Purification and characterization of human transcription factor IIIA.

The 5s gene-specific transcription factor, TFIIIA, was purified approximately 35,000-fold from HeLa cell extracts using a combination of conventional and affinity chromatographic methods. A single polypeptide of apparent molecular mass 42-kDa cofractionates with both 55 DNAbinding and SS transcription activities, and was conclusively identified as human TFIIIA by its ability to direct specific 55 gene transcription in vitro following elution and renaturation from SDS-polyacrylamide gels. The DNase I protection pattern of the purified human factor on a human 55 gene is similar to the pattern previously observed with Xenopus TFIIU. A Xenopus SS gene, but not a yeast 5s gene, is able to effectively compete for binding of the human factor. In addition, we report a previously undetected immunological cross-reactivity between human and Xenopus TFIIIk hTFIIIA is recognized specifically both by polyclonal antisera raised against Xenopus laevis TFIIIA as well as by a monoclonal antibody generated against the amphibian protein. These observations indicate that human TFIIIA is structurally related to the Xenopus oocyte factor and that the previous inability to detect human TFIIIA by immunological methods is due primarily to the low abundance of this factor in HeLa cell extracts. TFIIIA' is a 5s gene-specific regulatory factor which is required in addition to the general class I11 initiation factors TFIIIB and TFIIIC to direct 55 gene ~a n s ~p t i o n by RNA polymerase I11 in uitro (reviewed in Geiduschek and Tocchini- Valentini (19881, Murphy et al. (19891, and Gabrielsen and Sentenac (1991)). The sequential association of the class I11 transcription factors to form a functional 55 transcription complex was originally established by in vitro studies using partially purified human (Lassar et al., 1983; Bieker et al., 1985) or amphibian Direct binding of TFIIIA to the A-box and C-box elements of the 5s internal control region results in formation of a metastable binary complex which is further stablilized by the subsequent association of TFIIIC. This ternary complex is resistant to challenge by a second template in transcription competition assays, and is required for recruitment of TFIIIB