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 role of the C-terminal Phe882-Ala883 residues of bacteriophage T7 RNA polymerase in specific transcription has been investigated by means of site-directed mutagenesis. A mutant enzyme that lacks the C-terminal Phe882-Ala883 residues, denoted the "foot" mutant, has been cloned and overproduced, and the effects of the deletion on promoter recognition, initiation, and elongation have been determined. Gel retardation assays and DNase I footprinting show that the foot mutant specifically recognizes and binds to T7 promoters, although this binding appears to be approximately 30-fold weaker than that of the wild-type enzyme. Transcription assays using oligonucleotide templates that contain the consensus T7 promoter show a dramatic decrease in transcriptional activity for the foot mutant. With templates whose coding region begins CCC..., the mutant synthesizes poly(G) products even in the presence of all four nucleotides. The synthesis of poly(G) products from such templates has previously been observed for the wild-type enzyme when GTP is the sole nucleotide present in the reaction and is thought to occur by a novel mechanism involving slippage of the RNA chain 3' to 5' relative to the template [Martin, C.T., Muller, D.K., & Coleman, J.E. (1988) Biochemistry 27, 3966-3974]. These data suggest that the loss in transcriptional activity by the foot mutant results from a severe decrease in processivity as well as catalytic efficiency of the enzyme. Removal of the C-terminal Phe and Ala residues from the wild-type enzyme with carboxypeptidase A generates the phenotype of the mutant precisely, proving that all of the properties of the foot mutant derive from the loss of the Phe-Ala-COOH moiety.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Processivity of T7 RNA polymerase requires the C-terminal Phe882-Ala883-COO- or "foot". 205 36

The lysozyme of bacteriophage T7 is a bifunctional protein that cuts amide bonds in the bacterial cell wall and binds to and inhibits transcription by T7 RNA polymerase. The structure of a mutant T7 lysozyme has been determined by x-ray crystallography and refined at 2.2-A resolution. The protein folds into an alpha/beta-sheet structure that has a prominent cleft. A zinc atom is located in the cleft, bound directly to three amino acids and, through a water molecule, to a fourth. Zinc is required for amidase activity but not for inhibition of T7 RNA polymerase. Alignment of the zinc ligands of T7 lysozyme with those of carboxypeptidase A and thermolysin suggests structural similarity among the catalytic sites for the amidase and these zinc proteases. Mutational analysis identified presumed catalytic residues for amidase activity within the cleft and a surface that appears to be the site of binding to T7 RNA polymerase. Binding of T7 RNA polymerase inhibits amidase activity.
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PMID:The structure of bacteriophage T7 lysozyme, a zinc amidase and an inhibitor of T7 RNA polymerase. 817 Oct 31

Bacteriophage T7 lysozyme binds to T7 RNA polymerase (RNAP) and regulates its transcription by differentially repressing initiation from different T7 promoters. This selective repression is due in part to a lysozyme-induced increase in the KNTP of the initiation complex (IC) and to intrinsically different NTP concentration requirements for efficient initiation from different T7 promoters. While lysozyme represses initiation, once the enzyme has left the promoter and formed an elongation complex (EC) it is generally resistant to the effects of lysozyme. The mechanism by which the inhibitory effects of lysozyme are largely restricted to the initiation phase of transcription is not well understood. We find that T7 lysozyme destabilizes initial transcription complexes (ITCs) and increases the rate of release of transcripts from these complexes but does not destabilize ECs. However, if the RNA:RNAP interaction proposed to be important for EC stability is disrupted by proteolysis of the RNA-binding domain or use of templates which interfere with establishment of this RNA:RNAP interaction, the EC becomes sensitive to lysozyme. Comparison of the X-ray structures of T7RNAP and of a T7RNAP:T7 lysozyme complex reveals that lysozyme causes the C terminus of the polymerase to flip out of the active site. Experiments in which carboxypeptidase A is used to probe the lysozyme-induced exposure of the C terminus reveal a large decrease in carboxypeptidase sensitivity following transcription initiation, suggesting that interactions with the 3'-end of the RNA help stabilize the active site in a functional (carboxypeptidase protected) conformation. Thus, the resistance of the EC to lysozyme appears to be due to the consecutive establishment of two sets of RNA:RNAP interactions. The first is made with the 3'-end of the RNA and helps stabilize a functional conformation of the active site, thereby suppressing the effects of lysozyme on KNTP. The second is made with a more upstream element of the RNA and keeps the EC from being destabilized by lysozyme binding.
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PMID:Mechanisms by which T7 lysozyme specifically regulates T7 RNA polymerase during different phases of transcription. 1054 43