RNA Synthesis In Bacteria Research Articles

A Role for the RNA Polymerase Gene Specificity Factor σ 54 in the Uniform Colony Growth of Uropathogenic Escherichia coli

ABSTRACTThe canonical function of a bacterial sigma (σ) factor is to determine the gene specificity of the RNA polymerase (RNAP). In several diverse bacterial species, the σ54 factor uniquely confers distinct functional and regulatory properties on the RNAP. A hallmark feature of the σ54-RNAP is the obligatory requirement for an activator ATPase to allow transcription initiation. Different activator ATPases couple diverse environmental cues to the σ54-RNAP to mediate adaptive changes in gene expression. Hence, the genes that rely upon σ54 for their transcription have a wide range of different functions suggesting that the repertoire of functions performed by genes, directly or indirectly affected by σ54, is not yet exhaustive. By comparing the growth patterns of prototypical enteropathogenic, uropathogenic, and nonpathogenic Escherichia coli strains devoid of σ54, we uncovered that the absence of σ54 results in two differently sized colonies that appear at different times specifically in the uropathogenic E. coli (UPEC) strain. Notably, UPEC bacteria devoid of individual activator ATPases of the σ54-RNAP do not phenocopy the σ54 mutant strain. Thus, it seems that σ54’s role as a determinant of uniform colony appearance in UPEC bacteria represents a putative non-canonical function of σ54 in regulating genetic information flow.IMPORTANCE RNA synthesis is the first step of gene expression. The multisubunit RNA polymerase (RNAP) is the central enzyme responsible for RNA synthesis in bacteria. The dissociable sigma (σ) factor subunit directs the RNAP to different sets of genes to allow their expression in response to various cellular needs. Of the seven σ factors in Escherichia coli and related bacteria, σ54 exists in a class of its own. This study has uncovered that σ54 is a determinant of the uniform growth of uropathogenic E. coli on solid media. This finding suggests a role for this σ54 in gene regulation that extends beyond its known function as an RNAP gene specificity factor.

Structural basis of ribosomal RNA synthesis in bacteria

Structural basis of ribosomal RNA synthesis in bacteria

Cyanobacterial Metabolite Calothrixins: Recent Advances in Synthesis and Biological Evaluation

The marine environment is host to unparalleled biological and chemical diversity, making it an attractive resource for the discovery of new therapeutics for a plethora of diseases. Compounds that are extracted from cyanobacteria are of special interest due to their unique structural scaffolds and capacity to produce potent pharmaceutical and biotechnological traits. Calothrixins A and B are two cyanobacterial metabolites with a structural assembly of quinoline, quinone, and indole pharmacophores. This review surveys recent advances in the synthesis and evaluation of the biological activities of calothrixins. Due to the low isolation yields from the marine source and the promise this scaffold holds for anticancer and antimicrobial drugs, organic and medicinal chemists around the world have embarked on developing efficient synthetic routes to produce calothrixins. Since the first review appeared in 2009, 11 novel syntheses of calothrixins have been published in the efforts to develop methods that contain fewer steps and higher-yielding reactions. Calothrixins have shown their potential as topoisomerase I poisons for their cytotoxicity in cancer. They have also been observed to target various aspects of RNA synthesis in bacteria. Further investigation into the exact mechanism for their bioactivity is still required for many of its analogs.

Bacterial global regulators DksA/ppGpp increase fidelity of transcription.

Collisions between paused transcription elongation complexes and replication forks inevitably happen, which may lead to collapse of replication fork and could be detrimental to cells. Bacterial transcription factor DksA and its cofactor alarmone ppGpp were proposed to contribute to prevention of such collisions, although the mechanism of this activity remains elusive. Here we show that DksA/ppGpp do not destabilise transcription elongation complexes or inhibit their backtracking, as was proposed earlier. Instead, we show, both in vitro and in vivo, that DksA/ppGpp increase fidelity of transcription elongation by slowing down misincorporation events. As misincorporation events cause temporary pauses, contribution to fidelity suggests the mechanism by which DksA/ppGpp contribute to prevention of collisions of transcription elongation complexes with replication forks. DksA is only the second known accessory factor, after transcription factor Gre, that increases fidelity of RNA synthesis in bacteria.

Mechanisms of physiological regulation of RNA synthesis in bacteria: new discoveries breaking old schemes

Although in bacterial cells all genes are transcribed by RNA polymerase, there are 2 additional enzymes capable of catalyzing RNA synthesis: poly(A) polymerase I, which adds poly(A) residues to transcripts, and primase, which produces primers for DNA replication. Mechanisms of actions of these 3 RNA-synthesizing enzymes were investigated for many years, and schemes of their regulations have been proposed and generally accepted. Nevertheless, recent discoveries indicated that apart from well-understood mechanisms, there are additional regulatory processes, beyond the established schemes, which allow bacterial cells to respond to changing environmental and physiological conditions. These newly discovered mechanisms, which are discussed in this review, include: (i) specific regulation of gene expression by RNA polyadenylation, (ii) control of DNA replication by interactions of the starvation alarmones, guanosine pentaphosphate and guanosine tetraphosphate, (p)ppGpp, with DnaG primase, (iii) a role for the DksA protein in ppGpp-mediated regulation of transcription, (iv) allosteric modulation of the RNA polymerase catalytic reaction by specific inhibitors of transcription, rifamycins, (v) stimulation of transcription initiation by proteins binding downstream of the promoter sequences, and (vi) promoter-dependent control of transcription antitermination efficiency.

Initial nucleotide frequencies of bacterial RNA synthesized during amino-acid starvation or changes of carbon source.

THERE is growing evidence to indicate that RNA synthesis in bacteria is regulated through adjustment of the frequency of initiation of new RNA molecules. In this framework an understanding of the process of initiation of RNA synthesis takes on a special importance, and for this reason we have investigated in varying conditions the composition of the 5′ terminal or first-inserted nucleotide. It has been previously shown that such initiations do not occur randomly, either in vivo1 or in the proper conditions in vitro2,3, but that RNA chains are exclusively initiated with the purines, adenosine and guanosine. Additionally, there was a recent suggestion based on in vitro studies that the nucleotide guanosine-3′-diphosphate-5′-diphosphate (MS1), proposed to be a regulatory agent in RNA synthesis, functioned by specifically depressing the frequency of initiation of a large fraction of RNA molecules beginning with guanosine4. Here we report, however, that in vivo, in conditions in which regulation of RNA synthesis is manifest, the ratio of molecules initiated with adenosine and guanosine is not changed.

Role of host RNA polymerase for λ phage development

Rifampicin, a specific inhibitor of RNA synthesis in bacteria, markedly inhibits growth of λ phage on wild-type Escherichia coli. Synthesis of both early and late λ messenger RNA was inhibited by rifampicin even when the drug was added after phage messenger RNA synthesis already started. In contrast, normal phage growth was observed on any of the rifampicin-resistant mutants tested producing RNA polymerase that are themselves resistant to the drug. These results suggest that host RNA polymerase (at least components relating to rifampicin sensitivity) is involved in expression of λ genes throughout the phage development.

Studies on the amino acid regulation of RNA-synthesis in mammalian cells in tissue culture

In normal stringent control of RNA synthesis in bacteria, RNA formation is dependent on the internal concentration of amino acids in the cells. To find out if mammalian cells have a similar regulatory link between the availability of amino acids for protein synthesis and RNA formation, was the aim of the present investigation. RNA, protein and DNA synthesis was studied in monolayer cultures of mouse fibroblasts (strain L-929). Macromolecular synthesis was measured by the incorporation of 14C-uridine and radioactive amino acids respectively, into acid-insoluble product. Amino acid starvation was achieved by excluding all free amino acids and serum from the medium. Serum starvation for up to 12 h was found not to diminish DNA or protein synthesis. RNA synthesis was depressed to 20–50%. The rate of RNA synthesis in mouse fibroblasts was unimpaired under conditions where the rate of protein synthesis was decreased to 30% of normal by amino acid starvation. This protein synthesis in the absence of added amino acids was shown to be related to the rate of intracellular protein degradation. The uptake of 14C-uridine into the uridine nucleotide pools of the cells was similarly unimpaired by amino acid starvation. In contrast, DNA synthesis was markedly inhibited by amino acid starvation. The DNA synthesis of starved cells diminished gradually and seemed to stop after 4 to 6 h of starvation. This time period is comparable to the length of the DNA-synthesis period (S-phase) of this strain of fibroblasts.

Effect of steffimycin on cell-free polypeptide synthesis

Steffimycin interferes with amino acid incorporation mediated by synthetic polyribonucleotides in cell-free bacterial systems. This inhibition is a secondary activity of the antibiotic, which acts primarily as a suppressor of RNA synthesis in bacteria and bacterial cell-free systems. Inhibition of peptide biosynthesis is apparent only at a drug concentration higher than that necessary for inhibition of RNA synthesis in cell-free systems. The chelating properties of the antibiotic toward divalent cations do not appear to be responsible for inhibition of peptide biosynthesis. Reversal studies suggested that excess transfer RNA but not 30S fraction or polyuridylic acid reduces steffimycin inhibition of amino acid incorporation. Difference spectra obtained from antibiotic-transfer RNA mixtures indicate that steffimycin binds to transfer RNA. It is concluded that the binding of steffimycin to transfer RNA is the basis for its interference with amino acid incorporation.

Actinomycin inhibition of [formula omitted] protein synthesis in rat liver

Actinomycin inhibits the synthesis of RNA in bacteria and in mammalian cells in culture ( Kirk, 1960; Reich, Franklin, Shatkin and Tatum, 1962; Tamaoki and Mueller, 1962; Harbers and Muller, 1962; Hurwitz, Furth, Malamy and Alexander, 1962; Goldberg, Rabinowitz and Reich, 1962). At least three types of RNA are concerned in protein biosynthesis so that inhibition of RNA synthesis should be reflected in inhibition of protein synthesis (cf. Kirk, 1960). Meritz (1963) has recently shown that actinomycin inhibits all RNA synthesis in rat liver and the evidence presented below indicates that actinomycin inhibits protein synthesis in rat liver.

Regulation of Metabolic Breakdown and Synthesis of Messenger RNA in Bacteria

Regulation of Metabolic Breakdown and Synthesis of Messenger RNA in Bacteria

Use of chloramphenicol to study control of RNA synthesis in bacteria

With a view to deciding whether changes in the rate of synthesis of RNA (relative to that of protein or DNA) in bacteria are dependent on variations in amounts of enzymes concerned with this synthesis, use had been made of the fact that chloramphenicol addition to these cells blocks protein but not RNA synthesis. Measurements have been made of the rate of RNA synthesis before and after chloramphenicol addition (a) to cultures in balanced growth making RNA at different rates relative to their synthesis of protein or DNA, and (b) to cultures in unbalanced growth with changing rates of RNA synthesis. The findings were: 1. 1. Chloramphenicol addition to cultures in balanced growth usually results in an increase in the rate of RNA synthesis. 2. 2. In many cases the rate of RNA synthesis in the presence of chloramphenicol can be markedly increased by enrichment of the medium after chloramphenicol addition. 3. 3. With cultures early in the depressed phase of RNA synthesis during decelerating growth, the addition of chloramphenicol causes a resumption of fast RNA synthesis; later addition of chloramphenicol results in progressively less stimulation. These results are interpreted to mean that the rate of RNA synthesis in Aerobacter aerogenes is not primarily controlled by variations in the amount of enzymes concerned with this synthesis. The amounts of some of these enzymes, however, may be secondarily adjusted to match the actual rate of RNA synthesis.

Metabolic regulation of RNA synthesis in bacteria.

Bacteria are small, unicellular organisms whose ecological niche comprises an incredibly large number of microcosms which are subject to frequent and cataclysmic changes in their physical and chemical properties (Stanier, 1953). Evolutionary survival of these organisms has depended not only upon their ability to grow as rapidly as possible under a wide variety of ambient conditions, but also upon their ability to adjust as rapidly as possible to frequent changes in these conditions. Thus it is not surprising to find that bacteria possess many regulatory devices which render their metabolic processes adaptively responsive to the environment. These devices permit adjustment of the cells' internal affairs so as to allow a rapid, orderly synthesis of protoplasm in the face of inconstant ambient conditions.