The Fundamental Ribosomal RNA Transcription Initiation Factor-IB (TIF-IB, SL1, Factor D) Binds to the rRNA Core Promoter Primarily by Minor Groove Contacts

Abstract

Acanthamoeba castellanii transcription initiation factor-IB (TIF-IB) is the TATA-binding protein-containing transcription factor that binds the rRNA promoter to form the committed complex. Minor groove-specific drugs inhibit TIF-IB binding, with higher concentrations needed to disrupt preformed complexes because of drug exclusion by bound TIF-IB. TIF-IB/DNA interactions were mapped by hydroxyl radical and uranyl nitrate footprinting. TIF-IB contacts four minor grooves in its binding site. TIF-IB and DNA wrap around each other in a right-handed superhelix of high pitch, so the upstream and downstream contacts are on opposite faces of the helix. Dimethyl sulfate protection assays revealed limited contact with a few guanines in the major groove. This detailed analysis suggests significant DNA conformation dependence of the interaction.Extensive in vitro and in vivo analyses have shown that efficient transcription of the rRNA gene by RNA polymerase I requires formation of a stable transcription factor-promoter DNA complex, the committed complex (for review, see Refs. 1 and 2). In most species, two regions of the rRNA promoter, the core promoter element (CPE) 1 and upstream promoter element (UPE), participate in expression of the rRNA gene and formation of the committed complex. The CPE is located between Ϫ50 and ϩ10 relative to the transcription start site (position ϩ1), is absolutely required in all organisms, and is sufficient in some for initiation. It is the primary binding site for the TBPcontaining transcription factor. The UPE is located farther upstream, from approximately Ϫ150 to Ϫ110, and acts primarily to stimulate transcription. It has a variable requirement between species; at one end of the spectrum, Acanthamoeba castellanii has no UPE discernible in vitro (3). We (4) and others (5) have shown recently that the CPE can be subdivided into an element that interacts intimately with TIF-IB and an element that functions like the initiator element (Inr) of some RNA polymerase II promoters, interacting with a specific TAF I .The UPE and CPE are important because they serve as the binding sites for two RNA polymerase I transcription factors, UBF and TIF-IB (also known as SL1, factor D, Rib1, or corebinding factor) respectively. These two proteins interact, by mechanisms that are still unclear but apparently involve DNA looping (6), to form a stable initiation complex capable of directing specific RNA polymerase I recruitment through multiple rounds of transcription. The need for UBF is variable between species; UBF is required in human and Xenopus (7-9), but is dispensable in rat (10), mouse (11), yeast (12-14), and A. castellanii (15). Therefore, TIF-IB is, by elimination, the fundamental transcription factor for rRNA genes, recruiting RNA polymerase I in the next step of the initiation process (16).TIF-IB has recently been purified to homogeneity from several eukaryotic species, including human (17), mouse (18), yeast (12,14), and A. castellanii.2 Biochemical analysis has shown that TIF-IB consists of TBP an...

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

“…This step apparently requires a homolog of mouse TIF-IA that is associated with a small fraction of the pol I. 2 Footprinting and minor groove-binding drug inhibition studies (14) indicated that TIF-IB binds primarily in the minor groove. Thus, it seemed logical to test photocross-linking reagents that either targeted the minor groove or, better, were unbiased as to groove.…”

A Novel RNA Polymerase I Transcription Initiation Factor, TIF-IE, Commits rRNA Genes by Interaction with TIF-IB, Not by DNA Binding

“…The 5Ј-end-labeled 150-bp BamHI/SacI fragment of pGG4C and the 217-bp BamHI/PstI fragment of pEBH10 were used in EMSAs. Binding conditions were the same as those described by Geiss et al (18) except that bovine serum albumin was used at 0.5 mg/ml instead of 0.05 mg/ml. DNA was incubated with proteins for 20 min at 25°C.…”

“…Plasmid pGG4C and/or plasmid pEBH10 was used for preparation of the DNAs used in electrophoretic mobility shift assays (EMSAs), methidiumpropyl-EDTA ⅐ Fe(II) [MPE ⅐ Fe(II)] footprinting, and template binding order-of-addition assays. Plasmid pGG4C contains a 114-bp fragment of the A. castellanii core promoter, from Ϫ96 to ϩ18 relative to the transcription initiation site (tis) (ϩ1), subcloned into the NotI site of pBluescript II SK(Ϫ) (18). Plasmid pEBH10 contains the A. castellanii core promoter from Ϫ120 to ϩ80 cloned into the HincII site of pUC8 (5).…”

“…One microliter of a 70 M MPE and 50 M (NH 4 ) 2 Fe(SO 4 ) 2 solution and 1 l of 0.5 M dithiothreitol were added to the reaction mixtures, and incubation continued for another 15 min at 25°C. MPE ⅐ Fe(II) reactions were stopped, and reaction products were processed, as described by Geiss et al (18). DNA was analyzed on denaturing (7 M urea) 10% sequencing gels with 1ϫ TBE run buffer (53).…”

“…This fundamental factor is referred to as transcription initiation factor IB (TIF-IB) in the mouse and Acanthamoeba castellanii, SL1 in the human and the rat, Rib1 in Xenopus spp., factor D or TFID in the rat, and core factor (CF) in the yeast Saccharomyces cerevisiae. In the best-studied committed complex, A. castellanii TIF-IB binds to the minor groove (18) and to the ribosomal initiator element (50) of the core domain of the promoter. In all species, TIF-IB is a multisubunit protein complex composed of TATA-binding protein (TBP) and three or four additional subunits known as Pol I-specific TBP-associated factors (TAF I s), though the tenacity of TBP association with the TAF I s is variable between species (12,30).…”