Mechanisms for Fidelity of RNA synthesis by human RNA polymerase II Yuri A. Nedialkov, Xue Q. Gong, Chunfen Zhang and Zachary F. Burton, Department of Biochemistry and Molecular Biology, Michigan State University, E. Lansing, MI 48824-1319, 517-353-0859, firstname.lastname@example.org
Our laboratory pioneered millisecond phase, transient state kinetic analyses of elongation by human RNA polymerase II to understand mechanism and regulation. This effort has broadened, however, to encompass unexpected insights into the fidelity of templated biological polymerization reactions. Kinetic and structural analyses indicated that RNA polymerase II partitions into two dominant translocation states: pre-translocated and post-translocated. Our kinetic data, however, indicate that both translocation states bind NTP substrate and that binding of the NTP substrate to the pre-translocated elongation complex strongly stimulates forward translocation. The NTP-driven translocation model is a fidelity mechanism, because accurately paired NTPs drive translocation, and translocation is a prerequisite for NMP incorporation. Recently, T7 RNA polymerase has been shown to have a templated NTP pre-insertion site, indicating the generality of polymerases testing the accuracy of NTP loading prior to transfer of substrate into the active site for chemistry. DNA polymerases also have a template base pre-insertion site, distal to the active site, indicating that (d)NTP-driven substrate loading, first identified for human RNA polymerase II, may be a general mechanism. Studies with the mushroom toxin alpha-amanitin, a potent inhibitor of human RNA polymerase II, have given further insight into transcriptional fidelity. These studies demonstrate a conformationally tightened RNA polymerase II active site at the time of chemistry. Conformational tightening promotes fidelity, because an inaccurately loaded base pair blocks an isomerization step that is required before chemistry. (d)NTP-driven induced fit has been posited as the key fidelity mechanism of both DNA and RNA polymerases, and we provide functional evidence for active site tightening prior to chemistry in the human RNA polymerase II mechanism. To our surprise, studies with alpha-amanitin also allowed detection of dynamic phosphodiester bond reversal, in the presence of a translocation block: that is, rapid formation and then reversal of a specific phosphodiester bond, observed in the millisecond phase. Dynamic bond reversal is an unanticipated mechanism for correcting transcriptional errors during rapid RNA synthesis. We posit that human RNA polymerase II tests the accuracy of NTP loading, prior to translocation, during translocation, during active site tightening, during chemistry, and even after chemistry. Millisecond phase kinetic studies provide insights that could not be gained using traditional steady-state methods. Essentially, these experiments provide real time insight into formation and scission of specific phosphodiester bonds, to correlate functional dynamics with emerging structural studies and provide the most advanced insights into the fidelity and mechanism of RNA and DNA polymerases.