Abstract
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.
Highlights
Pre-steady state kinetic analysis allows the progress of an enzymatic reaction to be tracked in real time [1, 2], and coupling enzyme functional dynamics to the structure provides the clearest insight into the mechanism
The other NTP appears to be the substrate for addition of the subsequent bond. These observations lead to the following conclusions: 1) RNA polymerase II (RNAP II) elongates according to a substrate NTP-induced fit mechanism; 2) translocation can be induced by prior NTP binding
Translocation Can Be Driven by the Incoming Substrate NTP—We have tracked the progress of kinetically homogeneous populations of human RNAP II molecules through the sequence 40CAAAGG45, concentrating on synthesis of the G44 and G45 bonds
Summary
Pre-steady state kinetic analysis allows the progress of an enzymatic reaction to be tracked in real time [1, 2], and coupling enzyme functional dynamics to the structure provides the clearest insight into the mechanism. We compare the first transient state kinetic studies of human (Homo sapiens) RNAP II1 to the x-ray structure of the yeast (Saccharomyces cerevisiae) RNAP II elongation complex (EC) [3] These studies give new insight into the RNAP II mechanism and demonstrate the feasibility of a detailed kinetic study of a highly regulated enzyme that is at the hub of gene control in human cells. Translocation must occur at either the beginning or the end of each bond addition cycle, and in the presence of TFIIF, both are highly dependent on the templated NTP. HDAg eliminates the high NTP dependence of the slow step after phosphodiester bond formation, demonstrating the unusual nature of the RNAP II mechanism in the presence of TFIIF. As with TFIIF, the HDAgstimulated mechanism shows evidence of substrate NTPinduced fit during escape from a stall, but, unlike TFIIF, NTP dependence is not detected with HDAg in the normal processive transition between bonds
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