Structural Study Of The Interactions Between Eukaryotic TFIIS And Leo1 And Structural And Functional Study Of TbEsa1 Tudor Domain From Trypanosoma Brucei

This dissertation mainly focuses on the structural study of the interactions between eukaryotic transcription elongation factor TFIIS and Leo1 subunit of RNA polymerase-associated factor 1 complex(Paf1C),and the structural and functional study of Tudor domain of essential Sas-related acetyltransferase 1(Esal)from Trypanosoma brucei(T.brucei).1.Regulation of transcription is extremely complex in eukaryotes.Multiple protein complexes are involved in transcription initiation,elongation and termination.Paf1C complex is one of these protein complexes,which can regulate gene expression.In the process of regulation mediated by Pafl C complex,transcription elongation factor TFIIS was identified to cooperate with Paf1C complex,this interaction ensures transcription elongation and successive gene transcription.Although recent cryo-EM structural study of Paf1C-TFIIS-Pol ? complex from Saccharomyces cerevisiae reveals Paf1C-TFIIS interactions,structural basis and molecular mechanism of these interactions are still unclear due to the low resolution of the Pafl C complex structure.Here,we carried out the structural study on the basis of the interactions between TFIIS and Paf1C complex from three systems of eukaryotes:Trypanosoma brucei(a protozoan unicellular eukaryote causing sleeping sickness in human and nagana in cattle in sub-Saharan Africa),Saccharomyces cerevisiae and Homo sapiens.GST pull-down and ITC experiments indicated that eukaryotic TFIISs all interact with Leol subunit of Paf1C complex.We further revealed that TFIISs interact with core interaction fragment(including 'LFG' motif)of Leo1 through their LW domain,and the 'LFG' motif is conserved from trypanosoma,yeast to human.We further determined a crystal structure of TbTFIIS2-2 LW domain in complex with core interaction fragment of TbLeo1 from T.brucei.The TbTFIIS2-2 LW domain adopts a conformation with a compact six-helix bundle,and the a3a4a5a6 helices form a hydrophobic core,which interacts with the core fragment of TbLeo1.Meanwhile,we further solved the structures of LW domain of TFIIS from Saccharomyces cerevisiae(ScTFIIS)and Homo sapiens(HsTFIIS).The structural study indicated TFIISs LW domains are all comprised of conserved five or six ?-helices.Structural analysis,supported by chemical shift perturbation,ITC and site-directed mutagenesis experiment,revealed that LW domains of TFIIS are all responsible for recognition of a 'LFG' motif of Leo1 by a hydrophobic cleft,and further indicated that eukaryotic TFIIS LW domains all possess the key and conserved hydrophobic residues forming a conserved hydrophobic core to mediate Leol interaction.These results indicated that the interactions between Paf1C complex and TFIIS are conserved from lower to higher eukaryotes in evolution.Interestingly,the interactions between LW domain of TbTFIIS and TbLeo1 are much more stronger than the interactions between their homologues in yeast and human.Structural analysis showed that the C-terminus of TbTFIIS LW domain possesses a specific rigid a6 helix interacting with TbLeo1,and the terminus of ?6 helix and TbLeol form a specific hydrogen bond,thereby for better anchor on Leo1.The polycistronic transcription is present in T.brucei,which requires a strong affinity among TFIIS,Pafl C and RNA polymerase to stabilize a long chain of mRNA,this strong affinity may explain how transcription elongation is maintained in T.brucei.Overall,we revealed some common and unique molecular mechanism of the interactions between Pafl C complex and TFIIS in eukaryotes,which may contribute to our understanding of the origin and evolution of Paf1C complex and transcription factor TFIIS in transcription elongation regulation among eukaryotes.2.The essential SAS2-related acetyltransferase 1(Esal),as a acetyltransferase of MYST family,is indispensable for the cell cycle and transcriptional regulation.The Tudor domain consists of about 60 amino acids and belongs to the Royal family,which serves as a DNA or methylated histone binding module.Although Tudor domain has been widely studied in higher eukaryotes,its structure and function remain unclear in T.brucei.In T.brucei,only one presumed Tudor domain existing in Esal was found.Here,we determined a high-resolution structure of TbEsal Tudor domain from T.brucei by X-ray crystallography.TbEsal Tudor domain adopts a conserved Tudor-like fold,which is comprised of a five-stranded ?-barrel surrounded by two short ?-helices.Furthermore,we revealed a non-specific DNA binding pattern of TbEsal Tudor domain.However,TbEsal Tudor domain showed no methyl-histone binding ability,due to the absence of key aromatic residues forming a conserved aromatic cage.Overall,we revealed a non-specific binding mode of TbEsal Tudor domain to DNA and the structural basis for its lack of recognition of methylated histones.