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)

In order to test the proposal that most nucleotide polymerases share a common active site structure and folding topology, we have generated 22 mutations of residues within motifs A, B and C of T7 RNA polymerase (RNAP). Characterization of these T7 RNAP mutants showed the following: (i) most of the mutations resulted in moderate to drastic reductions in T7 RNAP transcriptional activity supporting the idea that motifs A, B and C identify part of the polymerase active site; (ii) the degree of conservation of an amino acid within these motifs correlated with the degree to which mutation of that amino acid reduced transcriptional activity, supporting the predictive ability of this alignment in identifying the most functionally critical residues; (iii) a comparison of DNAP I and T7 RNAP mutants revealed similarities (as well as differences) between corresponding mutant phenotypes; (iv) the Klenow fragment structure is shown to provide a reasonable basis for interpretation of the differential effects of mutating different amino acids within motifs A, B and C in T7 RNAP. These observations support the proposal that these polymerase active sites have similar three-dimensional structures.
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PMID:Mutations in T7 RNA polymerase that support the proposal for a common polymerase active site structure. 139 70

A general mechanism for polymerase translocation is elaborated. The central feature of this mechanism is that a rapid translocational equilibrium is established after each cycle of nucleoside monophosphate incorporation such that the polymerase distributes itself by diffusional sliding between all accessible positions on the template with relative occupancy determined by relative free energy. While alternative models for translocation have not been fully developed, much of the language currently used to describe this step suggests an active mechanism coupled to conformational transitions in the polymerase. For example, a recent study of force generation by Escherichia coli RNA polymerase during transcription suggests that it is a mechanoenzyme analogous to kinesin of myosin motor proteins. While the proposed mechanism does not rule out conformational transitions during polymerase translocation, it suggests that they may be unnecessary and that translocation can be explained in terms of the affinity of the active site for nucleoside triphosphate and the relative free energies of the polymerase bound at different positions on the template. This mechanism makes specific predictions which are borne out experimentally with polymerases as distinct as E. coli DNAP I, phage T7 RNAP, and E. coli RNAP.
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PMID:A model for the mechanism of polymerase translocation. 899 20

Molecular dynamics simulation of Thermus thermophilus (Tt) RNA polymerase (RNAP) in a catalytic conformation demonstrates that the active site dNMP-NTP base pair must be substantially dehydrated to support full active site closing and optimum conditions for phosphodiester bond synthesis. In silico mutant beta R428A RNAP, which was designed based on substitutions at the homologous position (Rpb2 R512) of Saccharomyces cerevisiae (Sc) RNAP II, was used as a reference structure to compare to Tt RNAP in simulations. Long range conformational coupling linking a dynamic segment of the bridge alpha-helix, the extended fork loop, the active site, and the trigger loop-trigger helix is apparent and adversely affected in beta R428A RNAP. Furthermore, bridge helix bending is detected in the catalytic structure, indicating that bridge helix dynamics may regulate phosphodiester bond synthesis as well as translocation. An active site "latch" assembly that includes a key trigger helix residue Tt beta' H1242 and highly conserved active site residues beta E445 and R557 appears to help regulate active site hydration/dehydration. The potential relevance of these observations in understanding RNAP and DNAP induced fit and fidelity is discussed.
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PMID:Conformational coupling, bridge helix dynamics and active site dehydration in catalysis by RNA polymerase. 2047 25