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Coming Events

8/8/2014 - Thesis Review - Kristin Dittenhafer-Reed
 
8/14/2014 - Beckman Rotor Seminar
 
9/12/2014 - IPiB Retreat
 

Record Lab - Characterizing the steps and large conformational changes in transcription initiation

To release the genetic information encoded in the DNA double helix, multi-subunit RNA polymerases unwind and open 12-14 bp of DNA, forming the open complex RPo. Despite the central biological importance of this process, the precise timing and extent of DNA opening during the steps of transcription initiation remain unknown. We are performing kinetic-mechanistic experiments to understand this fundamental biological process in detail. Recent kinetic studies reveal a startling role for nonconserved DNA sequences upstream of the promoter: they greatly accelerate the rate-limiting isomerization step in forming open complexes. Mapping the protection of the DNA backbone in the early initiation intermediate I1 onto the structure of RNA polymerase suggests a model (above) for how upstream DNA functions during initiation. We hypothesize that upstream DNA wraps around E. coli RNA polymerase, rearranging RNA polymerase domains (including the downstream jaw) that block the entry of downstream DNA into the active site channel (shown as steric clash in figure above). This conformational change allows DNA containing the start site of transcription to fully load into this channel. Although RNA polymerase interacts with over 80 base pairs of DNA in I1, the DNA remains largely closed and therefore must open in subsequent steps. We are currently using fast footprinting and rapid quench mixing methods to identify exactly when opening occurs and to what extent.

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