Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Kinetics and template-dependency of ribonucleic acid synthesis by bacterial ribonucleic acid polymerase.

ABSTRACT: The rate of RNA synthesis catalysed by DNA-dependent RNA polymerase shows a Michealis-Menten-type saturation curve with increasing template concentration. However, the apparent Km is proportional to enzyme concentration, indicating that the reaction does not obey a simple kinetic scheme. The action of inhibitors also indicates a more complex interaction between the enzyme and the DNA template; many inhibitors of RNA synthesis either decrease Vmax. without affecting Km, or increase Km without affecting Vmax. All of these observations can be accounted for quantitatively by a reaction pathway in which the non-specific binding sites of the viral DNA template inhibit competitively the binding of the enzyme to the initiation sites. In terms of this pathway the two classes of inhibitors of RNA synthesis must then act predominantly either on the rate of elongation or on the availability of the binding sites respectively.

SUBMITTER: Panayotatos N

PROVIDER: S-EPMC1184182 | biostudies-other | 1978 Feb

REPOSITORIES: biostudies-other

ACCESS DATA

Json Xml

Similar Datasets

Bluetongue virus VP1 polymerase activity in vitro: template dependency, dinucleotide priming and cap dependency.

Project description:BackgroundBluetongue virus (BTV) protein, VP1, is known to possess an intrinsic polymerase function, unlike rotavirus VP1, which requires the capsid protein VP2 for its catalytic activity. However, compared with the polymerases of other members of the Reoviridae family, BTV VP1 has not been characterized in detail.Methods and findingsUsing an in vitro polymerase assay system, we demonstrated that BTV VP1 could synthesize the ten dsRNAs simultaneously from BTV core-derived ssRNA templates in a single in vitro reaction as well as genomic dsRNA segments from rotavirus core-derived ssRNA templates that possess no sequence similarity with BTV. In contrast, dsRNAs were not synthesized from non-viral ssRNA templates by VP1, unless they were fused with specific BTV sequences. Further, we showed that synthesis of dsRNAs from capped ssRNA templates was significantly higher than that from uncapped ssRNA templates and the addition of dinucleotides enhanced activity as long as the last base of the dinucleotide complemented the 3' -terminal nucleotide of the ssRNA template.ConclusionsWe showed that the polymerase activity was stimulated by two different factors: cap structure, likely due to allosteric effect, and dinucleotides due to priming. Our results also suggested the possible presence of cis-acting elements shared by ssRNAs in the members of family Reoviridae.

| S-EPMC3216989 | biostudies-literature

Nonenzymatic polymerase-like template-directed synthesis of acyclic L-threoninol nucleic acid.

Project description:Evolution of xeno nucleic acid (XNA) world essentially requires template-directed synthesis of XNA polymers. In this study, we demonstrate template-directed synthesis of an acyclic XNA, acyclic L-threoninol nucleic acid (L-aTNA), via chemical ligation mediated by N-cyanoimidazole. The ligation of an L-aTNA fragment on an L-aTNA template is significantly faster and occurs in considerably higher yield than DNA ligation. Both L-aTNA ligation on a DNA template and DNA ligation on an L-aTNA template are also observed. High efficiency ligation of trimer L-aTNA fragments to a template-bound primer is achieved. Furthermore, a pseudo primer extension reaction is demonstrated using a pool of random L-aTNA trimers as substrates. To the best of our knowledge, this is the first example of polymerase-like primer extension of XNA with all four nucleobases, generating phosphodiester bonding without any special modification. This technique paves the way for a genetic system of the L-aTNA world.

| S-EPMC7864931 | biostudies-literature

Impact of microfluidic processing on bacterial ribonucleic acid expression.

Project description:Bacterial transcriptomics is widely used to investigate gene regulation, bacterial susceptibility to antibiotics, host-pathogen interactions, and pathogenesis. Transcriptomics is crucially dependent on suitable methods to isolate and detect bacterial RNA. Microfluidics offer ways of creating integrated point-of-care systems, analysing a sample from preparation, and RNA isolation to detection. A critical requirement for on-chip diagnostics to deliver on their promise is that mRNA expression is not altered via microfluidic sample processing. This article investigates the impact of the use of microfluidics upon RNA expression of bacteria isolated from blood, a key step towards proving the suitability of such systems for further development.

| S-EPMC4449352 | biostudies-literature

Creative template-dependent synthesis by human polymerase mu.

Project description:Among the many proteins used to repair DNA double-strand breaks by nonhomologous end joining (NHEJ) are two related family X DNA polymerases, Pol ? and Pol µ. Which of these two polymerases is preferentially used for filling DNA gaps during NHEJ partly depends on sequence complementarity at the break, with Pol ? and Pol µ repairing complementary and noncomplementary ends, respectively. To better understand these substrate preferences, we present crystal structures of Pol µ on a 2-nt gapped DNA substrate, representing three steps of the catalytic cycle. In striking contrast to Pol ?, Pol µ "skips" the first available template nucleotide, instead using the template base at the 5' end of the gap to direct nucleotide binding and incorporation. This remarkable divergence from canonical 3'-end gap filling is consistent with data on end-joining substrate specificity in cells, and provides insights into polymerase substrate choices during NHEJ.

| S-EPMC4547271 | biostudies-literature

Inhibition by soluble ribonucleic acid of stimulatory effect of liver template ribonucleic acid.

Project description:1. Liver soluble RNA (s-RNA) and, to a smaller extent, Escherichia coli s-RNA inhibited the stimulation of [(14)C]leucine incorporation into protein in an E. coli S-30 system brought about by liver microsomal RNA or polyuridylic acid as template. 2. The inhibitory activity was associated with the fraction of the s-RNA possessing transfer-RNA activity. 3. The inhibition was exercised at a stage after charging of the s-RNA with amino acid. 4. Neither the method of preparation of the s-RNA nor its state of amino acid acylation affected its inhibitory action. 5. Stimulation of [(14)C]phenylalanine incorporation by polyuridylic acid or by liver microsomal RNA was not inhibited by addition of s-RNA. 6. It appears that excess of s-RNA inhibits the ambiguous incorporation of leucine with polyuridylic acid and also that something similar occurs with a natural template. 7. Estimation of messenger activity of samples of RNA should be carried out only after removal of s-RNA.

| S-EPMC1270127 | biostudies-other

Substrate, metal and template effects on inhibition of bacteriophage-qbeta ribonucleic acid polymerase by ortho- and pyro-phosphate.

Project description:Two reactions of bacteriophage-Qbeta RNA polymerase with synthetic templates were characterized and used to study the effects of substrate, metal and template on inhibition by Pi and PPi. Analysis of the poly(C)-dependent reaction yielded results on kinetics, GTP-dependence, preference for Mn2+ over Mg2+, and Michaelis constants for template similar to those in the literature. New data are provided for the poly(U2,C)-dependent reaction. Our results suggest that GTP and Mn2+ can form relatively stable complexes with the polymerase and that such complexes change the interaction of the enzyme with the inhibitors, Pi and PPi.

| S-EPMC1184020 | biostudies-other

DNA template sequence control of bacterial RNA polymerase escape from the promoter.

Project description:Promoter escape involves breaking of the favourable contacts between RNA polymerase (RNAP) and the promoter to allow transition to an elongation complex. The sequence of DNA template that is transcribed during promoter escape (ITS; Initially Transcribed Sequence) can affect promoter escape by mechanisms that are not yet fully understood. We employed a highly parallel strategy utilizing Next Generation Sequencing (NGS) to collect data on escape properties of thousands of ITS variants. We show that ITS controls promoter escape through a combination of position-dependent effects (most prominently, sequence-directed RNAP pausing), and position-independent effects derived from sequence encoded physical properties of the template (for example, RNA/DNA duplex stability). ITS often functions as an independent unit affecting escape in the same manner regardless of the promoter from which transcription initiates. However, in some cases, a strong dependence of ITS effects on promoter context was observed suggesting that promoters may have 'allosteric' abilities to modulate ITS effects. Large effects of ITS on promoter output and the observed interplay between promoter sequence and ITS effects suggests that the definition of bacterial promoter should include ITS sequence.

| S-EPMC5961368 | biostudies-literature

Template strand scrunching during DNA gap repair synthesis by human polymerase lambda.

Project description:Family X polymerases such as DNA polymerase lambda (Pol lambda) are well suited for filling short gaps during DNA repair because they simultaneously bind both the 5' and 3' ends of short gaps. DNA binding and gap filling are well characterized for 1-nucleotide (nt) gaps, but the location of yet-to-be-copied template nucleotides in longer gaps is unknown. Here we present crystal structures revealing that, when bound to a 2-nt gap, Pol lambda scrunches the template strand and binds the additional uncopied template base in an extrahelical position within a binding pocket that comprises three conserved amino acids. Replacing these amino acids with alanine results in less processive gap filling and less efficient NHEJ when 2-nt gaps are involved. Thus, akin to scrunching by RNA polymerase during transcription initiation, scrunching occurs during gap filling DNA synthesis associated with DNA repair.

| S-EPMC2767187 | biostudies-literature

NCResNet: Noncoding Ribonucleic Acid Prediction Based on a Deep Resident Network of Ribonucleic Acid Sequences.

Project description:Noncoding RNA (ncRNA) is a kind of RNA that plays an important role in many biological processes, diseases, and cancers, while cannot translate into proteins. With the development of next-generation sequence technology, thousands of novel RNAs with long open reading frames (ORFs, longest ORF length > 303 nt) and short ORFs (longest ORF length ? 303 nt) have been discovered in a short time. How to identify ncRNAs more precisely from novel unannotated RNAs is an important step for RNA functional analysis, RNA regulation, etc. However, most previous methods only utilize the information of sequence features. Meanwhile, most of them have focused on long-ORF RNA sequences, but not adapted to short-ORF RNA sequences. In this paper, we propose a new reliable method called NCResNet. NCResNet employs 57 hybrid features of four categories as inputs, including sequence, protein, RNA structure, and RNA physicochemical properties, and introduces feature enhancement and deep feature learning policies in a neural net model to adapt to this problem. The experiments on benchmark datasets of 8 species shows NCResNet has higher accuracy and higher Matthews correlation coefficient (MCC) compared with other state-of-the-art methods. Particularly, on four short-ORF RNA sequence datasets, specifically mouse, Saccharomyces cerevisiae, zebrafish, and cow, NCResNet achieves greater than 10 and 15% improvements over other state-of-the-art methods in terms of accuracy and MCC. Meanwhile, for long-ORF RNA sequence datasets, NCResNet also has better accuracy and MCC than other state-of-the-art methods on most test datasets. Codes and data are available at https://github.com/abcair/NCResNet.

| S-EPMC7059790 | biostudies-literature

Nucleic acid enzymology of extremely halophilic bacteria. Halobacterium cutirubrum ribonucleic acid-dependent ribonucleic acid polymerase.

Project description:1. Crude extracts of the extreme halophile Halobacterium cutirubrum contain separable DNA-dependent and RNA-dependent RNA polymerases. 2. The RNA-dependent enzyme has been purified about 2800-fold. 3. It requires RNA, preferably of high molecular weight, and all four ribonucleoside triphosphates to incorporate (14)C-labelled nucleoside triphosphate into an acid-insoluble, ribonuclease-sensitive product. 4. Both the stability and activity of the RNA polymerase are relatively insensitive to changes in potassium chloride or sodium chloride concentration, but incorporation is stimulated by both Mg(2+) and Mn(2+). 5. The molecular weight of the enzyme is about 17000-18000.

| S-EPMC1176639 | biostudies-other

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data