Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Reverse transcriptase of the human immunodeficiency virus possesses DNA polymerase and ribonuclease (RNase) H activities. Although the nucleic acid binding cleft separating these domains can accommodate structurally diverse duplexes, it is currently unknown whether regular DNA/RNA hybrids can simultaneously contact both active sites. In this study, we demonstrate that ligands capable of trapping the 3'-end of the primer at the polymerase active site affect the specificity of RNase H cleavage without altering the efficiency of the reaction. Experiments under single-turnover conditions reveal that complexes with a bound nucleotide substrate show specific RNase H cleavage at template position -18, while complexes with the pyrophosphate analogue foscarnet show a specific cut at position -19. This pattern is indicative of post-translocated and pre-translocated conformations. The data are inconsistent with models postulating that the substrate toggles between both active sites, such that the primer 3'-terminus is disengaged from the polymerase active site when the template is in contact with the RNase H active site. In contrast, our findings provide strong evidence to suggest that the nucleic acid substrate can engage both active sites at the same time. As a consequence, the bound and intact DNA/RNA hybrid can restrict access of RNase H active site inhibitors. We have mapped the binding site of the recently discovered inhibitor beta-thujaplicinol between the RNase H active site and Y501 of the RNase H primer grip, and have shown that the inhibitor is unable to bind to a preformed reverse transcriptase-DNA/RNA complex. In conclusion, the bound nucleic acid substrate and in turn, active DNA synthesis can represent an obstacle to RNase H inhibition with compounds that bind to the RNase H active site.
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PMID:HIV-1 reverse transcriptase can simultaneously engage its DNA/RNA substrate at both DNA polymerase and RNase H active sites: implications for RNase H inhibition. 1928 31

How living cells deal with head-on collisions of the replication and transcription complexes has been debated for a long time. Even in the widely studied model bacteria Escherichia coli, the enzymes that take care of such collisions are still unknown. We report here that in vivo, the DinG, Rep and UvrD helicases are essential for efficient replication across highly transcribed regions. We show that when rRNA operons (rrn) are inverted to face replication, the viability of the dinG mutant is affected and over-expression of RNase H rescues the growth defect, showing that DinG acts in vivo to remove R-loops. In addition, DinG, Rep and UvrD exert a common function, which requires the presence of two of these three helicases. After replication blockage by an inverted rrn, Rep in conjunction with DinG or UvrD removes RNA polymerase, a task that is fulfilled in its absence by the SOS-induced DinG and UvrD helicases. Finally, Rep and UvrD also act at inverted sequences other than rrn, and promote replication through highly transcribed regions in wild-type E. coli.
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PMID:The helicases DinG, Rep and UvrD cooperate to promote replication across transcription units in vivo. 1985 Dec 82

A hallmark of metazoan RNA polymerase II transcripts is the presence of numerous small exons surrounded by large introns. Abundant evidence indicates that splicing to excise introns occurs co-transcriptionally, prior to release of the nascent transcript from RNAP II. Here, we established an efficient model system for co-transcriptional splicing in vitro. In this system, CMV-DNA constructs immobilized on beads generate RNAP II transcripts containing two exons and an intron. Consistent with previous work, our data indicate that elongating nascent transcripts are tethered to RNAP II on the immobilized DNA template. We show that nascent transcripts that reach full length, but are still attached to RNAP II, are efficiently spliced. When the nascent transcript is cleaved within the intron using RNase H, both the 5' and 3' cleavage fragments are detected in the bound fraction, where they undergo splicing. Together, our work establishes a system for co-transcriptional splicing in vitro, in which the spliceosome containing the 5' and 3' exons are tethered to RNAP II for splicing.
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PMID:A model in vitro system for co-transcriptional splicing. 2063 Oct 7

During translation of the genetic information of DNA into proteins, mRNA is synthesized by RNA polymerase and after the transcription process degraded by RNase H. The endoribonuclease RNase H is a member of the nucleotidyl-transferase (NT) superfamily and is known to hydrolyze the phosphodiester bonds of RNA which is hybridized to DNA. Retroviral RNase H is part of the viral reverse transcriptase enzyme that is indispensable for the proliferation of retroviruses, such as HIV. Inhibitors of this enzyme could therefore provide new drugs against diseases like AIDS. In our study we investigated the molecular mechanism of RNA cleavage by human RNase H using a comprehensive high level DFT/B3LYP QM/MM theoretical method for the calculation of the stationary points and nudged elastic band (NEB) and free energy calculations to identify the transition state structures, the rate limiting step and the reaction barrier. Our calculations reveal that the catalytic mechanism proceeds in two steps and that the nature of the nucleophile is a water molecule. In the first step, the water attack on the scissile phosphorous is followed by a proton transfer from the water to the O2P oxygen and a trigonal bipyramidal pentacoordinated phosphorane is formed. Subsequently, in the second step the proton is shuttled to the O3' oxygen to generate the product state. During the reaction mechanism two Mg(2+) ions support the formation of a stable associated in-line S(N)2-type phosphorane intermediate. Our calculated energy barrier of 19.3 kcal mol(-1) is in excellent agreement with experimental findings (20.5 kcal mol(-1)). These results may contribute to the clarification and understanding of the RNase H reaction mechanism and of further enzymes from the RNase family.
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PMID:Atomistic details of the associative phosphodiester cleavage in human ribonuclease H. 2067 57

Reverse transcriptase (RT) catalyzes the formation of dsDNA from single-stranded retroviral RNA genome. This enzyme is unique among DNA polymerases in its ability to use either RNA or DNA as a template. Moloney Murine Leukemia virus reverse transcriptase lacking RNase H activity (M-MLVH- RT) especially holds particular interest because of its ability to eliminate the deleterious effect of RNase H, which results in more efficient synthesis of full-length cDNA from mRNA. Therefore, the development of a simple purification method attracts the attention of retroviral drug and enzyme researchers and manufacturers. The present work is the first purification example of a non-tagged (native) RT by affinity chromatography using synthetic affinity ligands. In this study, the ligand was selected from a structure-biased combinatorial library of dNTP-mimetic ligands, and it was evaluated for its ability to bind and purify M-MLVH- RT from inclusion bodies of recombinant E. coli. The selected ligand (AEAd), bearing 9-aminoethyladenine and 1,6-diamine-hexane both linked on the same triazine scaffold, displayed the highest enzyme purifying ability after applying mild desorption conditions (6 mM MnCl(2) in 20 mM Tris-HCl buffer, pH 7.5). The binding capacity of immobilized AEAd with M-MLVH- RT was determined to be equal to approximately 1 mg enzyme/g moist weight gel. Adsorption studies with immobilized AEAd and soluble M-MLVH- RT demonstrated that the formation of the respective complex was perturbed by ATP. Quality control tests of the purified M-MLVH- RT essentially showed a single band (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and absence of nucleic acids and contaminating nuclease activities.
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PMID:Purification of M-MLVH- RT on a 9-aminoethyladenine-(1,6-diamine-hexane)-triazine selected from a combinatorial library of dNTP-mimetic ligands. 2082 67

The construction of synthetic biochemical circuits from simple components illuminates how complex behaviors can arise in chemistry and builds a foundation for future biological technologies. A simplified analog of genetic regulatory networks, in vitro transcriptional circuits, provides a modular platform for the systematic construction of arbitrary circuits and requires only two essential enzymes, bacteriophage T7 RNA polymerase and Escherichia coli ribonuclease H, to produce and degrade RNA signals. In this study, we design and experimentally demonstrate three transcriptional oscillators in vitro. First, a negative feedback oscillator comprising two switches, regulated by excitatory and inhibitory RNA signals, showed up to five complete cycles. To demonstrate modularity and to explore the design space further, a positive-feedback loop was added that modulates and extends the oscillatory regime. Finally, a three-switch ring oscillator was constructed and analyzed. Mathematical modeling guided the design process, identified experimental conditions likely to yield oscillations, and explained the system's robust response to interference by short degradation products. Synthetic transcriptional oscillators could prove valuable for systematic exploration of biochemical circuit design principles and for controlling nanoscale devices and orchestrating processes within artificial cells.
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PMID:Synthetic in vitro transcriptional oscillators. 2128 41

C7-Substituted 2-hydroxyisoquinoline-1,3-diones inhibit the strand transfer of HIV integrase (IN) and the reverse-transcriptase-associated ribonuclease H (RNH). Hepatitis C virus (HCV) NS5B polymerase shares a similar active site fold to RNH and IN, suggesting that N-hydroxyimides could be useful inhibitor scaffolds of HCV via targeting the NS5B. Herein we describe the design, chemical synthesis, replicon and biochemical assays, and molecular docking of C-6 or C-7 aryl substituted 2-hydroxyisoquinoline-1,3-diones as novel HCV inhibitors. The synthesis involved an improved and clean cyclization method, which allowed the convenient preparation of various analogs. Biological studies revealed that the C-6 analogs, a previously unknown chemotype, consistently inhibit both HCV replicon and recombinant NS5B at low micromolar range. Molecular modeling studies suggest that these inhibitors may bind to the NS5B active site.
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PMID:The design, synthesis and biological evaluations of C-6 or C-7 substituted 2-hydroxyisoquinoline-1,3-diones as inhibitors of hepatitis C virus. 2210 Feb 56

Microarrays of RNA aptamers are fabricated in a one-step, multiplexed enzymatic synthesis on gold thin films in a microfluidic format and then employed in the detection of protein biomarkers with surface plasmon resonance imaging (SPRI) measurements. Single-stranded RNA (ssRNA) oligonucleotides are transcribed on-chip from double-stranded DNA (dsDNA) templates attached to microarray elements (denoted as generator elements) by the surface transcription reaction of T7 RNA polymerase. As they are synthesized, the ssRNA oligonucleotides diffuse in the microfluidic channel and are quickly captured by hybridization adsorption onto adjacent single-stranded DNA (ssDNA) microarray elements (denoted as detector elements) that contain a sequence complementary to 5'-end of the ssRNA. The RNA aptamers attached to these detector elements are subsequently used in SPRI measurements for the bioaffinity detection of protein biomarkers. The microfluidic generator-detector element format permits the simultaneous fabrication of multiple ssRNA oligonucleotides with different capture sequences that can hybridize simultaneously to distinct detector elements and thus create a multiplexed aptamer microarray. In an initial set of demonstration experiments, SPRI measurements are used to monitor the bioaffinity adsorption of human thrombin (hTh) and vascular endothelial growth factor (VEGF) proteins onto RNA aptamer microarrays fabricated in situ with this on-chip RNA polymerase synthesis methodology. Additional SPRI measurements of the hydrolysis and desorption of the surface-bound ssRNA aptamers with a surface RNase H are used to verify the capture of ssRNA with RNA-DNA surface hybridization onto the detector elements. The on-chip RNA synthesis described here is an elegant, one-step multiplexed methodology for the rapid and contamination-free fabrication of RNA aptamer microarrays for protein biosensing with SPRI.
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PMID:On-chip synthesis of RNA aptamer microarrays for multiplexed protein biosensing with SPR imaging measurements. 2245 58

Reverse transcriptase of human immunodeficiency virus type 1 (HIV-1) has two enzymatic functions. One of the functions is ribonuclease (RNase) H activity concerning the digestion of only RNA of RNA/DNA hybrid. The RNase H activity is an attractive target for a new class of anti-HIV drugs because no approved inhibitor is available now. In our previous studies, an agent bearing 5-nitro-furan-2-carboxylic acid ester core was found from chemical screening and dozens of the derivatives were synthesized to improve compound potency. In this work, some parts of the chemical structure were modulated to deepen our understanding of the structure-activity relationship of the analogous compounds. Several derivatives having nitro-furan-phenyl-ester skeleton were shown to be potent RNase H inhibitors. Attaching methoxy-carbonyl and methoxy groups to the phenyl ring increased the inhibitory potency. No significant cytotoxicity was observed for these active derivatives. In contrast, the derivatives having nitro-furan-benzyl-ester skeleton showed modest inhibitory activities regardless of attaching diverse kinds of functional groups to the benzyl ring. Both the modulation of the 5-nitro-furan-2-carboxylic moiety and the conversion of the ester linkage resulted in a drastic decrease in inhibitory potency. These findings are informative for designing potent inhibitors of RNase H enzymatic activity of HIV-1.
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PMID:Structural modulation study of inhibitory compounds for ribonuclease H activity of human immunodeficiency virus type 1 reverse transcriptase. 2268 29

An overarching goal of synthetic and systems biology is to engineer and understand complex biochemical systems by rationally designing and analyzing their basic component interactions. Practically, the extent to which such reductionist approaches can be applied is unclear especially as the complexity of the system increases. Toward gradually increasing the complexity of systematically engineered systems, programmable synthetic circuits operating in cell-free in vitro environments offer a valuable testing ground for principles for the design, characterization, and analysis of complex biochemical systems. Here we illustrate this approach using in vitro transcriptional circuits ("genelets") while developing an activatable transcriptional switch motif and configuring it as a bistable autoregulatory circuit, using just four synthetic DNA strands and three essential enzymes, bacteriophage T7 RNA polymerase, Escherichia coli ribonuclease H, and ribonuclease R. Fulfilling the promise of predictable system design, the thermodynamic and kinetic constraints prescribed at the sequence level were enough to experimentally demonstrate intended bistable dynamics for the synthetic autoregulatory switch. A simple mathematical model was constructed based on the mechanistic understanding of elementary reactions, and a Monte Carlo Bayesian inference approach was employed to find parameter sets compatible with a training set of experimental results; this ensemble of parameter sets was then used to predict a test set of additional experiments with reasonable agreement and to provide a rigorous basis for confidence in the mechanistic model. Our work demonstrates that programmable in vitro biochemical circuits can serve as a testing ground for evaluating methods for the design and analysis of more complex biochemical systems such as living cells.
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PMID:Ensemble Bayesian analysis of bistability in a synthetic transcriptional switch. 2365 Dec 85


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