High resolution footprinting of the hepatitis C virus polymerase NS5B in complex with RNA.
The nucleic acid binding channel of the hepatitis C virus RNA polymerase remains to be defined. Here we employed complementary footprinting techniques and show that the enzyme binds to a newly synthesized duplex of approximately seven to eight base pairs. Comparative analysis of surface topologies of free enzyme versus the nucleoprotein complex revealed certain lysines and arginines that are protected from chemical modification upon RNA binding. The protection pattern helps to define the trajectory of the nucleic acid substrate. Lys(81), Lys(98), Lys(100), Lys(106), Arg(158), Arg(386), and Arg(394) probably interact with the bound RNA. The selective protection of amino acids of the arginine-rich region in helix T points to RNA-induced conformational rearrangements. Together, these findings suggest that RNA-protein interaction through the entire substrate binding channel can modulate intradomain contacts at the C terminus.
Deval
Jérôme
J
Department of Microbiology, McGill University, Montreal, Quebec H3A 2B4, Canada.
D'Abramo
Claudia M
CM
Zhao
Zhuojun
Z
McCormick
Suzanne
S
Coutsinos
Dimitrios
D
Hess
Sonja
S
Kvaratskhelia
Mamuka
M
Götte
Matthias
M
D000595:Amino Acid Sequence; D001120:Arginine / Q000737:chemistry; D001483:Base Sequence; D004926:Escherichia coli / Q000201:enzymology; D016174:Hepacivirus / Q000737:chemistry* / Q000235:genetics; D006868:Hydrolysis; D008239:Lysine / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D012367:RNA, Viral / Q000737:chemistry*; D016914:Ribonuclease H / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D017361:Viral Nonstructural Proteins / Q000737:chemistry*; D012324:RNA-Dependent RNA Polymerase
D001120:Arginine; D008239:Lysine; D012367:RNA, Viral; D016914:Ribonuclease H; D017361:Viral Nonstructural Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase