Architecture and mechanism of the human RNA polymerase II general transcription machinery

Gene specific regulation of human mRNA transcription requires a series of molecular events and assembly of numerous protein subunits at the promoter of the gene. The studies detailed in this thesis utilized kinetic and biophysical methods to characterize critical events during human RNA polymerase II transcription.;Formation of a stable elongation complex from an initiating complex requires a linear series of steps, with the rate-limiting step being promoter escape. Using a minimal in vitro human RNA polymerase II transcription system, we elucidated specifically where the rate-limiting step occurs. We found that the rate-limiting step occurs after synthesis of an eight nucleotide RNA during a stage that includes translocation of the active site to the ninth register. The position of promoter escape was found to be strictly dependent upon RNA length, did not rely any factors other than RNA polymerase II, and was not changed by the addition of TFIIE and TFIIH.;The initial and critical step in preinitiation complex assembly is the recruitment of TBP to the TATA-box. We showed that TBP bends the consensus TATA DNA at approximately 103° while addition of TFIIA to the complex decreases the bend angle to approximately 80°. At a nonconsenus TATA-box, however, the bend angle decreased to 70° when bound only by TBP but increased back to 80° upon addition of TFIIA. TBP was found to be transiently bound to promoter DNA under physiological salt conditions on consensus and nonconsensus TATA-boxes, however addition of TFIIA increased kinetic stability and attenuated the affect of high salt concentration. These results lead to a model in which TFIIA induces a conformational change in the TBP/TATA complex that results in enhanced kinetic stability and an increased tolerance for high salt concentrations.