NTP-driven translocation by human RNA polymerase II.
We report a "running start, two-bond" protocol to analyze elongation by human RNA polymerase II (RNAP II). In this procedure, the running start allowed us to measure rapid rates of elongation and provided detailed insight into the RNAP II mechanism. Formation of two bonds was tracked to ensure that at least one translocation event was analyzed. By using this method, RNAP II is stalled briefly at a defined template position before restoring the next NTP. Significantly, slow reaction steps are identified both before and after phosphodiester bond synthesis, and both of these steps can be highly dependent on the next templated NTP. The initial and final NTP-driven events, however, are not identical, because the slow step after chemistry, which includes translocation and pyrophosphate release, is regulated differently by elongation factors hepatitis delta antigen and transcription factor IIF. Because recovery from a stall and the processive transition from one bond to the next can be highly NTP-dependent, we conclude that translocation can be driven by the incoming substrate NTP, a model fully consistent with the RNAP II elongation complex structure.
Nedialkov
Yuri A
YA
Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824-1319, Japan.
Gong
Xue Q
XQ
Hovde
Stacy L
SL
Yamaguchi
Yuki
Y
Handa
Hiroshi
H
Geiger
James H
JH
Yan
Honggao
H
Burton
Zachary F
ZF
D000255:Adenosine Triphosphate / Q000378:metabolism; D001483:Base Sequence; D004305:Dose-Response Relationship, Drug; D006160:Guanosine Triphosphate / Q000378:metabolism; D006367:HeLa Cells; D035923:Hepatitis delta Antigens / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009711:Nucleotides / Q000737:chemistry; D011487:Protein Conformation; D021381:Protein Transport; D012319:RNA Polymerase II / Q000502:physiology*; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D013997:Time Factors
D035923:Hepatitis delta Antigens; D009711:Nucleotides; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D012319:RNA Polymerase II