DOA4 IN RELEASING UBIQUITIN FROM UBIQUITIN PROTEIN CONJUGATES

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The SPT4 and SPT5 genes of Saccharomyces cerevisiae were identified in several mutant hunts for proteins involved in transcription regulation. Recent biochemical and genetic analysis of Spt4 and Spt5 has shown that these proteins play a role in regulating the processivity of RNA polymerase II, most likely by managing the structure of chromatin over transcribed genes. Biochemical experiments show that Spt4 and Spt5 form a tight complex. Using a derivative of Spt5 tagged with the Flag epitope, we have partially purified this Spt4/Spt5 complex. Consistent with the idea that the Spt4/Spt5 complex intermittently interacts with other proteins during the repetitive cycle of transcription elongation, several other proteins co-purify with the Spt4/Spt5 complex in substoichiometric quantities. Among these co-purifying proteins is Rpb1, the largest subunit of RNA polymerase II. We will affinity purify the Spt4/Spt5 complex, and identify other copufiying proteins by mass spectrometry. Identification of these proteins should provide further insight into the nature of the machinery that is required to insure processive elongation of transcripts by RNA polymerase II.