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Literature summary for 2.7.7.6 extracted from

  • Seibold, S.A.; Singh, B.N.; Zhang, C.; Kireeva, M.; Domecq, C.; Bouchard, A.; Nazione, A.M.; Feig, M.; Cukier, R.I.; Coulombe, B.; Kashlev, M.; Hampsey, M.; Burton, Z.F.
    Conformational coupling, bridge helix dynamics and active site dehydration in catalysis by RNA polymerase (2010), Biochim. Biophys. Acta, 1799, 575-587.
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

Cloned (Comment) Organism
recombinant Rpb2 R512C, TAP-tagged at the C-terminus of the RNAP II Rpb9 subunit Saccharomyces cerevisiae

Protein Variants

Protein Variants Comment Organism
E529A the substitution mutant is are faster than the wild-type enzyme in RNA elongation Saccharomyces cerevisiae
E529D the substitution mutant is are faster than the wild-type enzyme in RNA elongation Saccharomyces cerevisiae
E529Q the substitution mutant is are slower than the wild-type enzyme in RNA elongation Saccharomyces cerevisiae
additional information simulation of diverse McJ25-resistant mutations and their effects on enzyme activity, overview Thermus thermophilus
R428A site-directed mutagenesis, designed based on substitutions at the homologous position (Rpb2 R512) of Saccharomyces cerevisiae RNAP II, used as a reference structure, molecular dynamics simulations with starting Tt RNAP TEC structure, PDB 205J, that is in a strained, catalytic conformation that responds very sensitively to the R428A substitution but is stable for wild-type enzyme, overview. 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. The R428A substitution is instable in the i+1 dTMP-ATP base pair, as indicated by fluctuations in the dTMP O4-ATP N6 base pairing distance in R428A Thermus thermophilus
R512C site-directed mutagenesis, the highly conserved residue is located about 20 A from Mg2+-I and just C-terminal to the fork loop, molecular dynamics simulations, overview. Mutant Sc Rpb2 R512C is slow in elongation and shows transcriptional defects. Rpb2 R512C may have a defect in CTP-Mg2+ sequestration Saccharomyces cerevisiae
R766A the substitution is lethal, consistent with an important role for this invariant latch residue Saccharomyces cerevisiae
R766Q the substitution is lethal, consistent with an important role for this invariant latch residue Saccharomyces cerevisiae

Inhibitors

Inhibitors Comment Organism Structure
alpha-Amanitin the potent Sc RNAP II inhibitor binds to a ternary elongation complex with an open wedged conformation of the trigger loop Saccharomyces cerevisiae
Streptolydigin the antibiotic binds to a Tt RNAP TEC with an open trigger loop Thermus thermophilus

Metals/Ions

Metals/Ions Comment Organism Structure
Mg2+ required Thermus thermophilus
Mg2+ required Saccharomyces cerevisiae

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
ATP + RNAn Thermus thermophilus
-
diphosphate + RNAn+1
-
?
ATP + RNAn Saccharomyces cerevisiae
-
diphosphate + RNAn+1
-
?
CTP + RNAn Thermus thermophilus
-
diphosphate + RNAn+1
-
?
CTP + RNAn Saccharomyces cerevisiae
-
diphosphate + RNAn+1
-
?
GTP + RNAn Thermus thermophilus
-
diphosphate + RNAn+1
-
?
GTP + RNAn Saccharomyces cerevisiae
-
diphosphate + RNAn+1
-
?

Organism

Organism UniProt Comment Textmining
Saccharomyces cerevisiae
-
strains YZS84 and YDP19
-
Thermus thermophilus
-
-
-

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
ATP + RNAn
-
Thermus thermophilus diphosphate + RNAn+1
-
?
ATP + RNAn
-
Saccharomyces cerevisiae diphosphate + RNAn+1
-
?
CTP + RNAn
-
Thermus thermophilus diphosphate + RNAn+1
-
?
CTP + RNAn
-
Saccharomyces cerevisiae diphosphate + RNAn+1
-
?
GTP + RNAn
-
Thermus thermophilus diphosphate + RNAn+1
-
?
GTP + RNAn
-
Saccharomyces cerevisiae diphosphate + RNAn+1
-
?

Synonyms

Synonyms Comment Organism
multi-subunit RNA polymerase
-
Thermus thermophilus
multi-subunit RNA polymerase
-
Saccharomyces cerevisiae
RNAP
-
Thermus thermophilus
RNAP II
-
Saccharomyces cerevisiae

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
25
-
assay at Thermus thermophilus
25
-
assay at Saccharomyces cerevisiae

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.6
-
assay at Thermus thermophilus
7.6
-
assay at Saccharomyces cerevisiae

General Information

General Information Comment Organism
malfunction mutant Sc Rpb2 R512C is slow in elongation Saccharomyces cerevisiae
malfunction R428A RNAP is instable Thermus thermophilus
additional information in vitro assembly of Sc RNAP II ternary elongation complexes, overview. RNA polymerase 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. An active site latch assembly that includes a key trigger helix residue beta' H1242 and highly conserved active site residues beta E445 and R557 appears to help regulate active site hydration/dehydration. Molecular dynamics simulations, overview Saccharomyces cerevisiae
additional information modeling of Tt RNAP TEC containing a closed, catalytic trigger helix conformation. RNA polymerase 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. An active site latch assembly that includes a key trigger helix residue beta' H1242 and highly conserved active site residues beta E445 and R557 appears to help regulate active site hydration/dehydration. Molecular dynamics simulations, overview Thermus thermophilus