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)

Rifampin-resistant mutants were isolated from Lactobacillus casei S1 and examined for possible simultaneous alteration in nutritional properties. Among the 36 mutants obtained either spontaneously or after mutagenesis with 2-aminopurine, 22 were found to be altered with respect to the specific growth requirements. The majority (20 of 22) of the latter mutants were shown to require L-glutamine in addition to the nutrients required by the parental strain for maximal growth, whereas the remaining mutants had apparently lost the requirement for L-aspartate. Further studies with one of the glutamine-requiring mutants revealed that the rifampin resistance of this strain is due to the resistance of ribonucleic acid polymerase itself and that a single mutation is responsible for both rifampin resistance and the glutamine requirement. These results strongly indicate that a structural alteration of the ribonucleic acid polymerase caused by the rifampin resistance mutation somehow affected glutamine metabolism, possibly through change in selective transcription of the genes involved.
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PMID:Altered nutritional requirements associated with mutations affecting the structures of ribonucleic acid polymerase in Lactobacillus casei. 0 79

The behavior of nucelotides with thioketo-substituted pyrimidine bases (4-thiouracil, 2-thiouracil and 2-thiocytosine) or amino-analogue purine bases (2-aminopurine and 2,6-diaminopurine) in transcription and translation was investigated. The experimental results obtained led to the following conclusions. 1. The stereochemical basis of substrate selection in transcription is the geometry of Watson-Crick base pairs A-U (or A-T) and G-C between substrate and template bases. 2. The topology of the active site of Escherichia coli RNA polymerase is precisely adopted to the geometry of Watson-Crick base pairs. 3. The enzyme active site discriminates between A-U (A-T) and G-C base pairs. An essential feature in this discrimination is the 6-NH2 group of the A-U (A-T) base pair and the 2-keto group of cytosine in the G-C base pair. 4. The codon properties of a nucleic acid base in messenger RNA can be predicted on the basis of its specificity in polynucleotide interactions. There seems to be no evidence for the participation of protein topological sites in the control of the specificity of codon-anticodon interactions in translation.
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PMID:The stereochemical basis of template function. 31

The term vitamin D includes various chemical species. Vitamin D3 a true endogenous or alimentary prohormone is converted into its main metabolite, calcitriol, by successive hydroxylations in the liver in position 25 and in the kidney in position 1, the production of which is controlled by several factors including parathyroid hormone, blood calcium and phosphorus or insulin as well as by the metabolites of the hormone itself. It controls the synthesis of numerous peptides by acting on gene expression. Indeed, several structural proteins are involved including procollagen alpha 1l, core protein of proteoglycans, diverse regulatory peptides such as protooncogene c-myc and growth factors, "Tumor Necrosis Factor or TNF" and "Nerve Growth Factor or NGF" or hormones such as parathyroid hormone, and finally constitutive proteins of the mineralized tissues such as osteonectin, osteocalcin, osteopontin and calbindins. Therefore, it modulates very different cellular processes. It acts via a nuclear receptor the structure and function of which have been investigated by genetic engineering (cloning of genes encoding for the receptor and hormono-dependent peptides, transfection assays, directed mutagenesis). Actual studies investigate its role in the formation of the complex for transcription initiation near ADN sites, the "Vitamin D Responsive Element or VDRE", located upstream vitamin D-responsive genes and approximately RNA polymerase II. The receptor, which is present in many cell types at various concentrations, would determine spatial and temporal patterns of calcitriol action during development in conjunction with chromatin factors.
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PMID:[Vitamin D: biosynthesis, metabolism and mechanism of action at the cellular level]. 164 84

The Tetrahymena ribozyme has been shown to catalyze an RNA polymerase-like reaction in which an RNA primer is extended by the sequential addition of pN nucleotides derived from GpN dinucleotides, where N = A, C, or U. Here, we show that this reaction is influenced by the presence of a template; bases that can form Watson-Crick base pairs with a template add as much as 25-fold more efficiently than mismatched bases. A mutant enzyme with an altered guanosine binding site can catalyze template-directed primer extension with all four bases when supplied with dinucleotides of the form 2-aminopurine-pN.
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PMID:Template-directed primer extension catalyzed by the Tetrahymena ribozyme. 203 41

The Rickettsia prowazekii citrate synthase (gltA) gene, previously cloned in Escherichia coli, was localized to a 2.0-kilobase chromosomal fragment. DNA sequence analysis of a portion of this fragment revealed an open reading frame of 1,308 base pairs that encodes a protein of 435 amino acids with a molecular weight of 49,171. This translation product is comparable in size to both the E. coli and pig heart citrate synthase monomers and to the protein synthesized in E. coli minicells containing the rickettsial gene. Comparisons between the deduced amino acid sequence of R. prowazekii citrate synthase and those of the E. coli and pig heart enzymes revealed extensive homology (59%) between the two bacterial proteins. In contrast, only 20% of the rickettsial enzyme residues were shared with the functionally similar pig heart enzyme residues. Upstream from the open reading frame and in close proximity to one another, sequences with homology to E. coli consensus sequences for RNA polymerase and ribosome binding were identified. S1 nuclease mapping experiments demonstrated that the start of transcription for this gene in E. coli was located in the upstream region. Codon usage in the rickettsial gltA gene was found to be very biased and differed from the pattern observed in E. coli. Adenine and uracil were used preferentially in the third base position of rickettsial codons.
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PMID:Nucleotide sequence of the Rickettsia prowazekii citrate synthase gene. 311 24

T7 phage RNA polymerase was used to transcribe a series of DNA templates bearing any of several precisely localized lesions. Lesions were positioned downstream of the T7 promoter on either strand of the DNA template to investigate the effects of these lesions on elongation of transcription. The following four types of DNA modifications were studied: 1) 3-hydroxy-2-hydroxymethyltetrahydrofuran (tetrahydrofuran), a synthetic apurinic/apyrimidinic site; 2) 8-oxoguanine (8-oxodG), an oxidized derivative of guanine; 3) N-acetyl-2-aminofluorene (AAF) modified guanine; 4) 2-aminofluorene (AF) modified guanine. None of these lesions blocked transcription elongation when they were located on the non-template strand. Lesions on the template strand blocked elongation with varied efficiency. The series of AAF-dG, AF-dG, and tetrahydrofuran lesions showed a progressively decreasing ability to block elongation, while 8-oxo-dG caused little, if any, premature termination. T7 RNA polymerase was able to read through all of the lesions with sufficient efficiency to permit chain termination sequencing using the read-through products as templates. AAF-dG and AF-dG adducts did not induce detectable misreading. Adenine and, more rarely, cytosine were incorporated opposite 8-oxo-dG, as observed for translesional synthesis by DNA polymerases. Adenine was most commonly inserted opposite the non-instructional abasic site analogue, although a minor fraction of guanine was incorporated.
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PMID:Effects of DNA lesions on transcription elongation by T7 RNA polymerase. 844 51

Previous steady state kinetic studies of the initiation of transcription by T7 RNA polymerase have shown that melting of the DNA helix near the transcription start site is not rate limiting [Maslak, M., & Martin, C. T. (1993) Biochemistry 32, 4281-4285]. In the current work, fluorescence changes in a nucleotide analog incorporated within the promoter are used to monitor changes in the DNA helix associated with polymerase binding. The fluorescence of 2-aminopurine has been previously shown to depend on the environment of the base, with fluorescence increasing in the transition from a double-stranded to a single-stranded environment [Xu, D., Evans, K.O., & Nordlund, T. M.(1994) Biochemistry 33, 9592-9599]. Fluorescence changes associated with polymerase binding to promoters incorporating 2-aminopurine at positions -4 through -1 support a model which includes melting, in the statically bound complex, of the region of the promoter near the start site. Equilibrium titrations at 25 degrees C with label at position -2 provide a thermodynamic measure of the dissociation constant (Kd = 4.8 nM) for promoter binding, while stopped-flow kinetic assays measure the apparent association (k1 = 5.6 x 10(7) M-1 s-1) and dissociation (k-1 = 0.20 s-1) rate constants for simple promoter binding (the ratio k-1/k1 = 3.6 nM, in good agreement with the thermodynamic measurement of Kd). These results suggest that binding is close to the diffusion-controlled limit and helix melting is extremely rapid. In studies of structurally altered promoters, a base functional group substitution at position -10 is shown to significantly decrease k1, with little effect on k-1. In contrast, removal of the nontemplate strand from position +1 downstream results in a large decrease in k-1, with no significant effect on k1.
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PMID:Thermodynamic and kinetic measurements of promoter binding by T7 RNA polymerase. 893 55

The mechanism of bacteriophage T7 RNA polymerase binding to its promoter DNA was investigated using stopped-flow and equilibrium methods. To measure the kinetics of protein-DNA interactions in real time, changes in tryptophan fluorescence in the polymerase and 2-aminopurine (2-AP) fluorescence in the promoter DNA upon binary complex formation were used as probes. The protein fluorescence changes measured conformational changes in the polymerase whereas the fluorescence changes of 2-AP base, substituted in place of dA in the initiation region (-4 to +4), measured structural changes in the promoter DNA, such as DNA melting. The kinetic studies, carried out in the absence of the initiating nucleotide, are consistent with a two-step DNA binding mechanism, [formula: see text] where the RNA polymerase forms an initial weak EDa complex rapidly with an equilibrium association constant K1. The EDa complex then undergoes a conformational change to EDb, wherein RNA polymerase is specifically and tightly bound to the promoter DNA. Both the polymerase and the promoter DNA may undergo structural changes during this isomerization step. The isomerization of EDa to EDb is a fast step relative to the rate of transcription initiation and its rate does not limit transcription initiation. To understand how T7 RNA polymerase modulates its transcriptional efficiency at various promoters at the level of DNA binding, comparative studies with two natural T7 promoters, Phi10 and Phi3.8, were conducted. The results indicate that kinetics, the bimolecular rate constant of DNA binding, kon (K1k2), and the dissociation rate constant, koff (k-2), and thermodynamics, the equilibrium constants of the two steps (K1 and k2/k-2) both play a role in modulating the transcriptional efficiency at the level of DNA binding. Thus, the 2-fold lower kon, the 4-fold higher koff, and the 2-5-fold weaker equilibrium interactions together make Phi3.8 a weaker promoter relative to Phi10.
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PMID:Equilibrium and stopped-flow kinetic studies of interaction between T7 RNA polymerase and its promoters measured by protein and 2-aminopurine fluorescence changes. 894 10

This article describes the development of an in vitro multicolor fluorescence assay system for studying protein/DNA complex formation, transcription bubble formation, and mRNA production. These studies were accomplished using three different fluorescent spectroscopic probes: rhodamine-labeled DNA (at the 5' position) to monitor protein/DNA complex formation, DNA internally labeled with the base analog 2-aminopurine in place of adenine to monitor transcription bubble formation, and gamma-fluorophore-labeled UTP nucleotide to measure mRNA transcription rates. Combining these three assay systems allows the simultaneous determination of protein/DNA binding, localized DNA melting transitions, and mRNA production at physiological concentrations of reagents (pM-nM) and millisecond timing resolution. The application of this multicolor fluorescence assay to Escherichia coli RNA polymerase reactions (binding, open complex formation, and mRNA production) demonstrates the importance of kinetically coupled events in gene transcription.
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PMID:Real-time fluorescence assay system for gene transcription: simultaneous observation of protein/DNA binding, localized DNA melting, and mRNA production. 895 55

Initiation of transcription occurs through a series of steps starting with the binding of RNA polymerase to a promoter DNA and formation of a closed complex. The closed complexes, then isomerize to open complexes. In the open complexes a portion of the promoter DNA is unwound. Using fluorescence spectroscopy, we have investigated in real-time the mechanism of unwinding of promoter DNA during the transition from closed to open complexes of T7 RNA polymerase. We synthesized DNA templates containing the fluorescent base analog 2-aminopurine in place of adenine at specific positions in a T7 RNA polymerase promoter. We located the 2-aminopurine residues in the presumed melting domain of the promoter at -1, -4, and at -6. The fluorescence of 2-aminopurine increases when the DNA goes from a double-stranded form to a single-stranded form. By spectroscopically monitoring the increase in fluorescence of 2-aminopurine in DNA-T7 RNA polymerase complexes, we obtained kinetic and thermodynamic information for DNA unwinding. In the presence of the initiating nucleotide GTP, conformational transitions in the polymerase-promoter complex leading to strand opening were slower than in its absence. The rate of base pair disruption at -1, -6, and at -4 was also slower in the presence of GTP than in its absence. At 37 degrees C, base pair disruption occurred first at -1 followed by -6 and finally at -4. Open complex formation was temperature-sensitive. Temperature effects at -1, -6, and at -4 were consistent with this order of base pair disruption. The apparent activation energies (Ea) for base pair disruption around -1 and -6 were 14 kcal mol-1 and 50 kcal mol-1, respectively, also suggesting this order of base pair disruption. Transcription initiation assays using G-ladder synthesis revealed that initiation rates were almost the same on all three templates containing the modified base. Unlike strand opening, we did not observe lag times for G-ladder synthesis. We suggest that facile base pair disruption at -1 is sufficient for transcription initiation. Based on these data, it is proposed that the polymerase makes contacts at or near -1 and -6 resulting in untwisting of these base pairs thus creating at least two base pair disruption events at -1 and at -6, which are followed by bidirectional propagation to -4.
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PMID:A direct real-time spectroscopic investigation of the mechanism of open complex formation by T7 RNA polymerase. 896 34


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