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)

Accurate transcription by RNA polymerase II has been shown to require multiple factors in addition to the purified polymerase. In this study, we use a reconstituted transcription system, consisting of purified RNA polymerase II and three essential HeLa cell chromatographic fractions, to study events leading to transcription from the adenovirus major late promoter. A preincubation-pulse-chase protocol resolves the reaction into events occurring before and after nucleotide addition. Preincubation of template with a mixture of RNA polymerase II and factors allows formation of "activated" complexes, which are defined by the ability to rapidly commence accurate transcription when presented nucleotides. Maximal activation requires that polymerase, template, and each of the three HeLa fractions be present during preincubation. The activated complexes are template associated, as shown by their inability to exchange onto a second template added during further preincubation. Similar protocols are used to define functional intermediates leading to the activated complex. A template-associated functional complex is formed during the preincubation of template with just two of the HeLa fractions. Polymerase can associate with this intermediate complex in the absence of the third HeLa fraction. In the accompanying paper, we describe a direct analysis of initiation by "activated" complexes.
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PMID:Interactions between RNA polymerase II, factors, and template leading to accurate transcription. 669 78

We report the nucleotide sequence of a promoter recognized by RNA polymerase from the gram-positive bacterium Bacillus subtilis. This promoter, which was isolated from B. subtilis phage SP01 DNA, is homologous to promoters for Escherichia coli RNA polymerase; the sequences of the "-35 region" and the "Pribnow box" were 5'TTGACT and 5'CATAAT, respectively (T is the thymine analog 5-hydroxymethyluracil in SP01 DNA). These sequences each differed by only a single base pair from the preferred sequences for E. coli promoters. Not surprisingly, the SP01 promoter was actively transcribed in vitro by E. coli RNA Polymerase as well as by B. subtilis RNA polymerase.
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PMID:Nucleotide sequence of a promoter recognized by Bacillus subtilis RNA polymerase. 677 32

In this paper we obtain thermodynamic and molecular information about the specific complexes formed between Escherichia coli RNA polymerase holoenzyme and a restriction fragment of T7 D111 DNA carrying the A1 and D promoters. Specific binding was observed at both 0 and 37 degrees C over a side range of pH values and ion concentrations [Strauss, H. S., Burgess, R. R., & Record, M. T., Jr. (1980) Biochemistry (first paper of four in this issue)]. The specific complexes formed at these two temperatures may correspond to the closed and open promoter complexes discussed by Chamberlin [Chamberlin, M. J. (1976) RNA Polymerase (Losick, R., & Chamberlin, M., Eds.) pp 159-161, Cold Spring Harbor Laboratory, cold Spring Harbor, NY]. Promoter binding constants KobsdRP are obtained from competition filter binding data by using a statistical analysis and previously determined values of the nonspecific holoenzyme-DNA binding constant KobsdRD. From the magnitudes of KobsdRP at 0 and 37 degrees C, and the dependences of these binding constants on pH and ion concentrations, we conclude that, under physiological ionic conditions, both the 0 and the 37 degrees C complexes are stabilized to a large extent by the formation of ionic interactions and the accompanying release of counterions and that one or two protonation events (pK approximately 7.4) are required for complex formation in both cases. However, the 0 and 37 degrees C complexes differ in their sensitivity to ion concentrations as well as in the magnitude of KobsdRP, and we conclude that the two complexes are distinct. (More counterion release accompanies formation of the 37 degrees C complex). Comparisons of the two complexes with one another and with nonspecific holoenzyme-DNA complexes are drawn from the binding data. We have also examined the equilibrium selectivity ratio (KobsdRP/DobsdRD) and find it to be a sensitive function of temperature and ionic conditions. Selectivity of holoenzyme for promoter sites on the promoter-containing fragment is higher at 37 degrees C than at 0 degrees C under the conditions investigated. Selectivity at either temperature is increased by reducing the pH (in the range 6.1-8.6). At 37 degrees C, selectivity is increased by reducing the salt concentration. Under approximately physiological conditions (0.2 M NaCl and 0.003 M MgCl2, pH 7.4, 37 degrees C), the equilibrium selectivity ratio is found to be of order of magnitude 10(4).
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PMID:Binding of Escherichia coli ribonucleic acid polymerase holoenzyme to a bacteriophage T7 promoter-containing fragment: evaluation of promoter binding constants as a function of solution conditions. 699 5

2-Chloro-2'-deoxyadenosine (cladribine [CldAdo]) represents one of the most promising therapeutic agents for the treatment of pediatric leukemias and adult hairy cell leukemia. We examined whether CldAdo incorporation into DNA inhibited subsequent transcription in vitro using purified phage RNA polymerases. Control (Ade-containing) and 2-chloroadenine (ClAde)-substituted DNA strands that contained a RNA polymerase promoter sequence were synthesized by a modified asymmetric polymerase chain reaction. Complementary (+) and (-) strands were annealed, incubated with phage RNA polymerase, and analyzed with denaturing PAGE. When ClAde was present in both strands, the yield of full-length transcripts (approximately equal to 100 bases) was reduced by approximately equal to 90% relative to control DNA. Transcription was also reduced to a slightly lesser degree when substitutions occurred in only one of two strands. The observed low transcript levels on ClAde-containing DNA were due in part to the presence of the analogue within the promoter region. With gel shift binding assays, we demonstrated that RNA polymerase did not bind as well to ClAde-containing promoters. Polymerase/DNA complex formation was decreased by approximately equal to 80% compared with that on control unsubstituted promoters. In addition, on binding to the substituted promoter, RNA polymerase had an altered conformation that led to enhanced proteolytic clipping by endoproteinase Glu-C. Transcript sequence analysis indicated that SP6 RNA polymerase read through template ClAde residues with no apparent misincorporation into RNA. Our results provide insight into a novel effect of this nucleoside analogue that may explain its cytotoxicity in nondividing cells.
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PMID:In vitro transcription of DNA containing 2-chloro-2'-deoxyadenosine monophosphate. 747 21

The force produced by a single molecule of Escherichia coli RNA polymerase during transcription was measured optically. Polymerase immobilized on a surface was used to transcribe a DNA template attached to a polystyrene bead 0.5 micrometer in diameter. The bead position was measured by interferometry while a force opposing translocation of the polymerase along the DNA was applied with an optical trap. At saturating nucleoside triphosphate concentrations, polymerase molecules stalled reversibly at a mean applied force estimated to be 14 piconewtons. This force is substantially larger than those measured for the cytoskeletal motors kinesin and myosin and exceeds mechanical loads that are estimated to oppose transcriptional elongation in vivo. The data are consistent with efficient conversion of the free energy liberated by RNA synthesis into mechanical work.
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PMID:Transcription against an applied force. 750 62

Enzymatically synthesized RNA samples (in vitro transcripts) were analysed by matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS). Spectra of RNA up to 150 kDA (461 nucleotides) are shown. Polymerase generated sample heterogeneity and its contribution to mass resolution are discussed. A time course exonuclease digest of a 55 nt in vitro transcript was analyzed to investigate the performance of MALDI-MS on complex mixtures. Based on these data, the analysis by MALDI-MS of DNA sequencing reactions, produced by the action of an RNA polymerase, is discussed.
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PMID:Matrix assisted laser desorption/ionization mass spectrometry of enzymatically synthesized RNA up to 150 kDa. 752 29

A recently reported comparison of stable RNA (rRNA, tRNA) and mRNA synthesis rates in ppGpp-synthesizing and ppGpp-deficient (delta relA delta spoT) bacteria has suggested that ppGpp inhibits transcription initiation from stable RNA promoters, as well as synthesis of (bulk) mRNA. Inhibition of stable RNA synthesis occurs mainly during slow growth of bacteria when cytoplasmic levels of ppGpp are high. In contrast, inhibition of mRNA occurs mainly during fast growth when ppGpp levels are low, and it is associated with a partial inactivation of RNA polymerase. To explain these observations it has been proposed that ppGpp causes transcriptional pausing and queuing during the synthesis of mRNA. Polymerase queuing requires high rates of transcription initiation in addition to polymerase pausing, and therefore high concentrations of free RNA polymerase. These conditions are found in fast growing bacteria. Furthermore, the RNA polymerase queues lead to a promoter blocking when RNA polymerase molecules stack up from the pause site back to the (mRNA) promoter. This occurs most frequently at pause sites close to the promoter. Blocking of mRNA promoters diverts RNA polymerase to stable RNA promoters. In this manner ppGpp could indirectly stimulate synthesis of stable RNA at high growth rates. In the present work a mathematical analysis, based on the theory of queuing, is presented and applied to the global control of transcription in bacteria. This model predicts the in vivo distribution of RNA polymerase over stable RNA and mRNA genes for both ppGpp-synthesizing and ppGpp-deficient bacteria in response to different environmental conditions. It also shows how small changes in basal ppGpp concentrations can produce large changes in the rate of stable RNA synthesis.
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PMID:Guanosine tetraphosphate as a global regulator of bacterial RNA synthesis: a model involving RNA polymerase pausing and queuing. 753 31

The genes CDKN2B (MTS2) and CDKN2 (MTS1) encoding the proteins p15 and p16 are both located on chromosomal band 9p21, a locus at which frequent homozygous and heterozygous deletions occur in many primary human tumors, including esophageal carcinoma. CDKN2 and CDKN2B belong to a family of cyclin-dependent kinase 4 inhibitors (INK41) and control cell proliferation during the G1 phase of the cell cycle. Their inactivation may contribute to uncontrolled growth in human cancers. To investigate whether CDKN2B and CDKN2 are involved in esophageal tumorigenesis, we studied homozygous deletion, intragenic mutation, and messenger RNA (mRNA) expression of CDKN2 and CDKN2B in nine esophageal squamous cancer cell lines. Polymerase chain reaction (PCR) amplification revealed that five of the nine cell lines (55%) manifested homozygous deletions of CDKN2B, CDKN2, and/or flanking loci on chromosomal band 9p21. Reverse transcriptase-PCR (RT-PCR) was used to examine CDKN2 and CDKN2B mRNA in the nine cell lines. Lack of CDKN2 and CDKN2B mRNA correlated perfectly with homozygous deletion involving these genes. No subtle intragenic mutations of CDKN2B or CDKN2 were detected by DNA sequencing of their entire coding sequences in any cell lines lacking homozygous deletion. Two of the cell lines manifested homozygous deletions excluding CDKN2; one of these two deletions also excluded CDKN2B. These results suggest that inactivation of CDKN2B and CDKN2 may contribute to the malignant phenotype in esophageal cells and that homozygous deletion may be the predominant mechanism for inactivation of CDKN2B and CDKN2. Alternatively, a gene or genes adjacent to CDKN2B/CDKN2 may constitute the target(s) of deletion at this locus.
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PMID:Genomic DNA and messenger RNA expression alterations of the CDKN2B and CDKN2 genes in esophageal squamous carcinoma cell lines. 754 37

As an improved strategy for producing functional macromolecules by in vitro evolutionary optimization, we propose an automated machine that can process up to 960 samples in parallel. It consists of a 960-well PCR machine with special sealed plastic reaction vessels and appropriate handling devices. We show that the heat-sealing technique does not significantly affect the activity of Taq DNA Polymerase or that of the temperature-sensitive Q beta RNA polymerase but avoids cross-contamination and evaporation of the samples. Initial experiments demonstrate the suitability of the apparatus to uniformly process the samples and to perform the thermocycling. Serial transfer of reaction products into fresh reaction solution was used to initiate further rounds of amplification as a typical experimental setup for an evolutionary biotechnology application.
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PMID:Multichannel PCR and serial transfer machine as a future tool in evolutionary biotechnology. 759

Cytogenetic analysis of peripheral primitive neuroectodermal tumors (PNETs) has demonstrated a consistent primary chromosomal change characterized by a reciprocal translocation t(11;22)(q24:q12). In the central nervous system PNETs, most frequent of which are the cerebellar medulloblastomas, the most prevalent chromosomal abnormalities include deletions and unbalanced translocations. The recent cloning of the t(11;22) breakpoint has revealed the fusion of the human FLI-1 gene on chromosome 11q24 with a gene EWS on chromosome 22q12 and permitted detection of fusion transcripts. Molecular genetic analysis for the presence of EWS/FLI-1 fusion transcripts by the reverse transcriptase-polymerase chain reaction has recently been applied to peripheral PNETs. In the present study, we analyzed eight central PNETs by reverse transcriptase-polymerase chain reaction for EWS/FLI-1 fusion transcripts. The tumors included six PNETs of the cerebellum, one supratentorial PNET of the frontal lobe and one PNET of the pineal region. Polymerase chain reaction analysis in all eight cases failed to reveal a t(11;22) translocation indicating that this is not a cytogenetic abnormality of the central PNETs. Reverse transcriptase-polymerase chain reaction analysis of EWS/FLI-1 fusion transcripts provides a novel adjunctive tool in the differentiation of central versus peripheral PNET.
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PMID:Primitive neuroectodermal tumors of the cerebrum and cerebellum: absence of t(11;22) translocation by RT-PCR analysis. 767 66


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