Photocross-linking of the RNA Polymerase I Preinitiation and Immediate Postinitiation Complexes

Abstract

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 photocrosslink 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.

Structural mechanism of ATP-independent transcription initiation by RNA polymerase I

“…In contrast, two consecutive kinks of ~35° and ~45° near position −16 that are approximately 5 bp apart are generated by multiple protein-DNA contacts (Figure 3A). This is unlikely due to the absence of TBP in our structure, because TBP does not contact rDNA promoter using its TATA-binding saddle (Bric et al, 2004) and there is no consensus TATA-box sequence within the Core Factor binding region. Furthermore, TBP is not required in vitro but rather stimulates Pol I transcription (Aprikian et al, 2000; Bedwell et al, 2012; Siddiqi et al, 2001).…”

A housekeeper with power of attorney: the rRNA genes in ribosome biogenesis

“…Acanthamoeba TBP was shown to contact the DNA at around -45 bp, and RPI in the pre-initiation complexes sits across the initiation site, protecting the DNA from the downstream edge of the TBP complex to around +20 (Fig. 3) [118,119].…”

Identification of EhTIF-IA: The putative E. histolytica orthologue of the human ribosomal RNA transcription initiation factor-IA

“…Corresponding information in protozoan parasites is rudimentary, and detailed pathways have not yet been worked out (Polakowski and Paule 2002;Bric et al 2004;Gogain and Paule 2005;Nguyen et al 2007Nguyen et al , 2012Park et al 2011). A previous study in our lab had focused on identification of RNA Pol I subunits and understanding of the nucleolar organization in E. histolytica (Jhingan et al 2009).…”

“…Although some detailed work on RNA Pol I transcription machinery has been done in Acanthamoe bacastellanni and Trypanosoma brucei (Radebaugh et al 1994;Al-Khouri and Paule 2002;Bric et al 2004;Gogain and Paule 2005;Brandenburg et al 2007;Park et al 2011;Nguyen et al 2012), TIF-IA homologs have not yet been identified in protozoan parasites. The A. castellanni TIF-IA has been biochemically purified from the cell extracts and shown to be essential for RNA Pol I transcription but its complete protein sequence has not been reported till date.…”