, phage-like template RNA. This 680 nucleotide transcript (N- RNA) contains specific sequences required for Q beta replicase function. Included within this template RNA is a 360-nucleotide sequence that is complementary to the messenger RNA for DNA bacteriophage lambda N protein. The active messenger RNA for lambda N protein is expressed only upon replication of N- RNA by Q beta replicase. By employing an E. coli host strain that requires lambda N protein for growth under specific selective conditions, we were able to select only those cells in which RNA-directed RNA replication occurred. This system provides a potential for in vivo amplification of other heterologous messenger RNAs and their protein products via RNA replication and will enable one to study the evolution of messenger RNA in the live cell under a large variety of physiological pressures.

Base Sequence↗
PubMed2006

A basal promoter element recognized by free RNA polymerase sigma subunit determines promoter recognition by RNA polymerase holoenzyme.

During transcription initiation by bacterial RNA polymerase, the sigma subunit recognizes the -35 and -10 promoter elements; free sigma, however, does not bind DNA. We selected ssDNA aptamers that strongly and specifically bound free sigma(A) from Thermus aquaticus. A consensus sequence, GTA(C/T)AATGGGA, was required for aptamer binding to sigma(A), with the TA(C/T)AAT segment making interactions similar to those made by the -10 promoter element (consensus sequence TATAAT) in the context of RNA polymerase holoenzyme. When in dsDNA form, the aptamers function as strong promoters for the T. aquaticus RNA polymerase sigma(A) holoenzyme. Recognition of the aptamer-based promoters depends on the downstream GGGA motif from the aptamers' common sequence, which is contacted by sigma(A) region 1.2 and directs transcription initiation even in the absence of the -35 promoter element. Thus, recognition of bacterial promoters is controlled by independent interactions of sigma with multiple basal promoter elements.

Aptamers, Nucleotide↗
PubMed2006

Is it easy to stop RNA polymerase?

Among transcription factors that bind to bacterial RNA polymerase (RNAP) and modulate its activity, a number of small molecules irreversibly inhibit RNAP thereby causing cell death. To be of clinical significance such inhibitors must (1) inhibit a broad range of bacterial RNAPs but not affect human cells, (2) penetrate bacterial cell walls and (3) circumvent bacterial resistance mechanisms. Rifamycins, the only class of RNAP inhibitors that have found their way into clinical practice, are widely used in the treatment of tuberculosis and leprosy. However, the practical value of this class of antibiotics is limited by a rapid rise in resistant bacterial isolates. In this review we focus on recent advances in studies of prokaryotic transcription that allow a detailed structural and functional characterization of a number of RNAP/rifamycins complexes, thereby opening new opportunities for the design of superior antibacterial agents.

Anti-Bacterial Agents↗
PubMed1999

Abortive initiation by Saccharomyces cerevisiae RNA polymerase III.

Promoter escape can be rate-limiting for transcription by bacterial RNA polymerases and RNA polymerase II of higher eukaryotes. Formation of a productive elongation complex requires disengagement of RNA polymerase from promoter-bound eukaryotic transcription factors or bacterial sigma factors. RNA polymerase III (pol III) stably associates with the TFIIIB-DNA complex even in the absence of localized DNA unwinding associated with the open promoter complex. To explore the role that release of pol III from the TFIIIB-DNA complex plays in limiting the overall rate of transcription, we have examined the early steps of RNA synthesis. We find that, on average, only three rounds of abortive initiation precede the formation of each elongation complex and that nearly all pol III molecules escape the abortive initiation phase of transcription without significant pausing or arrest. However, when elongation is limited to 5 nucleotides, the intrinsic exoribonuclease activity of pol III cleaves 5-mer RNA at a rate considerably faster than product release or reinitiation. This cleavage also occurs in the normal process of forming a productive elongation complex. The possible role of nucleolytic retraction in disengaging pol III from TFIIIB is discussed.

Base Sequence↗
PubMed2001

New insight into RNase P RNA structure from comparative analysis of the archaeal RNA.

A detailed comparative analysis of archaeal RNase P RNA structure and a comparison of the resulting structural information with that of the bacterial RNA reveals that the archaeal RNase P RNAs are strikingly similar to those of Bacteria. The differences between the secondary structure models of archaeal and bacterial RNase P RNA have largely disappeared, and even variation in the sequence and structure of the RNAs are similar in extent and type. The structure of the cruciform (P7-11) has been reevaluated on the basis of a total of 321 bacterial and archaeal sequences, leading to a model for the structure of this region of the RNA that includes an extension to P11 that consistently organizes the cruciform and adjacent highly-conserved sequences.

Base Sequence↗
PubMed1993

Amplification and sequencing of variable regions in bacterial 23S ribosomal RNA genes with conserved primer sequences.

Published bacterial 23S ribosomal RNA sequences were aligned, and universally conserved regions flanking highly variable regions were looked for. In strategically positioned conserved regions, six oligonucleotides suitable for polymerase chain reaction (PCR) and sequencing were designed, allowing fast sequencing of four of the most variable 23S rRNA regions. Two other primers were designed for PCR amplification of nearly complete 23S rRNA genes. All these primers successfully amplified fragments of 23S rRNA genes from seven unrelated bacteria. Four primers were used to determine 938 bp of sequence for Campylobacter jejuni subsp. jejuni. These results indicate that the oligonucleotide sequences presented here are useful for PCR amplification and sequence determination of variable 23S rRNA regions for a broad variety of eubacterial species.

Base Sequence↗
PubMed2000

The role of region II in the RNA polymerase sigma factor sigma(N) (sigma(54)).

Bacterial RNA polymerase holoenzymes containing the sigma subunit sigma(N) (sigma(54)) can form a stable closed complex with promoter DNA but only undergo transition to an open complex and transcription initiation when acted on by an activator protein. Proteins of the sigma(N) family have a conserved N-terminal region of 50 amino acids (Region I) that is separated from a conserved C-terminal region of around 360 amino acids (Region III) by a much more variable sequence of between 30 and 110 residues (Region II). We have investigated the role of Region II in Klebsiella pneumoniae sigma(N) by studying the properties of deletions of all or part of the region both in vivo and in vitro. We found that whilst Region II is not essential, deletion of all or part of it can significantly impair sigma(N) activity. Deletions have effects on DNA binding by the isolated sigma factor and on holoenzyme formation, but the most marked effects are on transition of the holoenzyme from the closed to the open complex in the presence of the activator protein.

Amino Acid Sequence↗
PubMed2004

A regulator that inhibits transcription by targeting an intersubunit interaction of the RNA polymerase holoenzyme.

The structures of the bacterial RNA polymerase holoenzyme have provided detailed information about the intersubunit interactions within the holoenzyme. Functional analysis indicates that one of these is critical in enabling the holoenzyme to recognize the major class of bacterial promoters. It has been suggested that this interaction, involving the flap domain of the beta subunit and conserved region 4 of the sigma subunit, is a potential target for regulation. Here we provide genetic and biochemical evidence that the sigma region 4/beta-flap interaction is targeted by the transcription factor AsiA. Specifically, we show that AsiA competes directly with the beta-flap for binding to sigma region 4, thereby inhibiting transcription initiation by disrupting the sigma region 4/beta-flap interaction.

DNA-Directed RNA Polymerases↗
PubMed2006

Target prediction for small, noncoding RNAs in bacteria.

Many small, noncoding RNAs in bacteria act as post-transcriptional regulators by basepairing with target mRNAs. While the number of characterized small RNAs (sRNAs) has steadily increased, only a limited number of the corresponding mRNA targets have been identified. Here we present a program, TargetRNA, that predicts the targets of these bacterial RNA regulators. The program was evaluated by assessing whether previously known targets could be identified. The program was then used to predict targets for the Escherichia coli RNAs RyhB, OmrA, OmrB and OxyS, and the predictions were compared with changes in whole genome expression patterns observed upon expression of the sRNAs. Our results show that TargetRNA is a useful tool for finding mRNA targets of sRNAs, although its rate of success varies between sRNAs.

Base Pairing↗
PubMed2003

Visualizing RNA extrusion and DNA wrapping in transcription elongation complexes of bacterial and eukaryotic RNA polymerases.

Transcription ternary complexes of Escherichia coli RNA polymerase and yeast RNA polymerase III have been analyzed by atomic force microscopy. Using the method of nucleotide omission and different DNA templates, E.coli RNAP has been stalled at position +24, +70 and +379 and RNAP III at position +377 from the starting site. Conformational analysis of E.coli RNAP elongation complexes reveals an average DNA compaction of 22nm and a DNA deformation compatible with approximately 180 degrees DNA wrapping against the enzyme. The extent of protein-DNA interaction attributed to wrapping, however, is less than that of corresponding open promoter complexes. DNA wrapping was also observed for RNAP III elongation complexes, which showed a DNA compaction of 30nm. When the RNA polymerases were stalled far from the promoter (+379 and +377), the growing RNA transcript was often visible and it was prevalently seen exiting from the enzyme on the opposite side relative to the smallest angle subtended by the upstream and downstream DNA arms. Surprisingly, we found that many complexes had a second RNAP, not involved in transcription, bound to the growing RNA of a ternary complex. DNA wrapping in the elongation complex suggests a possible mechanism by which the polymerase may overcome the physical barrier to transcription imposed by the nucleosomes.

Bacterial Proteins↗
PubMed2005

The interaction between sigma70 and the beta-flap of Escherichia coli RNA polymerase inhibits extension of nascent RNA during early elongation.

The sigma-subunit of bacterial RNA polymerase (RNAP) is required for promoter-specific transcription initiation. This function depends on specific intersubunit interactions that occur when sigma associates with the RNAP core enzyme to form RNAP holoenzyme. Among these interactions, that between conserved region 4 of sigma and the flap domain of the RNAP beta-subunit (beta-flap) is critical for recognition of the major class of bacterial promoters. Here, we describe the isolation of amino acid substitutions in region 4 of Escherichia coli sigma(70) that have specific effects on the sigma(70) region 4/beta-flap interaction, either weakening or strengthening it. Using these sigma(70) mutants, we demonstrate that the sigma region 4/beta-flap interaction also can affect events occurring downstream of transcription initiation during early elongation. Specifically, our results provide support for a structure-based proposal that, when bound to the beta-flap, sigma region 4 presents a barrier to the extension of the nascent RNA as it emerges from the RNA exit channel. Our findings support the view that the transition from initiation to elongation involves a staged disruption of sigma-core interactions.

Amino Acid Substitution↗
PubMed1998

Optimization of differential display of prokaryotic mRNA: application to pure culture and soil microcosms.

The differential display (DD) technique, which is widely used almost exclusively for eukaryotic gene discovery, was optimized to detect differential mRNA transcription from both pure-culture and soil-derived bacterial RNA. A model system which included toluene induction of todC1 in Pseudomonas putida F1 was used to optimize the procedure. At 24-h tod induction was determined to be approximately 8 x 10(7) transcripts/microg or 0.08% of the total mRNA. The primer concentration, primer length, annealing temperature, and template, deoxynucleoside triphosphate, and MgCl2 concentrations were varied to optimize amplification of a todC1 fragment. The limit of detection of todC1 by DD was found to be 0.015 ng of total RNA template or approximately 10(3) transcripts. Once optimized, a todC1C2 gene fragment from P. putida F1 RNA was detected by using an arbitrary primer for the reverse transcriptase step in conjunction with the same arbitrary primer and a Shine-Dalgarno primer in the PCR. To verify the results, an arbitrary primer was used to detect recovery of a new salicylate-inducible naphthalene dioxygenase in Burkholderia cepacia JS150. The method was then used to detect mRNA induction in both inoculated and uninoculated toluene-induced soil microcosms. Several putative differentially expressed partial gene sequences obtained from the uninoculated microcosms were examined, and one novel fragment was found to be differentially expressed.

Gene Expression Regulation, Bacterial↗
PubMed2003

The flap domain is required for pause RNA hairpin inhibition of catalysis by RNA polymerase and can modulate intrinsic termination.

Bacterial RNA polymerase (RNAP) responds to formation of RNA secondary structures (hairpins) in newly synthesized RNA. Depending on the spacing of the hairpin from the RNA 3' end and the intervening RNA sequence, the hairpin can prolong pausing or cause transcriptional termination. At the his pause site, the pause hairpin contacts a flexible domain on RNAP called the flap, which forms a critical part of a hairpin-interaction site on the enzyme. We report that pause hairpin-flap interaction stabilizes an inhibited configuration of RNAP's active site without changing RNAP's translocation register. The distal part of the flap (the flap tip) is required for the hairpin to affect the active site, but not for hairpin formation. In contrast, the flap tip is not required for intrinsic termination, but can modulate it at suboptimal termination signals.

Binding Sites↗
PubMed1998

Comparative structure analysis of vertebrate ribonuclease P RNA.

Ribonuclease P cleaves 5'-precursor sequences from pre-tRNAs. All cellular RNase P holoenzymes contain homologous RNA elements; the eucaryal RNase P RNA, in contrast to the bacterial RNA, is catalytically inactive in the absence of the protein component(s). To understand the function of eucaryal RNase P RNA, knowledge of its structure is needed. Considerable effort has been devoted to comparative studies of the structure of this RNA from diverse organisms, including eucaryotes, primarily fungi, but also a limited set of vertebrates. The substantial differences in the sequences and structures of the vertebrate RNAs from those of other organisms have made it difficult to align the vertebrate sequences, thus limiting comparative studies. To expand our understanding of the structure of diverse RNase P RNAs, we have isolated by PCR and sequenced 13 partial RNase P RNA genes from 11 additional vertebrate taxa representing most extant major vertebrate lineages. Based on a recently proposed structure of the core elements of RNase P RNA, we aligned the sequences and propose a minimum consensus secondary structure for the vertebrate RNase P RNA.

Animals↗
PubMed1993

Yeast RNA polymerase II subunit RPB11 is related to a subunit shared by RNA polymerase I and III.

The characterization of RNA polymerase subunit genes has revealed that some subunits are shared by the three nuclear enzymes, some are homologous, and some are unique to RNA polymerases I, II, or III. We report here the isolation and characterization of the yeast RNA polymerase II subunit RPB11, which is encoded by a single copy RPB11 gene located directly upstream of the topoisomerase I gene, TOPI, on chromosome XV. The sequence of the gene predicts an RPB11 subunit of 120 amino acids (13,600 daltons), only two amino acids shorter than the RPB9 polypeptide, that co-migrates with RPB11 under most SDS-PAGE conditions, RPB11 was found to be an essential gene that encodes a protein closely related to an essential subunit shared by RNA polymerases I and III, AC19. RPB11 contains a 19 amino acid segment found in three other yeast RNA polymerase subunits and the bacterial RNA polymerase subunit alpha. Some mutations that affect RNA polymerase assembly map within this segment, suggesting that this region may play a role in subunit interactions. As the isolation of RPB11 completes the isolation of known yeast RNA polymerase II subunit genes, we briefly summarize the salient features of these twelve genes and the polypeptides that they encode.

Amino Acid Sequence↗
PubMedDate not supplied

[Effect of Escherichia coli mutation affecting the RNA polymerase sigma factor on phage T4 development].

The bacterial RNA polymerase sigma factor is necessary throughout T4 development. T4 can develop in the E. coli RpoD800 mutant cells only at permissive temperature. RNA synthesis in T4-infected mutant cells remains temperature-sensitive throughout infection as in uninfected mutant bacteria. This shows that bacterial sigma factor is necessary for all types of RNA synthesis in infected E. coli. The data obtained suggest also that active sigma factor is necessary for early, but not for late T4 DNA replication.

DNA Replication↗