Preliminary Study On The Structures Of HMD And Plus3Domains Of Rtflunit Of Saccharomyces Cerevisiae Transcription ElongationfactorPaf1Complex And A PDZ Domain From Trypanosoma Brucei

Proteins are important components of the organisms, whose functions are mostly achieved via the interaction of the proteins with their ligands. Some domains play key roles in the interactions. It is critical to study the structure of these domains to elucidate the functioning mechanisms of them and further the full-length proteins. In this work, we preliminarily investigated the structures of a few important domains involved in protein interactions, including Rtfl-Plus3and Rtf1-HMD from the Saccharomyces cerevisiae and PDZ domain from the Trypanosoma brucei. Our results are supposed to help to resolve the high resolution structures of them and further reveal their functioning menchanism and their roles in the full-length proteins.(Ⅰ) Rtfl is a multifunctional component of the Pafl Complex that regulates gene expression by directing cotranscriptional. Rtfl influences transcription and chromatin structure through several independent functional domains. However, the mechanisms of Rtfl functions are undefined yet. In this work, we recombinantly cloned, expressed and purified Plus3and HMD domains, which are key for yeast Rtfl interaction with its ligands, for structural investigation. Currently, we have obtained an appropriate protocol for the recombinant production of the two domains, and determined basic conditions for their NMR studies. This hopfully will contribute to the further resolution of their three dimention structures and the eluctidation of their functioning mechanisms.(Ⅱ) PDZ domains are ubiquitious protein-protein interaction modules in various species. They always identify short motifs at the C-termini of target proteins. PDZ domains can take part in multiple processes such as intracellular transportation, ion channel signaling, and other cell singal transduction systems. Using bioinformatics method we found several PDZ domain-containing proteins in Trypanosoma brucei. In this work, we recombinantly cloned, expressed and purified the PDZ domains of these proteins, and one of them (named TbPDZ herein) was successfully produced. In order to understand the potential functions of TbPDZ domain and the full-length protein, we used NMR method toinvestigate its structure preliminary.. The backbone assignment of TbPDZ has been completed, and the chmeical shifts were used in CSI to assess secondary structure. Our results showed that TbPDZ probably contrains one a-helix and eight β-strands, some distinguished from the classical PDZ domains. As Trypanosoma brucei is lower eukaryote, besides contribute to the elucidation of TbPDZ functioning mechanism, the resolution of its structure would provide clues to discover the evolution mechanism of PDZ domain.