title: Donation of catalytic residues to RNA polymerase active center by transcription factor Gre.
abstract: During transcription elongation, RNA polymerase (RNAP) occasionally loses its grip on the growing RNA end and backtracks on the DNA template. Prokaryotic Gre factors rescue the backtracked ternary elongating complex through stimulation of an intrinsic endonuclease activity, which removes the disengaged 3' RNA segment. By using RNA-protein crosslinking in defined ternary elongating complexes, site-directed mutagenesis, discriminative biochemical assays, and docking of the two protein structures, we show that Gre acts by providing two carboxylate residues for coordination of catalytic Mg2+ ion in the RNAP active center. A similar mechanism is suggested for the functionally analogous eukaryotic SII factor. The results expand the general two-metal model of RNAP catalytic mechanism whereby one of the Mg2+ ions is permanently retained, whereas the other is recruited ad hoc by an auxiliary factor.
authors.affiliation: Public Health Research Institute, 225 Warren Street, Newark, NJ 07103, USA.
authors.forename: Ekaterina
authors.initials: E
authors.lastname: Sosunova
authors.forename: Vasily
authors.initials: V
authors.lastname: Sosunov
authors.forename: Maxim
authors.initials: M
authors.lastname: Kozlov
authors.forename: Vadim
authors.initials: V
authors.lastname: Nikiforov
authors.forename: Alex
authors.initials: A
authors.lastname: Goldfarb
authors.forename: Arkady
authors.initials: A
authors.lastname: Mustaev
mesh_terms: D000595:Amino Acid Sequence; D020134:Catalytic Domain; D017124:Conserved Sequence; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry* / Q000235:genetics; D029968:Escherichia coli Proteins; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism*; D014158:Transcription, Genetic; D035602:Transcriptional Elongation Factors
chemical_list: D029968:Escherichia coli Proteins; C079667:GreB protein, E coli; D011994:Recombinant Proteins; D014157:Transcription Factors; D035602:Transcriptional Elongation Factors; D012321:DNA-Directed RNA Polymerases