Abstract
Yeast RNA polymerase II general initiation factor g was purified to near homogeneity on the basis of its function in a reconstituted transcription system. Polypeptides of 30, 54, and 105 kDa co-purified with transcriptional activity, forming a complex with a mass of 300 kDa as judged by gel filtration, but only 100 kDa based on sedimentation in glycerol gradients, suggesting an elongated shape. Transcription activity could be reconstituted after separation of the three polypeptides under denaturing conditions; the 54- and 105-kDa subunits were both essential, while the 30-kDa subunit was slightly stimulatory. Factor g was required for initiation at all promoters tested, including those from Saccharomyces cerevisiae, Schizosaccharomyces pombe, and adenovirus. Factor g can stably associate with RNA polymerase II, as shown by cosedimentation in a glycerol gradient.
Highlights
Transcription assays, lysis buffer, and buffers A, B, C, D, E, and F were as described [27]
Subsequent manipulations at a promoter in vitro [1,2,3]. Efforts to isolate such factors from yeast nuclear extract yielded five fractions necessary to reconstitutepromoter-dependenttranscription, termed a-e [4].The b and d fractions restored transcription activity to a heated nuclear extract, andfactor b was purified were performed at 0-4 “C. Cells were suspended in 1 liter of 3 X lysis buffer, broken with glass beads as described [5], and centrifuged in a Beckman JA-10 rotor for 20 min at 8,000 rpm
+-DEAE-5PW Monos of factor g activity eluteadt 0.42 M potassium acetate.Active fractions were pooled, dialyzed against buffer G-0.025 tothe conductivity of buffer G-0.1, centrifuged ina microcentrifuge a t 13,000rpm for 15 min, and applied a t 0.3 ml/min to a TSK-heparin5-PW HPLC column (75 X 7.5 mm; Supelco) equilibrated in buffer G-0.1
Summary
Transcription assays, lysis buffer, and buffers A, B, C, D, E, and F were as described [27]. +-DEAE-5PW Monos of factor g activity eluteadt 0.42 M potassium acetate.Active fractions were pooled (fraction V), dialyzed against buffer G-0.025 tothe conductivity of buffer G-0.1, centrifuged ina microcentrifuge a t 13,000rpm for 15 min, and applied a t 0.3 ml/min to a TSK-heparin5-PW HPLC column (75 X 7.5 mm; Supelco) equilibrated in buffer G-0.1.
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