Structural Study Of LW Domains Of Eukaryotic Transcription Elongation Factor TFIISs

Regulation of transcription is extremely complex in eukaryotes.Multiple protein complexes are involved in transcription initiation,elongation and termination.Gene transcription regulation is very important for the cellular activities,which has been an important area of research for several decades.A number of studies revealed that transcription elongation factor TFIIS and Pafl complex were play an important role in transcription elongation.TFIIS is one of the conserved and best-characterized transcription elongation factors in eukaryotes which can relieve the arrest of RNA Pol ? and improve the efficiency of transcription significantly.Human transcription elongation factor TFIIS consists of three domains:domain ?,domain ? and domain ?.Domain ? is essential for RNA Pol ? to read through transcription pause sites and cleave the RNA transcripts.Domain ? is a nucleic acid-binding domain.So far,the specific biological function of domain ? has not been reported.Domain ? contains the conserved leucine(L)and tryptophan(W)residues,therefore its also called the LW domain.Three TbTFIIS homologues have been identified,named TbTFIIS1,TbTFIIS2-1 and TbTFIIS2-2 in Trypanosoma brucei.Previous study in the laboratory indicated that TbTFIIS2-1 and TbTFIIS2-2 can interact with TbPaf1 complex,and this interaction is invovled in the gene transcription regulation.Further experiments indicated that TbTFIIS2-1 and TbTFIIS2-2 both bind to Leol subunit of TbPaf1 complex through the LW domain.In order to further understand the molecular mechanism of the interactions between TbTFIIS LW domain and TbLeo1 subunit,we constructed multiple TbTFIIS2-2 LW-Leo1 fusion expression vectors by overlap extension PCR,and obtained multiple TbTFIIS2-2 LW-Leo1 complexes through expression and purification.Finally,we obtained a stable TbTFIIS2-2 LW-Leo1 complex.TbTFIIS2-2 LW-Leol complex was used in subsequent crystallographic studies.Subsequently,Gao Jie obtained the crystal structure of the complex through crystallographic experiments.The analysis of the crystal structure showed that TbTFIIS2-2 LW domain is composed of six ?-helices,and the four helices ?3,?4,?5 and ?6 form a hydrophobic pocket to bind to TbLeo1.The crystal structure of TbTFIIS2-2 LW-Leo1 complex provides a structural basis for studying the molecular mechanism of interactions between TbTFIIS2-2 LW domain and TbLeol subunit.In human,previous studies reported that transcription elongation factor TFIIS and Pafl complex coordinate the process of transcription elongation and HsTFIIS interacts with Leo1 subunit of HsPaf1 complex.We found that HsTFIIS interacts with HsLeol subunit through its LW domain.In order to understand the molecular mechanism of the interactions between HsTFIIS and HsLeol,we constructed multiple expression plasmids containing HsTFIIS LW domain with different C-terminal boundaries,and obtained these proteins by expression and purification in vitro.Subsequently,HsTFIIS LW domain that is the most suitable for NMR structural analysis was screened out.Finally,the solution structure of HsTFIIS LW domain was determined by NMR spectrometry.The analysis of the solution structure of HsTFIIS LW domain showed that the residues 2-77 at the N-terminus form a five-helix bundle and the residues 78-96 at the C-terminus form a disordered loop region.The five-helix bundle is composed of ?1(residues 2-18),?2(residues 21-33),?3(residues 38-44),?4(residues 46-56)and ?5(residues 60-77).Among them,N-terminal ?1 and C-terminal ?5 of the five-helix bundle are relatively longer,?2 and ?4 are slightly shorter,?3 is the shortest,and the orientation of ?3 is different from the other four helices.In order to further understand the molecular mechanism of the interactions between HsTFIIS LW domain and HsLeol subunit,Gao Jie later proved the key hydrophobic pockets and key residues for the interactions between HsTFIIS LW domain and HsLeol subunit through a series of experiments including NMR chemical shift perturbation,ITC and in vitro mutagenesis experiments.The result indicated ?3,?4 and ?5 of HsTFIIS LW domain form a hydrophobic pocket as the binding site of HsLeol subunit.Comparing the structure of TFIIS LW domain and its interaction with Leol subunit in T brucei and human,we revealed that the molecular mechanism of interactions between TFIIS LW domain and Leol subunit in eukaryotes is conserved.Meanwhile,some differences in the interactions were also found between these two species.