Serebrov Victor, Beran Rudolf K F, Pyle Anna Marie
Department of Molecular Biophysics and Biochemistry and Howard Hughes Medical Institute, Yale University, New Haven, Connecticut 06520, USA.
J Biol Chem. 2009 Jan 23;284(4):2512-21. doi: 10.1074/jbc.M805460200. Epub 2008 Nov 14.
The NS3 helicase from hepatitis C virus is a prototypical DEx(H/D) RNA helicase. NS3 has been shown to unwind RNA in a discontinuous manner, pausing after long apparent steps of unwinding. We systematically examined the effects of duplex stability and ionic conditions on the periodicity of the NS3 unwinding cycle. The kinetic step size for NS3 unwinding was examined on diverse substrate sequences. The kinetic step size (16 bp/step) was found to be independent of RNA duplex stability and composition, but it exhibited strong dependence on monovalent salt concentration, decreasing to approximately 11 bp/step at low [NaCl]. We addressed this behavior by analyzing the oligomeric state of NS3 at various salt concentrations. Whereas only NS3 oligomers are capable of processive unwinding, we found that monomeric NS3 is an active helicase that unwinds with low processivity. We demonstrate that low salt conditions enhance unwinding by monomeric NS3, which is likely to account for the reduction in apparent step size under low salt conditions. Based on results reported here, as well as available structural and single molecule data, we present an unwinding mechanism that addresses the apparent periodicity of NS3 unwinding, the magnitude of the step size, and that integrates the various stepwise motions observed for NS3. We propose that the large kinetic step size of NS3 unwinding reflects a delayed, periodic release of the separated RNA product strand from a secondary binding site that is located in the NTPase domain (Domain II) of NS3. These findings suggest that the mechanism of product release represents an important and unexplored feature of helicase mechanism.
丙型肝炎病毒的NS3解旋酶是一种典型的DEx(H/D) RNA解旋酶。NS3已被证明以不连续的方式解开RNA，在长时间的明显解旋步骤后会暂停。我们系统地研究了双链稳定性和离子条件对NS3解旋循环周期性的影响。在不同的底物序列上研究了NS3解旋的动力学步长。发现动力学步长（每步16个碱基对）与RNA双链稳定性和组成无关，但强烈依赖于单价盐浓度，在低[NaCl]浓度下减小到约每步11个碱基对。我们通过分析不同盐浓度下NS3的寡聚状态来解释这种行为。虽然只有NS3寡聚体能够进行连续解旋，但我们发现单体NS3是一种活性解旋酶，其解旋的持续性较低。我们证明低盐条件增强了单体NS3的解旋，这可能是低盐条件下明显步长减小的原因。基于本文报道的结果以及现有的结构和单分子数据，我们提出了一种解旋机制，该机制解释了NS3解旋的明显周期性、步长大小，并整合了观察到的NS3的各种逐步运动。我们提出，NS3解旋的大动力学步长反映了从位于NS3的NTPase结构域（结构域II）的二级结合位点延迟、周期性释放分离的RNA产物链。这些发现表明产物释放机制是解旋酶机制中一个重要且未被探索的特征。
