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)

The HIV-1 trans-activator protein, Tat, is a potent activator of transcriptional elongation. Tat is recruited to the elongating RNA polymerase during its transit through the trans-activation response region (TAR) because of its ability to bind directly to TAR RNA expressed on the nascent RNA chain. We have shown that transcription complexes that have acquired Tat produce 3-fold more full-length transcripts than complexes not exposed to Tat. Western blotting experiments demonstrated that Tat is tightly associated with the paused polymerases. To determine whether TAR RNA also becomes attached to the transcription complex, DNA oligonucleotides were annealed to the nascent chains on the arrested complexes and the RNA was cleaved by RNase H. After cleavage, the 5' end of the nascent chain, carrying TAR RNA, is quantitatively removed, but the 3' end of the transcript remains associated with the transcription complex. Even after the removal of TAR RNA, transcription complexes that have been activated by Tat show enhanced processivity. We conclude that Tat, together with cellular co-factors, becomes attached to the transcription complex and stimulates processivity, whereas TAR RNA does not play a direct role in the activation of elongation and is used simply to recruit Tat and cellular co-factors.
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PMID:Transfer of Tat and release of TAR RNA during the activation of the human immunodeficiency virus type-1 transcription elongation complex. 931 86

Reverse transcriptase (RT) is the key enzyme required for conversion of RNA to DNA. Cloning of Moloney murine leukemia virus (MMLV) RT has enable engineering an RT that lacks endogenous RNase H activity. RT catalyzes cDNA synthesis more efficiently in the absence of RNase H. We describe here a number of properties of MMLV RT and RNase H-minus MMLV RT not summarized in a single location elsewhere, providing a basis for best use of these enzymes in cDNA synthesis. In addition, general guidelines and detailed protocols are provided for use of MMLV RTs in one tube double-stranded cDNA synthesis, in [32P]cDNA synthesis, and in RT-PCR and long RT-PCR.
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PMID:Reverse transcriptase. The use of cloned Moloney murine leukemia virus reverse transcriptase to synthesize DNA from RNA. 932 98

Ribonucleases H (RNases H) are enzymes that specifically degrade the RNA of RNA-DNA hybrids. These enzymes are involved in DNA replication, reverse transcription (RT) and antisense oligodeoxyribonucleotide-mediated arrest of translation. One of the most valuable tools for assaying RNase H activity is the renaturation gel assay with which such activities can be detected using purified protein preparations or crude extracts. Radioactive substrates [32P labeled poly(rA)-poly(dT) hybrid] are commonly used with exposure of the gel to X-ray film; this is possible at any time without disturbing the renaturation-degradation process. Here, we describe a method using fluorescent-labeled substrates. RNA-DNA substrates are synthesized by first transcribing DNA with T7 RNA polymerase using Bodipy-TR-14-UTP and the four normal nucleoside triphosphates. The run-off transcript is annealed to a short oligomeric DNA complementary to the 3'-end of the transcript, and the DNA portion of the hybrid is formed by RT. This RNA-DNA is added to the polyacrylamide mixture before polymerization, and SDS-PAGE is performed as usual. After various periods of renaturation, the gel is scanned to detect fluorescent substrate using the red-excited laser of a fluorescence scanner. This fluorescence method has all of the advantages of using radio-labeled substrates and none of its disadvantages, and the sensitivities of the two methods are comparable. In addition, we show that the sensitivity of this procedure can be increased if damaging chemicals remaining in the gel after polymerization are eliminated by simultaneous electrophoresis of the RNase H and a protein with higher mobility.
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PMID:Ribonuclease H renaturation gel assay using a fluorescent-labeled substrate. 938 60

The BARE-1 copia-like retrotransposon constitutes nearly 7% of the barley (Hordeum vulgare L.) genome as a family of more than 2 x 10(4) mostly full-length copies dispersed on all chromosomes. BARE-1 elements are transcribed in barley tissues from promoters within the LTR (long terminal repeat). The predicted, translated polyprotein contains conserved domains for GAG, aspartic proteinase, integrase, reverse-transcriptase, and RNase H. Here, we have used inverse PCR with LTR-based primers to establish the consensus sequences for the terminal region of the LTR, the external dinucleotides of the cDNA integration intermediate, and the minus- and plus-strand priming sites. These key functional entities are well-conserved in the BARE-1 family, including wheat Wis2, but differ from those of other plant retrotransposons. The target site duplication was established as 5 bp. Of the 13 integration sites identified here, 8 were other BARE-1 elements and 1 another retrotransposon; 59% of the total 17 identified BARE-1 insertion sites are retrotransposons. This nested insertion pattern may represent a basic feature of plant retrotransposons.
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PMID:BARE-1 insertion site preferences and evolutionary conservation of RNA and cDNA processing sites. 944 Feb 75

Reverse transcriptase (RT)-associated ribonuclease H (RNase H) can cleave both the RNA template of DNA/RNA hybrids as well as double-stranded (ds) RNA. This report shows that human immunodeficiency virus (HIV)-RT can also cleave the template strand of dsDNA when Mg2+ is replaced by Fe2+ in the RNase H active site of HIV-RT. The cleavage mechanisms as well as the positions of the cut vary depending on whether RNA or DNA is used. While DNA is cleaved 17 base positions upstream of the primer 3'-end, RNA is cleaved 18 base positions upstream. Competition experiments show that Fe2+ replaces the catalytically active Mg2+ of RT-associated RNase H. The bound Fe2+ is the source of locally generated OH-radicals that cleave the most proximate base in the DNA. Electrophoretic mobility studies of the cleaved fragments suggest that DNA is cleaved by an oxidative mechanism, while RNA is cleaved by an enzymatic mechanism which is indistinguishable from the Mg2+-dependent cleavage. The Fe2+-dependent cuts can be used to trace the active site of RT-associated RNase H on dsDNA as well as on dsRNA and DNA/RNA hybrids. The observed 1 base difference in the cleavage positions on DNA and RNA templates can be attributed to conformational differences of the bound nucleic acids. We suggest that the lower pitch of dsRNA and DNA/RNA hybrids compared with dsDNA permits accommodation of an additional base pair in the region between the primer 3'-end and the Fe2+-dependent cleavage position at the RNase H active site.
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PMID:Localization of the active site of HIV-1 reverse transcriptase-associated RNase H domain on a DNA template using site-specific generated hydroxyl radicals. 955 61

Reverse transcriptase (RT) is a modular enzyme carrying polymerase and ribonuclease H (RNase H) activities in separable domains. Retroviral replication requires both of these activities. The RNase H domain is responsible for hydrolysis of the RNA portion of RNA x DNA hybrids, and this activity requires the presence of divalent cations (Mg2+ or Mn2+) that bind its active site. This domain is a part of a large family of homologous RNase H enzymes of which the RNase HI protein from Escherichia coli is the best characterized. Although the isolated RNase H domain from human immunodeficiency virus RT is inactive, the Moloney murine leukemia virus (MMLV) domain is active in the absence of the polymerase domain, making functional studies more accessible. Using circular dichroism spectroscopy, we characterized the stability and folding of two different fragments of MMLV RT that retain RNase H activity. The smaller fragment corresponding to the 157 C-terminal residues of RT is predominantly unfolded in the absence of divalent cations, but folding can be induced by the addition of metal. The larger fragment corresponding to the 175 C-terminal residues, however, is stably folded in the absence of metal. Thus, an 18 residue N-terminal extension outside the region homologous to E. coli RNase HI is important for the structural stability of the RNase H domain of MMLV RT. Therefore, this region should be considered part of the RNase H domain.
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PMID:Folding the ribonuclease H domain of Moloney murine leukemia virus reverse transcriptase requires metal binding or a short N-terminal extension. 974 51

We have isolated spontaneous rifampicin-resistant mutants from Escherichia coli that showed allele-specific suppression of the copy-number phenotype of ColE1 high-copy-number mutants in vivo. The key step in the regulatory circuitry of the initiation of ColE1 DNA replication is the formation of the persistent hybrid between the primer RNA and the DNA template around the replication origin. Three host-encoded enzymes, RNase H, DNA polymerase I, and RNA polymerase, are essential to the replication initiation in vitro. To decide whether the activity of RNA polymerase is involved directly in the formation of the persistent hybrid, we screened rifampicin-resistant colonies for suppressors of ColE1 copy-number mutants. Suppressor strain YY572 (rpoB572) changes the 572 residue of the beta subunit of RNA polymerase, encoded by the rpoB gene, from isoleucine to leucine. Another suppressor, YY513 (rpoB513), changes the 513 residue from glutamine to lysine. The other known rifampicin-resistant alleles located at residue 513, rpoB8 and rpoB101, did not show a significant suppression of the copy number of those ColE1 copy-number mutants as rpoB513. The suppression by rpoB513 on different ColE1 copy-number mutants showed allelic specificity. The possible roles of RNA polymerase in control of ColE1 copy number are discussed.
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PMID:Allele-specific suppression of ColE1 high-copy-number mutants by a rpoB mutation of Escherichia coli. 988 6

Reverse transcriptase enzymes (RT) convert single-stranded retroviral RNA genomes into double-stranded DNA. The RT enzyme can use both RNA and DNA primers, the former being used exclusively during initiation of minus- and plus-strand synthesis. Initiation of minus-strand DNA synthesis occurs by extension of a tRNA primer that is associated with the viral genome, and plus-strand DNA synthesis is initiated from an RNase H- resistant polypurine tract of the genomic RNA that remains bound to the newly synthesized minus-strand DNA. All other phases of reverse transcription represent elongation of a DNA primer. We demonstrate that the polymerase fidelity of RT enzymes is significantly higher in tRNA-primed reverse transcription compared with DNA-primed reactions. Two mechanistic explanations can be proposed. First, the type of template-primer (T- P) duplex (RNA-RNA versus RNA-DNA) may affect the RT enzyme conformation such that the discrimination against incorrect nucleotides is affected. Second, the tRNA primer may act as a fidelity co-factor through specific association with the RT enzyme. According to the latter hypothesis, the increased fidelity observed for an RNA-RNA T-P should persist at a distance from the initiation site, where the enzyme-bound nucleic acid duplex will consist of RNA-cDNA. However, we measured that the effect of tRNA on the fidelity is detectable only at a short distance from the initiation site. These results indicate that the type of T-P duplex influences the fidelity of reverse transcription, suggesting that two small segments of the viral genome downstream of the initiation sites for minus- and plus-strand DNA synthesis are copied with a fidelity that is greater than average.
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PMID:The fidelity of reverse transcription differs in reactions primed with RNA versus DNA primers. 1008 43

Availability of 4-thiouridine (4-thioU)-containing RNAs is the prerequisite for 4-thioU site-specific cross-linking studies. This paper presents a method for constructing such RNAs. A 5'- and a 3'-RNA are synthesized via phage RNA polymerase transcription and/or RNase H site-specific cleavage directed by 2'-O-methyl-RNA-DNA chimeras. These two half-RNAs in combination correspond to the sequence of full-length RNA, with a single nucleotide gap at the junction that will be filled in with a 4-thiouridylate. A single p4SUp, which is derived from 4SUpN (N can be any nucleotide) via 5'-phosphorylation (therefore, the phosphate can be radioactive) followed by RNase A digestion, is then ligated to the 3' end of the 5'-half RNA with T4 RNA ligase. The 3'-phosphate of the ligated product is subsequently removed by calf intestinal alkaline phosphatase to produce a 3'-hydroxyl group. The resulting 5'-half RNA and the 3'-half RNA with a 5'-phosphate group (which can also be radioactive) are then aligned with a bridging deoxyoligonucleotide and ligated with T4 DNA ligase. This method was previously applied to the P120 pre-mRNA that contains an AT-AC intron, yielding three RNAs each containing a single 4-thioU near the 5'-splice site. Subsequent cross-linking studies with these RNAs yielded detailed information regarding interactions between the 5'-splice site and other spliceosomal snRNAs and between the 5'-splice site and proteins during splicing. Because there is no sequence constraint surrounding the site of 4-thioU substitution, this method should be applicable to many other RNAs.
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PMID:Construction of 4-thiouridine site-specifically substituted RNAs for cross-linking studies. 1020 12

We have developed a reconstituted system which models the events associated with human immunodeficiency virus type 1 (HIV-1) plus-strand transfer. These events include synthesis of plus-strand strong-stop DNA [(+) SSDNA] from a minus-strand DNA donor template covalently attached to human tRNA3Lys, tRNA primer removal, and annealing of (+) SSDNA to the minus-strand DNA acceptor template. Termination of (+) SSDNA synthesis at the methyl A (nucleotide 58) near the 3' end of tRNA3Lys reconstitutes the 18-nucleotide primer binding site (PBS). Analysis of (+) SSDNA synthesis in vitro and in HIV-1 endogenous reactions indicated another major termination site: the pseudouridine at nucleotide 55. In certain HIV-1 strains, complementarity between nucleotides 56 to 58 and the first three bases downstream of the PBS could allow all of the (+) SSDNA products to be productively transferred. Undermodification of the tRNA may be responsible for termination beyond the methyl A. In studies of tRNA removal, we find that initial cleavage of the 3' rA by RNase H is not sufficient to achieve successful strand transfer. The RNA-DNA hybrid formed by the penultimate 17 bases of tRNA still annealed to (+) SSDNA must also be destabilized. This can occur by removal of additional 3'-terminal bases by RNase H (added either in cis or trans). Alternatively, the nucleic acid chaperone activity of nucleocapsid protein (NC) can catalyze this destabilization. NC stimulates annealing of the complementary PBS sequences in (+) SSDNA and the acceptor DNA template. Reverse transcriptase also promotes annealing but to a lesser extent than NC.
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PMID:Molecular requirements for human immunodeficiency virus type 1 plus-strand transfer: analysis in reconstituted and endogenous reverse transcription systems. 1023 40


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