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We have previously determined the crystal structure of a non-structural 5B (NS5B) protein, an RNA-dependent RNA polymerase (RdRp) of hepatitis C virus (HCV). NS5B protein with the hydrophobic C-terminal 21 amino acid residues truncated, designated NS5B 570 , shows a typical nucleotide polymerase structure resembling a right-hand shape. In the crystal structure, a C-terminal region between Leu545 and His562 occupies a putative RNA-binding cleft of this polymerase and seems to inhibit the polymerase activity. Varieties of recombinant NS5B proteins (NS5B 552 , NS5B 544 , NS5B 536 or NS5B 531 , with C-terminal 39, 47, 55 or 60 amino acid residues truncated, respectively) were systematically constructed to elucidate effects of the region on the polymerase activity. NS5B 544 , NS5B 536 and NS5B 531 showed markedly higher RdRp activities compared to the activities of NS5B 570 or NS5B 552 . Furthermore, when the hydrophobic amino acid residues Leu547, Trp550 and Phe551 (LWF) in NS5B 570 and NS5B 552 were changed to alanine, their activities were higher than that of the original NS5B 570 . The crystal structures of the various recombinant NS5B proteins were also determined. Structural comparison of the NS5B proteins indicates that the activation was caused by elimination of a unique hydrophobic interaction between the three C-terminal residues and a shallowly concave pocket consisting of thumb and palm domains.