A: Discovery of TFIIF

TFIIF was not initially identified when nuclear proteins were fractionated by Pll ion-exchange chromatography simply into four (A, B, C, and D) fractions (Matsui et al., 1980). The discovery of TFIIF was made possible only after further purification of the C fraction and following the identification of TFIIE (Flores et al., 1988). Human TFIIF was found to be composed of previously identified RNA polymerase

II-associated protein 30 (RAP30) and 74 (RAP74) isolated from calf thymus extracts as well as from human and mouse cell lines (Sopta et al., 1985; Flores et al., 1988). Further characterization of TFIIF by size exclusion column chromatography suggests that TFIIF is a heterotetramer composed of 2 subunits each of RAP30 and RAP74 (Flores et al., 1990). The structure-of the RAP30-RAP74 heterodimer from respective protein-protein interaction domains was resolved by X-ray crystallography (Gaiser et al., 2000). The cDNAs for RAP30 and RAP74 have already been cloned from human, Drosophila, and yeast (Sopta et al., 1989; Aso et al., 1992; Finkelstein et al., 1992; Kephart et al., 1993; Henry et al., 1994; Frank et al, 1995; Gong et al., 1995).

B: Subunits of TFIIF

Human RAP30 (calculated 26 kDa, apparent mass by SDS-PAGE -30 kDa) shares two significant sequence homology regions with bacterial a factors: a central domain that interacts with pol II and a cryptic DNA-binding domain at the C-terminus (Sopta et al., 1989; McCraken and Greenblatt, 1991; Garrett et al., 1992; Tan et al., 1994). RAP30 also contains an N-terminal domain important for binding to RAP74 and TFIIB (Fang and Burton, 1996). Interestingly, human TFIIF prebound to E.coli RNA polymerase core enzyme (a2(3(3' ) can be displaced by bacterial c ' . factor

0 0

Post a comment