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 5.8 kb RNA genome of potato leafroll luteovirus (PLRV) contains two overlapping open reading frames, ORF2a and ORF2b, which are characterized by helicase and RNA polymerase motifs, respectively, and possibly represent the viral replicase. Within the overlap, ORF2b lacks an AUG translational start codon and is therefore presumably translated by -1 ribosomal frameshifting as a transframe protein with ORF2a. This hypothesis was studied by introducing the putative frameshift region into an internal position of the beta-glucuronidase (GUS) gene and testing for the occurrence of frameshifting in vivo by transient expression of GUS activity in potato protoplasts as well as in vitro by translation in the reticulocyte system. Both experimental approaches demonstrate that a -1 frameshift occurs at a frequency of approximately 1%. Site-directed mutagenesis identified the frameshift region and the involvement of the novel heptanucleotide motif UUUAAAU in conjunction with an adjacent stem-loop structure. Part of this stem-loop encodes a basic region in the ORF2b moiety of the transframe protein which was shown by binding experiments with PLRV RNA to represent a nucleic acid-binding domain. These data support a possible biological significance of the frameshift to occur at this position of the large overlap by including the putative RNA template-binding site of the PLRV replicase in the ORF2a/ORF2b transframe protein.
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PMID:Ribosomal frameshifting in plants: a novel signal directs the -1 frameshift in the synthesis of the putative viral replicase of potato leafroll luteovirus. 154 75

An examination of the genomic strategy of pea enation mosaic virus (PEMV) RNA 1 has verified strong organizational and sequence relationships between PEMV and the beet western yellows-potato leafroll luteovirus subgroup. Sequence analysis of RNA 1 demonstrated five predominant open reading frames (ORFs). The extreme 5' ORF encodes a 34K product of unknown function. The second ORF encodes an 84K product which overlaps 90% of ORF 1 (in a unique reading frame) and is expressed by internal initiation beginning at the second start codon from the 5' terminus. This protein contains a protease-like motif characteristic of serine- and cysteine-based proteases, suggesting involvement in post-translational processing of viral translation products. The third ORF is characterized by a number of RNA polymerase motifs and a helicase-like motif typical of RNA-dependent RNA polymerases. It overlaps (out of frame) the ORF 2 product and is proposed to be expressed by a frameshift fusion of the ORF 2 and ORF 3 products. The fourth ORF encodes the viral coat protein, and is immediately followed in frame by a 33K ORF thought to represent the aphid transmission subunit of the PEMV virion. Northern blot analysis of polysome-associated RNA suggests that both products are expressed from an 1800 nucleotide subgenomic mRNA, with the 33K product expressed as a read-through fusion with the coat protein monomer.
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PMID:The nucleotide sequence and luteovirus-like nature of RNA 1 of an aphid non-transmissible strain of pea enation mosaic virus. 187 94

DNA containing the plasmid origin of bacteriophage P1 is replicated in vitro by a protein fraction prepared from uninfected Escherichia coli supplemented with purified P1 RepA protein. It has previously been shown that the reaction required the E. coli DnaA initiator protein, the DnaB helicase, DnaC protein, RNA polymerase, and DNA gyrase. I show here that three E. coli heat shock proteins, DnaJ, DnaK, and GrpE, are directly involved in P1 plasmid replication. Purified DnaJ, DnaK, and GrpE proteins were required to stimulate P1 plasmid ori DNA-dependent replication in in vitro complementation assays in which the host protein fractions were prepared from cells mutated in the corresponding gene. I have also found that the DnaJ and RepA proteins form a complex. This complex exists in crude cell extracts and can be isolated as a molecular species of about 160,000 Da containing one dimer of DnaJ protein and one dimer of RepA. The complex can also be reconstituted by mixing purified DnaJ and RepA proteins. These results imply that the DnaJ-RepA complex, DnaK, and GrpE are directly involved in P1 plasmid replication.
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PMID:Three Escherichia coli heat shock proteins are required for P1 plasmid DNA replication: formation of an active complex between E. coli DnaJ protein and the P1 initiator protein. 218 45

Barley stripe mosaic hordeivirus (BSMV) has a tripartite positive-sense RNA genome which encodes seven major polypeptides. Infectious in vitro transcripts derived from full-length wild-type and mutant cDNA clones have been used to investigate the contribution made by various BSMV gene products to viral RNA replication and systemic movement. We show that whereas all three of the BSMV RNA components are required for plant infection, RNAs alpha and gamma can replicate together in barley protoplasts, and therefore RNA beta must encode functions required for systemic invasion of plants. The alpha a and gamma a proteins, which contain helicase and RNA polymerase sequence motifs, together comprise the essential virus-encoded components of BSMV RNA replicase. A second BSMV protein (beta b) which contains a helicase motif is not required for RNA replication. A small cysteine-rich protein (gamma b) is dispensable for infection of plants, but in its absence the accumulation of viral coat (beta a) and beta b proteins is significantly reduced. In addition, mutations in both the gamma b and gamma a (replicase) proteins can affect the systemic movement phenotype.
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PMID:Identification of barley stripe mosaic virus genes involved in viral RNA replication and systemic movement. 220 52

Initiation of bacteriophage lambda DNA replication in vivo and in crude in vitro systems is strongly dependent on transcription at or near the lambda replication origin (ori lambda). Through its capacity to prevent RNA polymerase-mediated 'transcriptional activation' of lambda DNA replication, the lambda cI repressor is capable of negatively regulating initiation of lambda DNA replication, even when all required replication proteins are present. Surprisingly, the strict requirement for transcriptional activation of lambda DNA replication was lost when lambda replication was initiated in an in vitro system composed of nine purified replication proteins [Mensa-Wilmot et al. (1989) J. Biol. Chem., 264, 2853-2861]. We have found that crude extracts of Escherichia coli contain proteins that are capable of restoring the physiological linkage between transcription and ori lambda-dependent replication when they are added to the nine-protein replication system. The protein primarily responsible for this effect has been purified and identified as protein HU, a histone-like protein that is a major constituent of the bacterial nucleoid. HU, when present at a 1:1 weight ratio with supercoiled ori lambda plasmid, is a potent inhibitor of lambda DNA replication in the nine-protein replication system. However, when the ori lambda template is transcribed by E. coli RNA polymerase, the HU-mediated inhibition of lambda DNA replication is abolished. HU does not inhibit propagation of lambda replication forks. Instead, HU apparently interferes with the assembly or function of nucleoprotein structures containing the E. coli DnaB helicase that are formed at ori lambda prior to priming and DNA synthesis. We suggest that the chromatin structure of the template DNA in the region surrounding ori lambda plays a central role in the negative regulation of the initiation of lambda DNA replication in vivo.
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PMID:Transcriptional activation of bacteriophage lambda DNA replication in vitro: regulatory role of histone-like protein HU of Escherichia coli. 252 19

We have established an in vitro system, composed of highly purified bacteriophage lambda and Escherichia coli proteins, that specifically replicates supercoiled templates bearing the lambda replication origin (ori lambda). The complete system is composed of three groups of proteins: the virus-encoded initiator proteins (the lambda O and P proteins), the E. coli replication fork propagation machinery (single-stranded DNA-binding protein, dnaB helicase, dnaG primase, DNA polymerase III holoenzyme, and DNA gyrase), and two bacterial heat shock proteins (dnaJ and dnaK proteins). DNA replication in this system is initiated at or near ori lambda and proceeds unidirectionally rightwards through theta-structure intermediates, ultimately yielding a pair of intertwined daughter circles as the final product. In striking contrast to the situation in vivo and in crude in vitro systems, initiation of lambda DNA replication in the purified protein system does not require "transcriptional activation" of the origin region by E. coli RNA polymerase. We conclude that E. coli primase generates the primers for all leading and lagging strand DNA chains synthesized in this reconstituted lambda replication system.
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PMID:Reconstitution of a nine-protein system that initiates bacteriophage lambda DNA replication. 253 26

Bacteriophage T4 gene 41 protein is an essential replication protein, part of the primase-helicase required for lagging strand DNA synthesis. In a T4+ infection, 41 RNA is first expressed as a polycistronic transcript attached to the upstream RNA of genes uvsX (recombination protein) and 40 (stimulates head formation (Hinton, D. M. (1989) J. Biol. Chem. 264, 14432-14439). As infection proceeds, less of the upstream RNA extends into gene 41 due to an RNA 3' end, approximately equal to 60 bases downstream of uvsX. DNA sequence analysis of this region positions this end within gene 40, immediately after a GC-rich hairpin. This end probably arises from host factor-dependent transcription termination or RNA processing since it is observed in RNA expressed by a uvsX-40-41 plasmid in vivo, but is not seen after in vitro transcription with purified Escherichia coli RNA polymerase. The E. coli transcription termination (rho) mutant rho026 has been characterized as a rho mutation whose terminating activity is not effectively overcome by phage lambda antitermination (Das, A., Gottesman, M. E., Wardwell, J., Trisler, P., and Gottesman, S. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 5530-5534). During a T4+ abortive infection of rho026, the levels of some phage proteins, including 41, are depressed; a T4 phage mutant in goF gives wild type protein patterns in rho026 (Stitt, B. L., and Mosig, G. (1989) J. Bacteriol., in press). The RNA analyses presented here demonstrate that the severalfold decrease in 41 protein in rho026 is accompanied by a similar decrease in 41 RNA. There is both a general reduction in polycistronic uvsX-40-41 RNA and a 2-2.5-fold increase in the proportion of uvsX RNA ending at the 3' end. Infection of rho026 by T4 goF1 returns the relative amount of RNA reading into 41 versus that stopped to near a wild type level. These results suggest that host rho and the T4 goF are involved in the expression of T4 41 RNA.
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PMID:Altered expression of the bacteriophage T4 gene 41 (primase-helicase) in an Escherichia coli rho mutant. 266 90

We have examined the mechanism by which ribavirin inhibits the multiplication of reovirus. At a concentration of 12.5 microM (3 micrograms/ml) ribavirin inhibits viral multiplication, ssRNA formation, dsRNA formation, and protein synthesis by about 90%; when much higher concentrations are used for brief periods of time, the primary target of ribavirin is seen to be viral ssRNA synthesis. When the effect of ribavirin triphosphate (RTP) was tested on the in vitro transcription by cores of the dsRNA genome segments into plus-stranded RNA, elongation, that is, the formation of intact mRNA molecules, was found to be inhibited to the greatest extent; initiation was at least 2.5 times less sensitive, and cap formation and methylation were almost unaffected. The inhibition of elongation and initiation was not competitive with respect to any of the four nucleoside triphosphates. Remarkably, the transcription of plus strands into minus strands by immature reovirus particles (the replicase reaction) was insensitive to RTP. A model is proposed that envisages RTP binding to a site close to the catalytic site of the transcriptase. This binding is postulated to inhibit the helicase function of the transcriptase and lower its affinity for template RNA so that the likelihood of premature termination is greatly increased. The helicase activity is not, of course, necessary for the transcription of plus strands into minus strands, which would account for the differential sensitivity of the transcriptase and the replicase to RTP.
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PMID:Studies on the mechanism of the antiviral activity of ribavirin against reovirus. 290 88

DNA-dependent ATPase IV has been purified to near homogeneity from the Novikoff rat hepatoma. The enzyme is devoid of DNA polymerase, RNA polymerase, exonuclease, endonuclease, phosphomonoesterase, 3'- or 5'-phosphodiesterase, polynucleotide kinase, protein kinase, topoisomerase, helicase or DNA reannealing activities at a detection level of 10(-5) to 10(-7) relative to the ATPase activity. The enzyme is a monomer of Mr 110,000, has a sedimentation coefficient of 5.9 S, a Stokes radius of 40 A and a frictional coefficient of 1.32. In the presence of Mg2+ ion and a polynucleotide effector, ATPase IV hydrolyzes either ATP or dATP to the nucleoside diphosphate plus Pi. Other ribo- or deoxyribonucleoside triphosphates are not substrates. ATPase IV utilizes double-stranded DNA and single-stranded DNA as effector; however, it does not utilize poly(dT). The Km for dsDNA or ssDNA is 2.2 microM (nucleotide). A variety of ATP analogues were found to be competitive inhibitors of ATPase IV.
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PMID:Purification and enzymological characterization of DNA-dependent ATPase IV from the Novikoff hepatoma. 296 5

In vivo, T7 DNA replication is initiated 15% of the distance from the genetic left end of the chromosome. This site, the primary origin of replication, consists of a 200-base pair (bp) intergenic segment from 14.5 to 15.0% within which are located two tandem T7 RNA polymerase promoters (phi 1.1A and phi 1.1B) followed by a 61-bp AT-rich (79% A + T) region. A fragment of T7 DNA containing the primary origin has been inserted into plasmids in order to facilitate studies on initiation in vitro. Initiation of DNA synthesis can be reconstituted using T7 RNA polymerase, T7 DNA polymerase, and T7 origin-containing plasmid DNAs. DNA synthesis is stimulated greatly by the T7 gene 4 protein, an enzyme that has helicase and primase activities. When T7 gene 4 protein is present, replication primarily yields partially replicated Y-form molecules as observed by electron microscopy. Synthesis is unidirectional and the branches of the Y-form molecules are uniform in size, with the branch point of the Y located at the origin. Using restriction enzyme analysis, DNA synthesis has been shown to proceed in the same direction (rightward with respect to the T7 genetic map) as transcription from the two promoters located at the origin. Initiation of DNA synthesis in the opposite direction requires the addition of a single-stranded DNA-binding protein (Fuller, C.W., and Richardson, C.C. (1985) J. Biol. Chem. 260, 3197-3206). The initial products of DNA synthesis have been analyzed by polyacrylamide gel electrophoresis. These DNAs have 10 to 60 ribonucleotides covalently linked to their 5' termini. These RNA primers arise by transcription from each of the two promoters, phi 1.1A and phi 1.1B, located within the primary origin.
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PMID:Initiation of DNA replication at the primary origin of bacteriophage T7 by purified proteins. Site and direction of initial DNA synthesis. 298 51


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