SITE-DIRECTED PHOTO-CROSS-LINKING OF RIBOSOMAL-RNA TRANSCRIPTION INITIATION-COMPLEXES

Site-specific photo-cross-linking of the rRNA committed transcription complex was carried out by using [N-(p-azidobenzoyl)-3-aminoallyl]-dUM P-derivatized promoter DNA. Putative TAF(1)s of 145, 99, 96, and 91 kD a, as well as TATA-binding protein (TBP), were found to specifically p hoto-cross-link to different positions along the promoter. These had b een identified as potential subunits of the fundamental transcription initiation factor TIF-IB (also known as SL1, factor D, and TFID) from Acanthamoeba castellanii by purification to apparent homogeneity, No o ther polypeptides attributable to the rRNA architectural transcription factor UBF were identified, suggesting that this protein is not part of the committed complex, Scanning transmission electron microscopy of the complexes was used to estimate the mass of the complex and the co ntour length of the DNA in the complex. This showed that a single mole cule of TIF-IB is in each committed complex and that the DNA is not lo oped around the protein, as would be expected if UBF were in the compl ex, A circular permutation analysis of DNA bending resulting from TIF- IB binding revealed a 45 +/- 3.1 degrees (n = 14) bend centered 23 bp upstream of the transcription initiation site, This degree of bending and the position of the bend relative to the site of TBP photo-cross-l inking are consistent with earlier data showing that the TBP TATA box- binding domain is not utilized in the assembly of the rRNA committed c omplex (C. A. Radebaugh, J. L. Matthews, G. K. Geiss, F. Liu, J. Wong, E. Bateman, S. Camier, A. Sentenac, and M. R. Paule, Mol. Cell. Biol. 14:597-605, 1994).