Abstract
Both 45- and 47-kDa subunits of TFIIK, a subcomplex of RNA polymerase II general transcription factor TFIIH, are encoded by the yeast cyclin gene CCL1. In all likelihood, these two subunits individually form cyclin-dependent kinase/cyclin dimers with Kin28 protein, a key enzyme in phosphorylation of the C-terminal domain of RNA polymerase II concomitant with transcription.
Highlights
Three approaches were taken to reveal CCL1 gene products in TFIIK. Both the 45- and the 47-kDa subunits of TFIIK were identified in this way
Affinity-purified antiCcl1 antibodies, raised against recombinant Ccl1 protein produced in bacteria, reacted with the 45- and 47-kDa polypeptides in immunoblots (Fig. 1)
Tryptic peptides derived from both 45- and 47-kDa subunits corresponded identically with the deduced amino acid sequence of the CCL1 gene (Table I). How might both 45- and 47-kDa subunits of TFIIK derive from the single CCL1 gene? Two possibilities, phosphorylation and proteolytic degradation, were rendered unlikely by further analysis
Summary
The HindIII-XhoI fragment was cloned into the corresponding sites of the bacterial expression plasmid pET-20b (Novagen) introducing a leader peptide and a six-histidine tag at the 5Ј and 3Ј ends of the open reading frame, respectively. Recombinant Ccl1 was only partially soluble and was purified from the insoluble fraction essentially as described previously for recombinant Kin28 protein (Feaver et al, 1994b).
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