| Transcription by RNA Polymerase II (RNApII) in eukaryotic organisms is an intricate, dynamic process that involves extensive coordination between multiple protein complexes. Synthesis of mRNA by RNApII requires holoenzyme assembly, initiation, promoter clearance, elongation, and termination. Initiation is a particularly complex process, requiring the orchestrated recruitment of many proteins. It is not well understood how many of these factors are recruited, how they are affected by other members of the transcription machinery, or how the promoter sequence drives factor selection.;This dissertation describes a strategy to isolate and identify components of the initiation machinery from nuclear extracts with immobilized DNA templates and quantitative mass spectrometry. This strategy was used to perform a global analysis of proteins that specifically associate with promoter sequence from the HIS4 gene, both in the presence and absence of the Gal4-VP16 transcriptional activator. The approach identified Suppressor of IIB (Sub1) and Replication Factor A (Rfa1) as proteins that associate specifically with transcription complexes. Further characterization of these single-stranded DNA (ssDNA) binding factors revealed that Sub1 localizes near the transcription bubble in vitro and binds to active promoters in vivo dependent upon the TATA-binding protein (TBP). Rfa1 localizes primarily to transcribed regions of active gene, independently of replication. Furthermore, Rfa1 levels increase at active promoters in strains lacking Sub1 or carrying a Sub1 ssDNA binding mutant, suggesting that these two proteins compete for a common ssDNA binding site within the transcription complex.;Taken together, this work demonstrates a novel role for ssDNA binding in the transcription holoenzyme, and identifies the transcription bubble as a scaffold for protein binding. These data also suggest a new replication-independent role for Rfa1. More broadly, this work establishes a proteomic strategy for using immobilized templates to identify proteins that associate with specific DNA sequences. |
