Transcriptional Regulation

Many proteins have been implicated genetically or biochemically in the regulation of elongation and termination by RNA polymerase II (RNAPII) in the yeast Saccharomyces cerevisiae. To identify the subunit composition of these proteins as they exist in vivo, we have modified chromosomal genes, tagging the carboxyl termini of subunits of these complexes with protein A and a calmodulin-binding peptide. We then purified the complexes on immunoglobulin G and calmodulin columns and identified subunits by matrix-assisted laser desorption/ionization-time of flight (MALDI-ToF) mass spectrometry and tandem electrospray mass spectrometry. We found the elongator complex, previously shown to be associated with elongating RNAPII and thought to have three subunits encoded by the Elp1, -2, -3 genes, to have six subunits that are dissociated into two three-subunit complexes at high salt concentrations. Spt4 was associated with three phosphoisoforms of Spt5 in the complex known as DRB-sensitivity-inducing factors (DSIF) in humans, but Spt5 was also associated with Spt6 in a different complex. We found that Spt6 exists in three phosphoisoforms and in three different assemblies: as a free polypeptide; in association with Spt5; and in association with another, essential polypeptide of unknown function. We found Spt16 and Pob3, which were known to associate with histones and regulate both RNAPII and DNA polymerase a, to associate in humans only with each other, in the form known as FACT (facilitates chromatin transcription), when purified at high salt concentrations. When purified at more physiological salt concentrations, Spt16 and Pob3 were associated with histones, the elongation factor Rtf1, three polypeptides known to associate with RNAPII, and other polypeptides of unknown function. These and similar experiments with protein complexes that control termination by RNAPII, as well as cleavage and polyadenylation, indicate that the factors that control elongation and termination by RNAPII in vivo sometimes have multiple forms and are usually multisubunit complexes that easily become disassembled during multicolumn purification procedures.
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© 2002 Greenblatt Lab