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)

Rabbit globin complementary DNA made with RNA-dependent DNA polymerase (reverse transcriptase) was used as a template for in vitro synthesis of 32P-labeled RNA and deoxysubstituted RNA. The sequences of the nucleotides in most of the fragments resulting from combined ribonuclease T1 and alkaline phosphatase digestion have been determined. In addition, the 3' nearest neighbor was determined for several fragments resulting from digestion with T1 ribonuclease. The utility of the deoxysubstitution technique was demonstrated by the ease with which the sequences of pyrimidine-rich fragments could be determined. Many sequences thus determined were long enough to fit uniquely with the alpha- or beta-globin amino acid sequences. The positions of these fits were found to be clustered, leading us to believe that only certain regions of the complementary DNA are transcribed by Escherichia coli RNA polymerase. Other unique characteristics of RNA synthesis from a complementary DNA template include a high yield of free poly(A) and the fact that one must use low rather than high salt buffers to obtain transcripts of high molecular weight.
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PMID:Rabbit globin mRNA: analysis of T1 RNAse digestion fragments. 6 35

Sequence analysis studies were carried out on human beta-globin mRNA (beta-mRNA) prepared from alpha-thalassemic, sickle cell, and Hb A reticulocytes. Highly purified beta-mRNA served as substrate for the preparation of cDNA by RNA-dependent DNA polymerase. The cDNA was transcribed by Escherichia coli RNA polymerase and the resulting cRNA was analyzed. Over 300 nucleotides were assigned to the beta-mRNA coding region and 37 nucleotides were assigned to the 3'-terminal noncoding region. The normal termination codon is UAA which is separated by 28 nucleotides from an out of phase UAA triplet. The origin of each of the abnormally long beta-globin variants Tak and Cranston is consistent with reduplication of dinucleotides prior to the normal termination codon, and both globin variants can terminate at the out of phase UAA.
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PMID:Human beta-globin messenger RNA. I. Nucleotide sequences derived from complementary RNA. 87 28

We have used the globin family of genes in chicken to study developmental regulation of gene expression, both at the level of individual interaction of trans-acting factors with local promoters and enhancers, and at the level of chromatin structure. Regulation of all members of the alpha- and beta-globin clusters is affected by the erythroid regulatory factor GATA-1. Separate mechanisms exist for regulation of individual members of the family. As an example, we describe the control mechanisms that play a role in the expression of the rho-globin gene, which is expressed only in primitive lineage erythroid cells. In addressing the involvement of chromatin structure in gene activation, we have examined the role of locus control elements, and also considered the way in which RNA polymerase molecules might accommodate to the presence of nucleosomes on transcribed genes.
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PMID:Developmental regulation of globin gene expression. 129 48

A common mutation causing thalassemia in Mediterranean populations is an amber (UAG) nonsense mutation at the 39th codon of the human beta-globin gene, the beta-39 mutation. Studies of mRNA metabolism in erythroblasts from patients with beta-39 thalassemia and studies using heterologous transfection systems have suggested the possibility that this mutation not only affects protein synthesis but also alters mRNA metabolism. The effects of this mutation on several steps in the metabolism of mRNA have been investigated by transfection of the gene into permanent cell lines bearing a temperature-sensitive RNA polymerase II. Several RNA expression studies were performed, including analysis of transcription, mRNA stability, mRNA splicing accuracy, and mRNA polyadenylation. The results suggest that the defect in expression of the beta-39 mRNA occurs at a step prior to the accumulation of mRNA in the cytoplasm.
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PMID:Beta-globin nonsense mutation: deficient accumulation of mRNA occurs despite normal cytoplasmic stability. 155 99

Infection of cells with poliovirus results in a rapid inhibition of host RNA and protein synthesis. Concordant with this shutoff, the p220 subunit of the cap-binding protein complex is cleaved, probably indirectly, by the poliovirus proteinase p2A (2Apro). To elucidate the mechanism of action of 2Apro in inhibiting protein synthesis in vivo, we studied the effect of transient expression of 2Apro in COS-1 monkey kidney cells. In cells transfected with a 2Apro expression plasmid, p220 was cleaved and the 2Apro mRNA was reduced 30-fold compared to an identical plasmid containing a translation termination codon within the 2Apro coding region. The reduced expression from the 2Apro vector results from a 4-fold reduction in DNA replication and 22-fold reduction in transcription by RNA polymerase II from the adenovirus major late promoter/SV40 enhancer utilized in this vector. In contrast, no decrease in transcription of the adenovirus virus-associated I RNA gene by RNA polymerase III was observed. The effect of 2Apro expression on cap-dependent mRNA translation was studied by producing a dicistronic beta-globin mRNA harboring the encephalomyocarditis virus leader and 2Apro coding region within the 3' end of the mRNA to mediate cap-independent translation of 2Apro. Expression of this mRNA was also reduced 25-fold compared to an identical plasmid harboring a termination codon within the 2Apro coding region. Translation of the beta-globin marker gene from this mRNA was reduced 3-fold when corrected for mRNA level. These results suggest that p220 cleavage itself is not sufficient for complete inhibition of host translation and that an important effect of 2Apro expression on host protein synthesis is a reduction in RNA polymerase II transcription and to a lesser extent, DNA replication. This reduction could be a primary effect of 2Apro, or a secondary effect caused by the inhibition of translation.
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PMID:The effect of poliovirus proteinase 2Apro expression on cellular metabolism. Inhibition of DNA replication, RNA polymerase II transcription, and translation. 171 90

The transcriptase associated with Germiston virus was assayed in an in vitro reaction in which transcription was coupled to translation by adding reticulocyte lysate under the appropriate salt conditions. When analyzed in polyacrylamide gels, the major transcripts migrated like authentic S mRNAs and possessed 12- to 18-base-long nontemplated 5' extensions similar to the 5' end of viral mRNAs. These transcripts were functional for the synthesis of at least proteins N and NSS. When translation was inhibited by adding protein synthesis inhibitors such as puromycin, cycloheximide, and anisomycin, a drastic inhibitory effect was observed on the synthesis of the complete S mRNA transcripts. However, initiation and part of the elongation process were still active, since short and incomplete RNA molecules with RNA primers at their 5' ends were synthesized. On the other hand, we found that edeine, another inhibitor of protein synthesis, stimulated not only synthesis of S mRNAs but also that of the full-length S cRNAs. Taking into account the mode of action of this antibiotic, we discuss the results, which emphasize the crucial role of active ribosomes during bunyavirus transcription and confirm the observations reported on La Crosse virions. Moreover, we showed that the RNA transcripts synthesized in a transcription-translation reaction were capped and that most of them have acquired the 5' terminal sequences of the alpha- or beta-globin mRNA.
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PMID:Germiston virus transcriptase requires active 40S ribosomal subunits and utilizes capped cellular RNAs. 173 Nov 8

Mammalian gene promoters for transcription by RNA polymerase II are typically organized in the following order: upstream sequence motif(s)/TATA box/initiation site. Here we report studies in which the order, orientation and DNA sequences of these three elements are varied to determine how these affect polarity of transcription. We have constructed promoters with an 'octamer' upstream sequence ATTTGCAT (or its complement ATGCAAAT) in combination with several different TATA boxes and initiation (cap) sites, and tested these promoters in transfection experiments with cultured cells. TATA boxes derived from the adenovirus major late promoter (TATAAAA), immunoglobulin kappa light chain (TTATATA) and heavy chain (TAAATATA) promoter functioned equally well or even better when inverted. Only the beta-globin TATA box (CATAAAA) was poorly active when inverted. In addition, a symmetrical TATA box (TATATATA) derived from a casein gene was very active. Our results suggest that the asymmetry of most TATA boxes (consensus TATAAAA) is not a primary determinant of the polarity of transcription. We also found that the initiation (cap) site, which usually consists of an adenine embedded in a pyrimidine-rich region (PyPyCAPyPyPyPyPy), was permissive towards sequence alterations; even a randomly composed sequence worked well. However, an inverted, hence purine-rich, cap site reduced transcript levels to 1/7th, as did an oligo G sequence. Irrespective of the presence of a cap site, the configuration: 'TATA box/octamer' yielded a strong leftward, rather than rightward transcription. From this, we conclude that the polarity of transcription is primarily determined by the linear order of an upstream sequence relative to a TATA box, rather than by the individual orientations of either of these two elements.
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PMID:Upstream box/TATA box order is the major determinant of the direction of transcription. 176

Transcription of the rabbit alpha-globin gene begins primarily at the cap site, although some upstream start sites are also observed. Analysis by RNA polymerase run-on assays in nuclei shows that transcription continues at a high level past the polyadenylation site, after which the polymerase density actually increases in a region of about 400 nucleotides, followed by a gradual decline over the 700 nucleotides. These features are also observed in the transcription unit of the rabbit beta-globin gene. The region with the unexpectedly high nascent RNA hybridization signal in the 3' flank contains a conserved sequence, KGCAGCWGGR (K = G or T, W = A or T, R = A or G), followed by an inverted repeat. The inverted repeat (perhaps with the conserved sequence) may be a pause site for RNA polymerase II, thus accounting for the increase in polymerase density. This sequence and inverted repeat are found in the 3' flank of several globin genes and the simian virus 40 (SV40) early genes, as well as in the regions implicated in pausing or termination of transcription of eight different genes. Deletion of the conserved sequence and inverted repeat from the 3' flank of the SV40 early region causes a small increase in the levels of transcription downstream from this site. Replacement with the conserved sequence and inverted repeat from the rabbit alpha-globin gene causes an accumulation of polymerases, supporting the hypothesis that polymerases pause at this site. This proposed pause site may affect the efficiency of termination at some sites further downstream, perhaps by loss of a processivity factor.
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PMID:An apparent pause site in the transcription unit of the rabbit alpha-globin gene. 185 60

Expression systems based on the selective transcription of genes cloned behind a T7 promoter, by T7 RNA polymerase, display a non-negligible basal expression when the T7 RNA polymerase gene is present within the host organism before induction of the system. This is a problem, especially for cloning and controlled expression of genes toxic to the host organism. We have circumvented this problem by taking advantage of abortive T7 infection of E. coli (P1), in the course of which T7 RNA polymerase is synthesized but bacterial growth is not quantitatively impaired. We have tested this system with three reporter genes, the 6-phospho-beta-galactosidase gene of Staphylococcus aureus, the luciferase operon of Vibrio harveyi, and the rabbit beta-globin gene; we have found very low basal levels, while, upon T7 infection, transcription is at least as efficient as in other in vivo T7 RNA polymerase systems in use.
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PMID:T7 infection-dependent selective expression of cloned genes in P1-lysogenic Escherichia coli. 193 Jan 65

The existence of torsional stress in eukaryotic chromatin has been controversial. To determine whether it could be detected, we probed the structure of an alternating AT tract. These sequences adopt cruciform geometry when the DNA helix is torsionally strained by negative supercoiling. The single-strand-specific nuclease P1 was used to determine the structure of an alternating AT sequence upstream of the Xenopus beta-globin gene when assembled into chromatin in microinjected Xenopus oocytes. The pattern of cleavage by P1 nuclease strongly suggests that the DNA in this chromatin template is under torsional stress. The cruciform was detected specifically in the most fully reconstituted templates at later stages of chromatin assembly, suggesting that negative supercoiling is associated with chromatin maturation. Furthermore, the number of torsionally strained templates increased dramatically at the time when transcription of assembled chromatin templates began. Transcription itself has been shown to induce supercoiling, but the requisite negative supercoiling for cruciform extrusion by (AT)n in oocytes was not generated in this way since the characteristic P1 cutting pattern was retained even when RNA polymerase elongation was blocked with alpha-amanitin. Thus, torsional stress is associated with transcriptional activation of chromatin templates in the absence of ongoing transcription.
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PMID:Evidence for torsional stress in transcriptionally activated chromatin. 194 80


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