A small post-translocation energy bias aids nucleotide selection in T7 RNA polymerase transcription.
The RNA polymerase (RNAP) of bacteriophage T7 is a single subunit enzyme that can transcribe DNA to RNA in the absence of additional protein factors. In this work, we present a model of T7 RNAP translocation during elongation. Based on structural information and experimental data from single-molecule force measurements, we show that a small component of facilitated translocation or power stroke coexists with the Brownian-ratchet-driven motions, and plays a crucial role in nucleotide selection at pre-insertion. The facilitated translocation is carried out by the conserved Tyr(639) that moves its side chain into the active site, pushing aside the 3'-end of the RNA, and forming a locally stabilized post-translocation intermediate. Pre-insertion of an incoming nucleotide into this stabilized intermediate state ensures that Tyr(639) closely participates in selecting correct nucleotides. A similar translocation mechanism has been suggested for multi-subunit RNAPs involving the bridge-helix bending. Nevertheless, the bent bridge-helix sterically prohibits nucleotide binding in the post-transolocation intermediate analog; moreover, the analog is not stabilized unless an inhibitory protein factor binds to the enzyme. Using our scheme, we also compared the efficiencies of different strategies for nucleotide selection, and examined effects of facilitated translocation on forward tracking.
Department of Molecular and Cell Biology, University of California, Berkeley, California, USA. jinyu@berkeley.edu
Jin
J
Yu
George
G
Oster
D004247:DNA / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D009068:Movement; D009690:Nucleic Acid Conformation; D009711:Nucleotides / Q000378:metabolism*; D011487:Protein Conformation; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D013816:Thermodynamics; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000737:chemistry / Q000378:metabolism*
D009711:Nucleotides; D021122:Protein Subunits; D014764:Viral Proteins; D004247:DNA; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases