Amino acid substitutions in the switch 2 region of the Rpb1 subunit of S. cerevisiae RNAPII confer downstream shifts in transcription start site selection.
Robert C. Majovski, Alfred S. Ponticelli
Biochemistry, University at Buffalo, 3435 Main St., Buffalo, NY, 14214, United States
We have utilized alanine-scanning site-directed mutagenesis and plasmid-shuffling to systematically probe the function of individual residues in the vicinity of the S. cerevisiae RNA polymerase II active center. We will report on the generation, identification and characterization of Rpb1 mutants exhibiting altered growth properties, and the demonstration that several amino acid substitutions in the 'switch 2' region of Rpb1 confer downstream shifts in transcription start site selection in vivo. It has previously been proposed that the switch 2 region undergoes a structural change upon the transition to a transcribing complex, and that this change is associated with the movement of this region into the active center to potentially contact the RNA-DNA hybrid. We will present the results from additional genetic and biochemical analyses that test the hypothesis that the downstream shifts in transcription start site selection that are conferred by mutations in the Rpb1 switch 2 region, or by previously reported mutations in the highly-conserved TFIIB 'finger', are due to impaired stabilization of an RNA-DNA hybrid in the polymerase active center.
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