Photocross-linking of the RNA polymerase I preinitiation and immediate postinitiation complexes: implications for promoter recruitment.
The architecture of eukaryotic rRNA transcription complexes was analyzed, revealing facts significant to the RNA polymerase (pol) I initiation process. Functional initiation and elongation complexes were mapped by site-specific photocross-linking to template DNA. Polymerase I is recruited to the promoter via protein-protein interactions with DNA-bound transcription initiation factor-IB. The latter's TATA-binding protein (TBP) and TAFs photocross-link to the promoter from -78 to +10 relative to the tis (+1). Although TBP does not bind DNA using its TATA-binding saddle, it does photocross-link to a 22-bp sequence that does not resemble a TATA box. Only TAF(I)96 (the mammalian TAF(I) 68, yeast Rrn7p homolog) overlaps significantly with the DNA interaction cleft of pol I based on modeling to the pol II crystal structure. None of the pol I-specific subunits that are localized on the lips of the cleft (A49 and A34.5) or the pol I-specific stalk (A43 and A14) cross-link to DNA. Pol I does not extend significantly upstream of the promoter-proximal border of the factor complex (-11 to -14), and similarly in the promoter proximal elongation complex, the enzyme does not contact DNA upstream of its normal exit from the cleft.
Bric
Anka
A
Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, Colorado 80523-1870, USA.
Radebaugh
Catherine A
CA
Paule
Marvin R
MR
D000048:Acanthamoeba / Q000201:enzymology / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003432:Cross-Linking Reagents; D016054:DNA, Protozoan / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010777:Photochemistry; D011401:Promoter Regions, Genetic; D011487:Protein Conformation; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012318:RNA Polymerase I / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016053:RNA, Protozoan / Q000235:genetics / Q000378:metabolism; D012335:RNA, Ribosomal / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic
D003432:Cross-Linking Reagents; D016054:DNA, Protozoan; D015800:Protozoan Proteins; D016053:RNA, Protozoan; D012335:RNA, Ribosomal; D012318:RNA Polymerase I