PROXIMAL SEQUENCE ELEMENT-BINDING TRANSCRIPTION FACTOR (PTF) IS A MULTISUBUNIT COMPLEX REQUIRED FOR TRANSCRIPTION OF BOTH RNA POLYMERASE-II-DEPENDENT AND RNA POLYMERASE-III-DEPENDENT SMALL NUCLEAR-RNA GENES

The proximal sequence element (PSE), found in both RNA polymerase II ( Pol II)- and RNA Pol III-transcribed small nuclear RNA (snRNA) genes, is specifically bound by the PSE-binding transcription factor (PTF). W e have purified PTF to near homogeneity from HeLa cell extracts by usi ng a combination of conventional and affinity chromatographic methods. Purified PTF is composed of four polypeptides with apparent molecular masses of 180, 55, 45, and 44 kDa. A combination of preparative elect rophoretic mobility shift and sodium dodecyl sulfate-polyacrylamide ge l electrophoresis analyses has conclusively identified these four poly peptides as subunits of human PTF, while UV cross-linking experiments demonstrate that the largest subunit of PTF is in close contact with t he PSE. The purified PTF activates transcription from promoters of bot h Pot II- and Pol II-transcribed snRNA genes in a PSE-dependent manner . In addition, we have investigated Factor requirements in transcripti on of Pol III-dependent snRNA genes. We show that in extracts that hav e been depleted of TATA-binding protein (TBP) and associated factors, recombinant TBP restores transcription from U6 and 7SK promoters but n ot from the VAI promoter, whereas the highly purified TBP-TBP-associat ed factor complex TFIIIB restores transcription from the VAI but not t he U6 or 7SK promoter. Furthermore, by complementation of heat-treated extracts tacking TFIIIC activity, we show that TFIIIC1 is required fo r transcription of both the 7SK and VAI genes, whereas TFIIIC2 is requ ired only for transcription of the VAI gene. From these observations, we conclude (i) that PTF and TFIIIC2 function as gene-specific factors for PSE- and B-box-containing Pol III genes, respectively, (ii) that the form of TBP used by class III genes with upstream promoter element s differs from the form used by class III genes with internal promoter s, and (iii) that TFIIIC1 is required for both internal and external P ol III promoters.