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)

Procedures for the synthesis, purification, and characterization of beta, gamma-peroxy analogues of the eight common ribo- and deoxyribonucleoside triphosphates have been developed. Although adenosine 5'-(beta, gamma-peroxytriphosphate) was stable to conditions in most biochemical systems, incubation of a solution of the analogue at 100 degrees C led to formation of AMP and ATP, as well as ADP. NAD+ pyrophosphorylase was the only enzyme among 13 tested for which adenosine 5'-(beta, gamma-peroxytriphosphate) was a good substrate, but the analogue was an effective inhibitor for a number of kinases. The peroxy compounds tested inactive with Escherichia coli RNA polymerase and DNA polymerase I, as well as with wheat germ RNA polymerase II.
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PMID:Nucleoside 5'-(beta, gamma-peroxytriphosphates). 631 17

The T4 mot gene regulates middle mode RNA synthesis in phage-infected cells. The mot gene product has been identified in two ways. (i) Infections with amber and temperature-sensitive mot mutants both lead to the disappearance of a number of protein bands on SDS-polyacrylamide gels. These are middle mode proteins whose synthesis depends on mot function. The mot protein disappears from such gels after infection with a mot amber mutant, but not with the mot missense mutant. (ii) This same protein is the only one to have a charge alteration when proteins from wild-type phage and mot missense mutant infections are compared by two-dimensional gel electrophoresis. Mot protein is basic and has a mol. wt. of 24 000. It migrates between the positions of gp 1 and gp IPIII on 15% SDS-polyacrylamide gels. Mot protein synthesis begins immediately after infection and continues until 4 min after infection at 30 degrees C, after which time it is strongly inhibited. This inhibition depends neither on T4 DNA synthesis nor on ADP ribosylation of the alpha subunits of the Escherichia coli RNA polymerase. The mot protein does not regulate its own biosynthesis. It is stable throughout the course of infection.
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PMID:Identification and biosynthesis of the bacteriophage T4 mot regulatory protein. 635 9

The RNA polymerase from bacteriophage T4-infected Escherichia coli, which specifically initiates transcription at phage T4 late promoters, is extensively modified by ADP-ribosylation of core subunits and by binding several virus-encoded subunits. We show here that one of these subunits, the phage T4 gene 55 protein, designated gp55, alone endows unmodified RNA polymerase core enzyme from uninfected E. coli with the ability to selectively initiate transcription at the phage T4 late promoters, without participation by E. coli RNA polymerase o- subunit.
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PMID:Defining a bacteriophage T4 late promoter: bacteriophage T4 gene 55 protein suffices for directing late promoter recognition. 638 59

After infection of Escherichia coli with bacteriophage T4, the host RNA polymerase acquires several small phage-induced polypeptides (Stevens, A. (1974) Biochemistry 13, 493-503) and its alpha subunits get ADP-ribosylated by a virus-specific enzyme (Zillig, W., Mailhammer, R., Skorko, R., and Rohrer, H. (1977) Curr. Top. Cell. Regul. 12, 263-271). The modified polymerase displays changed enzymatic properties including sensitivity to increased salt concentration and a higher transition temperature of open promoter complex formation (promoter melting temperature). In order to assess the role of individual modifications in the changed enzyme properties, we isolated RNA polymerase from cells infected with T4 mutant defective in the ADP-ribosylating enzyme. We also purified one of the associated polypeptides, the 15,000-dalton protein which is invariably present in stoichiometric amounts in different RNA polymerase preparations. In an in vitro transcription system using T4 DNA as template, we demonstrate that the 15-kDa protein is the cause of the elevated promoter melting temperature and can induce this property when added to host RNA polymerase. We also show that the increased salt sensitivity of T4-modified polymerase is primarily the result of ADP-ribosylation of its alpha subunits.
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PMID:The effect of a bacteriophage T4-induced polypeptide on host RNA polymerase interaction with promoters. 638 13

Bacteriophage T4 infection rapidly and almost completely inhibits transcription of host and other phage DNAs. Two processes have been implicated to date in this inhibition: (1) ADP ribosylation of the alpha subunits of the RNA polymerase, involving gpalt (which is injected with the phage DNA) and, later, gpmod; and (2) the action of the T4 alc/unf gene product, synthesized immediately after infection. The latter unfolds the host genome and also blocks transcription of cytosine-containing DNA. Here, we describe the identification on two-dimensional polyacrylamide gels of gpalc/unf, the more precise mapping of the gene and the identification and analysis of the appropriate DNA sequence from an Unf+ alc mutant.
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PMID:Identification and characterization of the alc gene product of bacteriophage T4. 638 56

P3-[(2,4-Dinitrophenyl)amino]ethyl (DNPNHEt) and P3-methyl phosphate esters of nucleoside 5'-triphosphates have been synthesized. Their properties as substrates in the initiation and elongation steps of transcription have been examined by using RNA polymerase from Escherichia coli and poly[d(A-T)] or T7 DNA as templates. It is shown that transcription can be initiated by ATP-EtNHDNP and that 2,4-dinitrophenyl residues are incorporated at the 5' end of the RNA molecules. Steady-state kinetic experiments of abortive initation on promoters A1 and A3 of T7 DNA revealed that ATP-EtNHDNP, ADP-EtNHDNP, and ATP-OCH3 have lower Km values and markedly reduced Vmax values compared to those of ATP. The two classes of esters, NTR-EtNHDNP and NTP-OCH3, were found to differ regarding their utilization as substrates for elongation. Both ATP-OCH3 and UTP-OCH3 are substrates for transcription. However, only the pyrimidine derivatives of NTP-EtNHDNP are elongation substrates which release DNPNHEt-PP upon utilization. This dramatic difference between the purine and pyrimidine derivatives of NTP-EtNHDNP reflects a selective process in the transcriptional complex for purines and pyrimidines.
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PMID:Properties of P3 esters of nucleoside triphosphates as substrates for RNA polymerase from Escherichia coli. 702 6

After infection of Escherichia coli cells, bacteriophage T4 induces several changes in the host DNA-dependent RNA polymerase. A well-characterized chemical change is a two-step ADP-ribosylation of the enzyme's alpha subunit (1). In order to investigate the effect of this change on RNA polymerase transcriptional properties in an in vitro system, we have reconstituted the enzyme from separated individual subunits which were obtained from normal or T4-modified RNA polymerases. It is demonstrated that the enzymes containing T4-modified alpha differ from the enzymes with normal alpha in two respects: (i) their overall activity on T4 DNA is reduced and (ii) they fail to utilize certain T4 promotors while efficiently utilizing other promoters. Among the promoters which are switched off by alpha modification are the two promoters of the D region and one of the two promoters of the T4 tRNA gene cluster. The differential effect of alpha modification on the expression of the tRNA and the D regions in vitro correlates with the previously established pattern of their transcription in vivo. It is suggested that the T4-induced ADP-ribosylation of RNA polymerase alpha subunit is involved in the shutoff of the early bacteriophage genes at the late stage of phage development.
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PMID:Control of promoter utilization by bacteriophage T4-induced modification of RNA polymerase alpha subunit. 703 2

The isolation and purification of DNA-dependent RNA polymerase I (EC 2.7.7.6) from parsley (Petroselinum crispum) callus cells grown in suspension culture is described. The enzyme was solubilized from isolated chromatin. Purification was achieved by using DEAE- and phospho-cellulose in batches, followed by column chromatography on DEAE- and phospho-cellulose (two columns) and density-gradient centrifugation. The highly purified enzyme was stable over several months. The properties of purified parsley RNA polymerase I were investigated. Optimum concentration for Mn2+ was 1 mM, and for Mg2+ 4-6 mM, Mn2+ was slightly more stimulatory than Mg2+. The enzyme was most active at low ionic strengths [10-20 mM-(NH4)SO4]. The influence of various phosphates was tested: pyrophosphate inhibited RNA polymerase at low concentrations, whereas orthophosphate had no effect on the enzyme activity. ADP was slightly inhibitory, and AMP had no effect on the enzyme reaction. Nucleoside triphosphates and bivalent cations in equimolar concentrations in the range 4-11 mM did not influence the RNA synthesis in vitro. Free nucleoside triphosphates in excess of this 1:1 ratio inhibited the enzyme activity, unlike free bivalent cations, which stimulated RNA polymerase I.
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PMID:Purification and characterization of chromatin-bound DNA-dependent RNA polymerase I from parsley (Petroselinum crispum). Influence of nucleoside triphosphates. 747 92

Catalyzed polymerization reactions represent a primary anabolic activity of all cells. It can be assumed that early cells carried out such reactions, in which macromolecular catalysts were encapsulated within some type of boundary membrane. In the experiments described here, we show that a template-independent RNA polymerase (polynucleotide phosphorylase) can be encapsulated in dimyristoyl phosphatidylcholine vesicles without substrate. When the substrate adenosine diphosphate (ADP) was provided externally, long-chain RNA polymers were synthesized within the vesicles. Substrate flux was maximized by maintaining the vesicles at the phase transition temperature of the component lipid. A protease was introduced externally as an additional control. Free enzyme was inactivated under identical conditions. RNA products were visualized in situ by ethidium bromide fluorescence. The products were harvested from the liposomes, radiolabeled, and analyzed by polyacrylamide gel electrophoresis. Encapsulated catalysts represent a model for primitive cellular systems in which an RNA polymerase was entrapped within a protected microenvironment.
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PMID:Production of RNA by a polymerase protein encapsulated within phospholipid vesicles. 752 10

Initiation of in vitro ColE2 DNA replication requires the plasmid-specified Rep protein and DNA polymerase I but not RNA polymerase and DnaG primase. The ColE2 Rep protein binds specifically to the origin where replication initiates. Leading-strand synthesis initiates at a unique site in the origin and lagging-strand DNA synthesis terminates at another unique site in the origin. Here we show that the primer RNA for leading-strand synthesis at the origin has a unique structure of 5'-ppApGpA. We reconstituted the initiation reaction of leading-strand DNA synthesis by using purified proteins, the ColE2 Rep protein, Escherichia coli DNA polymerase I and SSB, and we showed that the ColE2 Rep protein is a priming enzyme, primase, which is specific for the ColE2 origin. The ColE2 Rep protein is unique among other primases in that it recognizes the origin region and synthesizes the primer RNA at a fixed site in the origin region. Specific requirement for ADP as a substrate and its direct incorporation into the 5' end of the primer RNA are also unique properties of the ColE2 Rep protein.
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PMID:Primer RNA synthesis by plasmid-specified Rep protein for initiation of ColE2 DNA replication. 758 42


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