Structural studies on the Initiator protein, IBP39.

University Of Tx Md Anderson Can Ctr, Houston TX

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Abstract

EXCEED THE SPACE PROVIDED. The promoters of protein-encoding genes in eukaryotes have a bipartite structure consisting of a core promoter and binding sites for gene specific regulators. Transcription initiation is mediated at the core promoter, which encompasses the transcription start site. Despite extensive studies on eukaryotic transcription, the mechanistic details of the initiation process are largely unknown. Even less well understood are the mechanisms of transcription regulation and initiation in single-celled parasitic protozoa. The promoters in these organisms appear to lack typical metazoan core promoter elements such as the TATA box or Initiator (Inr). However, recent studies have demonstrated that all core promoters in Trichomonas vaginalis, although TATA-less, contain Inr elements. The T. vaginalis Inr element is responsible for transcription start site selection in all genes and, unlike higher eukaryotes where numerous proteins have only been implicated in Inr function, the T. vaginalis/nr is bound by a single 39 kDa "initiator binding protein", IBP39. IBP39, which shows no sequence homology to any other protein, contains two domains, an N-terminal 14.5 kDa Inr binding domain (IBD) attached via a proteolyticaUy sensitive linker to a C-terminal 24.5 kDa domain (C-domain) of unknown function. To date, the structural basis for Inr recognition by an Inr-binding protein, what constitutes a functional Inr, and how the Inr functions to mediate transcription initiation remain unclear. Because T. vaginalis appears to have a simplified transcriptional mechanism, relying solely on the Inr, it provides an excellent model system for studying the structural basis of transcription initiation through structure/function studies on IBP39 and its interaction with the Inr. Our recent IBP39 C-domain structure determination and analysis and subsequent biochemical studies has demonstrated the exciting finding that this domain binds the C-terminal domain (CTD) of the T. vaginalis RNA polymerase (RNAP U) large subunit, suggesting it might function to recruit RNAP II to the Inr site. These studies have, therefore, provided key initial insight into IBP39 function. Thus, to provide a full mechanistic understanding of T. vaginalis Inr mediated transcription we propose the following aims: (1) determine the crystal structure of the IBD in the presence and absence of the Inr and delineate the nucleotide elements that constitute a functional T. vaginalis Inr via fluorescence polarization (FP) experiments. (2) determine the structure of the IBP39 C-domain-RNAP II CTD complex. (3) To biochemically characterize the C-domain-CTD interaction and to examine the effects of CTD phosphorylation on this interaction. The essential role that IBP39 plays in transcription of all protein-encoding genes in T. vaginalis suggests it is a possible target for the treatment of trichomoniasis, which afflicts over 170 million individuals each _,ear,