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Structure‐based kinetics of bacteriophage N4 RNA polymerase transcription initiation by trap‐freeze X‐ray crystallography
Author(s) -
Murakami Katsu,
Basu Ritwika,
Gleghorn Michael L
Publication year - 2011
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.25.1_supplement.504.1
Subject(s) - kinetics , bacteriophage , transcription (linguistics) , rna polymerase , chemistry , microbiology and biotechnology , polymerase , biophysics , rna polymerase i , rna , biology , dna , physics , biochemistry , gene , escherichia coli , linguistics , philosophy , quantum mechanics
RNA polymerase carries out two types of RNA synthesis including initiation and elongation. While the mechanism of transcription elongation has been characterized for both single‐ and multi‐subunit RNA polymerases, the mechanism of transcription initiation where two nucleotide substrates are loaded at the active site followed by nucleotidyl transfer has not been elucidated. We have determined the X‐ray crystal structures of the pre‐ and post‐catalytic forms of transcription initiation complex of bacteriophage N4 RNA polymerase that provide the first and complete set of atomic images depicting the process of transcription initiation by any RNA polymerase. Substrate loading drives a conformational change of O‐helix, but only the correct base pairing between +2 substrate and DNA base is able to complete the O‐helix conformational transition. Substrate binding also facilitates catalytic metal binding leading to alignment of the reactive groups of substrates and the catalytically essential carboxylates of Asp residues for the nucleotidyl transfer reaction. The temporal order of RNA polymerase conformational changes, monitored by time‐dependent “trap‐freeze” X‐ray crystallography, show that conformational change of Fingers and metal binding at the active site comprise very early and late stages of transcription initiation, respectively.