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)

Transcription termination by vaccinia virus RNA polymerase during synthesis of early mRNAs requires a virus-encoded termination factor (VTF). VTF is but one of many activities associated with the vaccinia virus mRNA capping enzyme, a heterodimer of 95- and 33-kDa subunits encoded by the D1 and D12 genes, respectively. Although the three catalytic domains involved in cap formation have been assigned to individual subunits or portions thereof, the structural requirements for VTF activity are unknown. We now report that both full-length subunits are required for transcription termination. The 844-amino acid D1 subunit by itself, which is fully active in triphosphatase and guanylyltransferase functions, has no demonstrable VTF activity in vitro. Neither does the D12 subunit by itself. The heterodimeric methyltransferase domain of D1 (residues 498 to 844) and D12 subunits also has no VTF activity. VTF is not affected by a K-to-M mutation of the guanylyltransferase active site at position 260 (K260M) that abolishes enzyme-GMP complex formation or by a H682A/Y683A double mutation of the D1 subunit, which abrogates methyltransferase activity. Thus, the structural requirements for termination are distinct from those for nucleotidyl transfer and methyl transfer.
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PMID:The D1 and D12 subunits are both essential for the transcription termination factor activity of vaccinia virus capping enzyme. 774 34

Avian reovirus S1133 was shown to contain all the enzymatic activities required for the synthesis of mature viral transcripts, including a dsRNA-dependent RNA polymerase, a nucleoside triphosphate phosphohydrolase, an mRNA guanylyltransferase, and two mRNA methyltransferases. The virus used these enzymes both in vitro and in vivo to catalyze the synthesis of viral mRNAs containing a type-1 cap at their 5' ends. Incubation of reovirions with GTP led to the formation of an intermediate structure consisting of GMP bound to the viral core protein lambda 3 through a phosphoamide linkage. The reaction was specific for GTP and required the presence of both Mg2+ and inorganic pyrophosphatase. The GMP moiety can be transferred from the lambda 3-GMP complex to acceptors such as GDP and GTP, yielding GpppG and GppppG, respectively. Our results demonstrate that lambda 3 is the avian reovirus guanylyltransferase.
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PMID:Endogenous enzymatic activities of the avian reovirus S1133: identification of the viral capping enzyme. 785 76

The double-stranded RNA bacteriophage phi 6 contains a virion-associated RNA-dependent RNA polymerase complex. Removal of the virus envelope and the nucleocapsid surface protein, P8, reveals a nucleocapsid core particle (proteins P1, P2, P4, P7) which is the viral polymerase complex, capable of synthesizing RNA strands of positive polarity. The in vitro plus strand synthesis (transcription) reaction of the particle obtained from the mature virion was optimized and its activation and inactivation were investigated. Purine nucleoside triphosphates (NTPs), binding to a low-affinity binding site in the polymerase complex, activated plus strand synthesis. GTP was the preferred NTP, but dGTP, ddGTP, and the noncleavable analog GMP-PCP could also switch on transcription. This NTP-binding site is probably different from that of the unspecific viral NTPase found in protein P4 and also from that of the rNTP-specific RNA polymerase active site. Binding of purine NTPs was sufficient for the switch-on; hydrolysis of the NTP was not required. Besides nucleotides, divalent cations had an effect on phi 6 in vitro plus strand synthesis. Magnesium ions are required for the activity but calcium ions inhibit the reaction. Manganese ions are shown to dissipate the effect of magnesium and calcium ions, leading to uncontrolled, exceptionally high level plus strand synthesis.
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PMID:In vitro transcription of the double-stranded RNA bacteriophage phi 6 is influenced by purine NTPs and calcium. 788 44

The Escherichia coli GreA and GreB proteins induce cleavage of 3' fragments from nascent transcripts in halted transcription complexes. We have overproduced and purified the GreA protein and tested how it affects initiation, pausing, and termination by E. coli RNA polymerase. Recombinant GreA induced cleavage of two to three nucleotide fragments in two promoter-proximal complexes, whereas an apparently endogenous cleavage removed a single larger fragment. Both types of cleavage stopped once the transcript was shortened to approximately 10 nucleotides. However, during initiation, GreA induced cleavage of transcripts as short as four nucleotides, inhibiting their release as abortive products and stimulating both productive initiation and "primer-shifting" at a weak promoter. GreA induced repetitive cleavage over a long distance in complexes containing a long G-less nascent transcript. However, reverse translocation was inhibited in transcription complexes that contained a G-rich, C-less nascent transcript. Substituting IMP for GMP in the transcript relieved inhibition. Finally, GreA had little effect on transcription through the his and trp leader pause sites or on termination at nine different p-independent terminators. We propose that transcript cleavage and reverse translocation are controlled in part by backsliding of the nascent transcript through an RNA-binding site.
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PMID:GreA-induced transcript cleavage in transcription complexes containing Escherichia coli RNA polymerase is controlled by multiple factors, including nascent transcript location and structure. 807 55

Incubation of purified infectious pancreatic necrosis virus (IPNV) in the presence of [alpha 32P]GTP resulted in the formation of VP1-GMP. The GMP is linked to VP1 by a phosphodiester bond and its formation does not require the presence of divalent cations. In contrast to reovirus guanylyl transferase, the formation of IPNV VP1-GMP is not reversible and the guanylylation reaction is not inhibited by inorganic pyrophosphate. Furthermore, the IPNV VP1-GMP cannot transfer the GMP to an acceptor molecule (such as GTP) indicating that VP1 is not a capping enzyme. Time-course experiments revealed that after the initial guanylylation of VP1 to form VP1-pG, a second GMP is added to form VP1-pGpG, the formation of which is template-dependent. Since VP1 is present in the virion both as a free polypeptide and in a genome-linked form as VPg, and it is also the virion-associated RNA polymerase, the results suggest that VP1 may function as a primer during in vitro RNA synthesis.
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PMID:In vitro guanylylation of infectious pancreatic necrosis virus polypeptide VP1. 838 4

Influenza virus M1 protein has been shown to inhibit the transcription catalyzed by viral ribonucleoprotein complexes isolated from virions. Here, this inhibition mechanism was studied with the recombinant M1 protein purified from Escherichia coli expressing it from cDNA. RNA mobility shift assays indicated that both soluble and aggregate forms of the recombinant M1, which were separated by the glycerol density gradient, were bound to RNA. Once an M1-RNA complex was formed, free M1 was bound to the M1-RNA complex cooperatively rather than to free RNA. In addition, the recombinant M1 was capable of binding to preformed RNA-nucleocapsid protein complexes. The mechanism for inhibition of the viral RNA polymerase activity was analyzed by the in vitro RNA synthesis systems that depend on an exogenously added RNA template. These systems were more sensitive for evaluating the inhibition by M1 than the RNA synthesis system depending on an endogenous RNA template. The RNA synthesis inhibition was examined at four steps: cleavage of capped RNA; incorporation of the first nucleotide, GMP; limited elongation; and synthesis of full-size product. M1 inhibited RNA synthesis mainly at the early steps. The experiments with M1 mutant proteins containing amino acid deletions suggested that the M1 region between amino acid residues 91 and 111 was essential for anti-RNA synthesis activity, RNA binding, and oligomerization of M1 on RNA.
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PMID:Mechanism for inhibition of influenza virus RNA polymerase activity by matrix protein. 852 32

The toxin alpha-amanitin is frequently employed to completely block RNA synthesis by RNA polymerase II. However, we find that polymerase II ternary transcription complexes stalled by the absence of NTPs resume RNA synthesis when NTPs and amanitin are added. Chain elongation with amanitin can continue for hours at approximately 1% of the normal rate. Amanitin also greatly slows pyrophosphorolysis by elongation-competent complexes. Complexes which are arrested (that is, which have paused in transcription for long periods in the presence of excess NTPs) are essentially incapable of resuming transcription in the presence of alpha-amanitin. Complexes traversing sequences that can provoke arrest are much more likely to stop transcription in the presence of the toxin. The substitution of IMP for GMP at the 3' end of the nascent RNA greatly increases the sensitivity of stalled transcription complexes to amanitin. Neither arrested nor stalled complexes display detectable SII-mediated transcript cleavage following amanitin treatment. However, arrested complexes possess a low level, intrinsic transcript cleavage activity which is completely amanitin-resistant; furthermore, pyrophosphorolytic transcript cleavage in arrested complexes is not affected by amanitin.
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PMID:Amanitin greatly reduces the rate of transcription by RNA polymerase II ternary complexes but fails to inhibit some transcript cleavage modes. 870 41

Vaccinia virus RNA polymerase terminates transcription downstream of a UUUUUNU signal in the nascent RNA. Transduction of the RNA signal to the elongating polymerase requires a termination factor (vaccinia termination factor/capping enzyme) and is coupled to the hydrolysis of ATP. It was shown previously that incorporation of 5-bromouracil or 5-iodouracil within the UUUUUNU element abolishes termination by preventing factor-dependent release of the nascent chain from the polymerase elongation complex. Here, we report that termination is prevented by phosphorothioate substitution at UMP residues in the nascent RNA. In contrast, phosphorothioate substitution at AMP, CMP, and GMP nucleotides does not inhibit termination. Thus, the action of a eukaryotic termination factor entails recognition of the nucleotide bases and the phosphate groups of the target sequence in nascent RNA.
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PMID:Transcription termination by vaccinia RNA polymerase entails recognition of specific phosphates in the nascent RNA. 899 14

A central step in promoter activation by RNA polymerase (RNAP) is the localized separation of the DNA strands to form the transcription bubble. We have used potassium permanganate footprinting to monitor DNA strand-separation by the Bacillus subtilis sigmaD RNAP at the strong promoter, Phag, directing transcription of flagellin. The susceptibility of individual thymine bases to permanganate oxidation is influenced by temperature, Mg2+, nucleotides, and the RNAP delta subunit. In the absence of delta, sigmaD RNAP establishes a partially opened complex even at 0 degrees C with permanganate reactivity localized between -11 and -4 (RP(-4)). The region of strand separation expands to near -1 at 20 degrees C (RP(-1)) and to +3 at 40 degrees C (RP(+3)). The delta subunit inhibits the downstream propagation of the transcription bubble and thereby increases the concentration of early intermediates in the melting pathway. Indeed, E delta sigmaD forms a distinct nucleated complex (RPn) at 0 degrees C with a structural distortion localized to an AT base step within the -10 element. We propose a model for promoter melting in which strand separation nucleates within the conserved -10 consensus and subsequently propagates downstream. Mg2+ and nucleoside triphosphates (NTPs) favor the downstream propagation of the transcription bubble and strongly stimulate the RP(-1) to RP(+3) conversion. The NTP effects are apparently mediated by binding of substrate to the initiating NTP site: purines are more effective than pyrimidines and GMP alone can greatly increase the level of DNA-melting. The binding of substrates, but not Mg2+ alone, can effectively overcome the anti-melting effect of delta.
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PMID:DNA-melting at the Bacillus subtilis flagellin promoter nucleates near -10 and expands unidirectionally. 909 6

We have addressed whether the intrinsic 3'-->5' nuclease activity of human RNA polymerase II (pol II) can proofread during transcription in vitro. In the presence of SII, a protein that stimulates the nuclease activity, pol II quantitatively removed misincorporated nucleotides from the nascent transcript during rapid chain extension. The basis of discrimination between the correct and incorrect base was the slow addition of the next nucleotide to the mismatched terminus. Incorporation of inosine monophosphate inhibited next nucleotide addition by a similar magnitude as a mismatched base. We used this finding to demonstrate that addition of SII to a transcription reaction dramatically altered the RNA base content, reflecting the stable incorporation of more "correct" (GMP) and fewer "incorrect" (IMP) nucleotides.
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PMID:Transcriptional fidelity and proofreading by RNA polymerase II. 960 37


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