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)

The alpha, beta2, and alphabeta forms of the RNA-dependent DNA polymerase of avian sarcoma virus B77 grown in duck embryo fibroblasts have been compared with respect to several kinetic properties. The following results were obtained. 1. The Km values for dTTP and dGTP for enzyme forms alpha, beta2, and alphabeta were 77, 39, and 74, and 6.8, 3.1, and 6.1 micronM, respectively. 2. The affinity of 70 S Rous sarcoma virus RNA for enzyme form alphabeta was about twice that for the other two forms. 3. The relative specific activities of the three enzyme forms on synthetic primer-templates such as poly(rA)-poly(dT) were almost the same. The viral 70 S RNA-dependent specific activities were 2 to 3 orders of magnitude lower and in the ratio of 1:3:5 for enzyme forms alpha:beta2:alphabeta. Addition of exogenous oligo(dT) stimulated the 70 S viral RNA-dependent activity of enzyme forms alphabeta and beta2 by a factor of 3, and that of enzyme form alpha by a factor of 30, so that it then became the most active transcriptase of viral 70 S RNA. 4. The largest transcripts formed by the three enzyme forms with 70 S viral RNA as primer-template were about 4,500 nucleotides long. About one-third of the total amount of polynucleotides polymerized by the alphabeta enzyme was in the form of such transcripts. This proportion was far higher than for the other two enzyme forms. 5. All three enzyme forms were capable of transcribing single-stranded into double-stranded DNA. 6. The 3-propylcyclohexyl piperidyl derivative of rifamycin SV, at a concentration of 100 microng/ml, inhibited enzyme forms beta2 and alphabeta by over 99.5 and 96%, respectively, but enzyme form alpha by only about 60%. 7. The beta2 and alphabeta forms of the enzyme were processive DNA polymerases, the alpha form a nonprocessive polymerase. 8. In general, these results indicate that in most respects the properties of the dimeric enzyme forms resemble each other much more closely than those of the alpha form. In some very important respects, such as affinity for viral RNA and the size of transcripts formed from it, the alphabeta enzyme form performs significantly better than either of the other two enzyme forms.
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PMID:RNA-dependent DNA polymerase of avian sarcoma virus B77. II. Comparison of the catalytic properties of the alpha, beta2, and alphabeta enzyme forms. 6 35

An electron microscopic method for demonstrating the presence of and mapping the positions of proteins specifically bound to nucleic acids is described. The nucleic acid-protein complex is treated with dinitrofluorobenzene under conditions such that dinitrophenyl (DNP) groups are attached to nucleophilic groups on the protein, with only a low level of random attachment to the nuclei acid. This product is treated with rabbit anti-DNP IgG. The position of the protein-(DNP)n(IgG)m complex on the nucleic acid strand can be observed by electron microscopy by protein free spreading methods and, in many cases, by cytochrome-c spreading. If necessary for visualization by the latter method, the size of the labeled region can be increased by treatment with goat anti-rabbit IgG. High efficiency of electron microscopic labeling is achieved. Examples studied are: the adenovirus-2 DNA terminal protein, a protein covalently bound to SV40 DNA, DNA polymerase I bound to DNA, E. coli RNA polymerase bound to T7 DNA, and proteins UV crosslinked to avian sarcoma virus RNA.
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PMID:An electron microscopic method for the mapping of proteins attached to nucleic acids. 21 86

Reverse transcriptase of the avian sarcoma and leukosis retroviruses is a heterodimer composed of a 63-kDa alpha and a 95-kDa beta polypeptide chain, both of which are encoded in the pol gene and are produced by proteolytic processing of a larger precursor. We previously constructed a bacterial expression clone of the entire pol coding region that produces a protein 4 kDa larger than the mature viral beta subunit. By use of this clone and synthetic oligonucleotides to introduce stop codons, two derivatives have been constructed: one that directs synthesis of a protein equivalent to the mature beta subunit and the other that directs synthesis of a protein equivalent to alpha subunit. Predicted amino acid sequences of these proteins differ from their viral counterparts only by an initiator methionine that was added to the N termini for expression in Escherichia coli. Both bacterially expressed proteins exhibit reverse transcriptase activity and appear to function as homodimers. The properties of these proteins resemble those of the viral reverse transcriptase heterodimer; however, the bacterially produced alpha dimer protein could be distinguished from the other proteins by its increased sensitivity to heat inactivation, which also has been reported for the corresponding viral product. These results show that correct folding and expression of enzymatic function does not require formation of a precursor. The alpha and beta clones provide a convenient source of individual pol gene products for further evaluation of their roles in the synthesis and integration of retroviral DNA.
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PMID:The alpha and beta chains of avian retrovirus reverse transcriptase independently expressed in Escherichia coli: characterization of enzymatic activities. 245 57

A new fractionation procedure separates a whole-cell HeLa extract into three components required for accurate in vitro transcription. One component (Sp1) is a promoter-specific factor required for transcription of the SV40 early and late promoters, but not for transcription of other promoters we have tested. The second component (Sp2) is a general factor required for transcription of the SV40 promoters and a series of others, including the adeno-virus 2 major late promoter, the human beta-globin promoter and the avian sarcoma virus promoter. The third component is a fraction containing the endogenous RNA polymerase II. When SV40 and adenovirus templates were present simultaneously in an in vitro transcription reaction, addition of the Sp1 factor stimulated SV40 early promoter transcription 40-fold, and inhibited adenovirus major late promoter transcription by 40%. This finding suggests that Sp1 is involved in promoter selection, and is not merely a general transcription stimulatory factor.
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PMID:Isolation of transcription factors that discriminate between different promoters recognized by RNA polymerase II. 618 69

The nucleotide sequences in the long terminal repeat of avian sarcoma virus that are recognized in vitro by HeLa cell RNA polymerase II have been identified. For this purpose, various 5' and 3' deletions were introduced into a cloned long terminal repeat fragment. The effects of these deletions on transcription initiation in HeLa whole-cell extracts were then studied. Three specific transcripts have been identified. The major transcript is initiated at nucleotide +1 (relative to the cap site). Deletion of the upstream sequence between -299 and -55 has no effect on the level of transcription from this start site, whereas deletion of the sequence downstream of -14 drastically reduces the levels of transcription. In contrast, deletion of the sequence downstream from the TATA box has no effect on the initiation or efficiency of synthesis of the two minor RNA species, which are initiated at around nucleotides -260 and -105. The transcription of these RNA products, however, is abolished by an upstream deletion between -299 and -55. These results suggest that HeLa cell RNA polymerase II recognizes in vitro more than one promoter site present in the long terminal repeat of the avian sarcoma virus genome and defines the sequences required for initiation of the major transcript.
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PMID:Localization of active promoters for eucaryotic RNA polymerase II in the long terminal repeat of avian sarcoma virus DNA. 630 47

The chromosomal features that influence retroviral integration site selection are not well understood. Here, we report the mapping of 226 avian sarcoma virus (ASV) integration sites in the human genome. The results show that the sites are distributed over all chromosomes, and no global bias for integration site selection was detected. However, RNA polymerase II transcription units (protein-encoding genes) appear to be favored targets of ASV integration. The integration frequency within genes is similar to that previously described for murine leukemia virus but distinct from the higher frequency observed with human immunodeficiency virus type 1. We found no evidence for preferred ASV integration sites over the length of genes and immediate flanking regions. Microarray analysis of uninfected HeLa cells revealed that the expression levels of ASV target genes were similar to the median level for all genes represented in the array. Although expressed genes were targets for integration, we found no preference for integration into highly expressed genes. Our results provide a more detailed description of the chromosomal features that may influence ASV integration and support the idea that distinct, virus-specific mechanisms mediate integration site selection. Such differences may be relevant to viral pathogenesis and provide utility in retroviral vector design.
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PMID:Genome-wide analyses of avian sarcoma virus integration sites. 1547 7