The involvement of the aspartate triad of the active center in all catalytic activities of multisubunit RNA polymerase.
Three conserved aspartate residues in the largest subunit of multisubunit RNA polymerases (RNAPs) coordinate two Mg2+ ions involved in the catalysis of phosphodiester bond synthesis. A structural model based on the stereochemistry of nucleotidyl transfer reaction as well as recent crystallographic data predict that these Mg2+ ions should also be involved in the reverse reaction of pyrophosphorolysis as well as in the endo- and exonucleolytic cleavage of the nascent RNA. Here, we check these predictions by constructing point substitutions of each of the three Asp residues in the beta' subunit of Escherichia coli RNAP and testing the mutant enzymes' functions. Using artificially assembled elongation complexes, we demonstrate that substitutions of any of the three aspartates dramatically reduce all known RNAP catalytic activities, supporting the model's predictions that same amino acids participate in all RNAP catalytic reactions. We demonstrate that though substitutions in the DFDGD motif decrease Mg2+ binding to free RNAP below detection limits, the apparent affinity to Mg2+ in transcription complexes formed by the mutant and wild-type RNAPs is similar, suggesting that NTP substrates and/or nucleic acids actively contribute to the retention of active center Mg2+.
Sosunov
Vasily
V
Public Health Research Institute 225 Warren Street, Newark, NJ 07103, USA.
Zorov
Savva
S
Sosunova
Ekaterina
E
Nikolaev
Anatoly
A
Zakeyeva
Irina
I
Bass
Irina
I
Goldfarb
Alex
A
Nikiforov
Vadim
V
Severinov
Konstantin
K
Mustaev
Arkady
A
D019943:Amino Acid Substitution; D001224:Aspartic Acid / Q000737:chemistry* / Q000235:genetics; D001483:Base Sequence; D002384:Catalysis; D020134:Catalytic Domain; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008274:Magnesium / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D014158:Transcription, Genetic
D021122:Protein Subunits; D001224:Aspartic Acid; C108570:beta' subunit of RNA polymerase; D012321:DNA-Directed RNA Polymerases; D008274:Magnesium