Site-directed photo-cross-linking of rRNA transcription initiation complexes

Abstract

Site-specific photo-cross-linking of the rRNA committed transcription complex was carried out by using 5-[N-(p-azidobenzoyl)-3-aminoallyl]-dUMP-derivatized promoter DNA. Putative TAFIs of 145, 99, 96, and 91 kDa, as well as TATA-binding protein (TBP), were found to specifically photo-cross-link to different positions along the promoter. These had been 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 other 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 contour length of the DNA in the complex. This showed that a single molecule of TIF-IB is in each committed complex and that the DNA is not looped around the protein, as would be expected if UBF were in the complex. 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-linking are consistent with earlier data showing that the TBP TATA box-binding domain is not utilized in the assembly of the rRNA committed complex (C. A. Radebaugh, J. L. Mathews, G. K. Geiss, F. Liu, J. Wong, E. Bateman, S. Camier, A. Sentenac, and M. R. Paule, Mol. Cell. Biol. 14:597-605, 1994).