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

Abstract

In the small, free-living amoeba Acanthamoeba castellanii, rRNA transcription requires, in addition to RNA polymerase I, a single DNA-binding factor, transcription initiation factor IB (TIF-IB). TIF-IB is a multimeric protein that contains TATA-binding protein (TBP) and four TBP-associated factors that are specific for polymerase I transcription. TIF-IB is required for accurate and promoter-specific initiation of rRNA transcription, recruiting and positioning the polymerase on the start site by protein-protein interaction. In A. castellanii, partially purified TIF-IB can form a persistent complex with the ribosomal DNA (rDNA) promoter while homogeneous TIF-IB cannot. An additional factor, TIF-IE, is required along with homogeneous TIF-IB for the formation of a stable complex on the rDNA core promoter. We show that TIF-IE by itself, however, does not bind to the rDNA promoter and thus differs in its mechanism from the upstream binding factor and upstream activating factor, which carry out similar complex-stabilizing functions in vertebrates and yeast, respectively. In addition to its presence in impure TIF-IB, TIF-IE is found in highly purified fractions of polymerase I, with which it associates. Renaturation of polypeptides excised from sodium dodecyl sulfate-polyacrylamide gels showed that a 141-kDa polypeptide possesses all the known activities of TIF-IE.rRNA transcription initiation in eukaryotic cells is a multistep process (20,40,45,47,52; reviewed in reference 46). Initially, an unusually stable complex of transcription factors, designated the committed complex, forms on the promoter. This complex is resistant to challenge by a second ribosomal DNA (rDNA) template and persists through multiple rounds of transcription. RNA polymerase I (Pol I), probably in association with other factors, is then recruited to the promoter by protein-protein interactions with the committed complex to form the preinitiation complex (PIC) (31,37,48,57). PIC formation is followed by melting of the double-stranded DNA, formation of the first few phosphodiester bonds, and promoter clearance (4,27,35). Following promoter clearance, the committed complex remains promoter bound and functional to recruit additional RNA polymerases for multiple rounds of transcription (45,47), though a new study suggests there may be differences between species in what components of the committed complex remain bound to the promoter (1).A single DNA-binding transcription initiation factor is essential and sufficient for the accurate initiation of transcription in vitro (14,33,36,38,62,67). 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 m...

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

“…3). The data from Acanthamoeba is probably the best in terms of the details of the early steps in initiation [109][110][111][112][113][114][115][116][117]. The system has shown that the exact site of RPI initiation is determined by the positioning of the TBP complex and is relatively sequence insensitive [110].…”

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

“…3-5). TIF-IE does not cross-link to the DNA, in accord with its not binding DNA (32). TAF photocross-linking was identical whether or not RNA polymerase I was present, so only the data for the quaternary complex are shown.…”

Acanthamoebaspp. as Agents of Disease in Humans

“…Acanthamoeba has been used as a model system to study eucaryotic RNA transcription, RNA polymerase functions, and cellular differentiation in trophozoites and cysts (29,63,79,190,203,338,396). Paule and coworkers (346) have purified RNA polymerases from Acanthamoeba and have performed extensive studies on initiation and regulation of RNA transcription in Acanthamoeba (8,194,347,348,355,364).…”