Structure And Function Of Transcription Elongation Factor TFIIS And PAF1 Complex From Trypanosoma Brucei

The normal gene transcription is very important for the cellular activities, which has been an important area of research for several decades. Although the processes of transcription in some higher eukaryptes have been well characterized, transcription of some unicellular eukaruotes such as Trypanosoma brucei (T. brucei) is still not well understood. T. brucei represents an early branch in the eukaryotic evolutionary tree and displays unusual features compared with other well-studied eukaryotic systems. For example, the mRNA of T. brucei is mostly transcribed polycistronically and most transcription factors have not been identified. In this dissertation, we mainly focus on structure and function of transcription elongation factor TFIIS and PAF1 complex from T. brucei.TFIIS is one of the conserved and best-characterized transcription elongation factors in eukaryotes which can relieve the arrest of RNAP II and significantly improve the efficiency of transcription. Three TFIIS homologues (TbTFIISl, TbTFIIS2-1 and TbTFIIS2-2) have been identified previously in T. brucei, however, the specific function of TFIIS involved in transcription regulation remains still unknown. In this work, when TbTFIIS2-1 or TbTFIIS2-2 was deleted using RNA interference (RNAi) technique in procylic-form T. brucei, the cell growth was not affected. However, the cell growth was affected significantly when TbTFIIS2-1 and TbTFIIS2-2 were deleted together, implying that TbTFIIS2-1 and TbTFIIS2-2 might play a redundant role in T. brucei. Interestingly, compared with other TFIIS factors by sequence alignments, TbTFIIS2-1 and TbTFIIS2-2 both contain an N-terminal PWWP domain (TFIIS2-1 PWWP and TFIIS2-2 PWWP), which was the first time for PWWP domains identified in transcription factors. We determined the structures of the two PWWP domains by NMR and they adopt the conserved PWWP fold containing a five-strand P-barrel and a α-helix in the C-terminus, which is similar to other PWWP domains. The PWWP domain has been reported not only to be a DNA binding domain, but also to bind methyllysine histones. AT-and GC-rich ssDNA and dsDNA sequences were used to titrate 15N-labeled TFIIS2-1 PWWP and TFIIS2-2 PWWP domains and we found that neither of the two PWWP domains has the DNA binding affinity. We also used NMR titration to investigate the abilities of TFIIS2-1 PWWP and TFIIS2-2 PWWP domains to bind histone peptides and found that TFIIS2-2 PWWP domain specifically recognized trimethylated H4K17 and H3K32 by the conserved aromatic cage in a similar binding pattern to other PWWP domains. However, TFIIS2-1 PWWP domain has no ability of binding to methylated histone. Sequence analysis of TbTFIIS2-1 PWWP and TbTFIIS2-2 PWWP revealed that the three aromatic residues formed the conserved cage might be essential for the binding ability of methylated-histone of PWWP domain. Overall, TFIIS2-2 PWWP domain is the first PWWP domain identified in T. brucei which can bind to methylated histones, which will provide a theoretical basis for the study of transcription of T. brucei.Moreover, we collected the interacting proteins of TbTFIIS2-1 by the tandem affinity purification of tagged TbTFIIS2-l and found the interactions between TFIIS and PAF1 complex. PAF1 complex, conserved in many eukaryotes, comprises CTR9, LEO1, PAF1, CDC73, RTF1 subunits and is important for many cellular activities, including gene expression and silencing, DNA repair, cell cycle regulation and so on. Three subunits of PAF1 complex in T. brucei have been identified previously including CTR9, CDC73 and LEO1, but PAF1 and RTF1 have not been identified because of low sequence similarity. In our work, the results of bioinformatics analysis and tandem affinity purification implied that two proteins of unknown function (Tb927.3.5070 and Tb927.7.4030) which have no recognized functional domains might be the PAF1 and RTF1 subunits of T. brucei and the interaction network of PAF1 complex was confirmed with the yeast two hybrids at the same time. The PAF1 complex is essential for the normal growth of procyclic trypanosomes revealed by RNA interference. In addition, we used GST pull down to confirm that domain I (LW domain) in TbTFIIS2-1 and the C-terminus of TbTFIIS2-2 with the representative residue of LW domain directly bind to LEO1. High-throughput sequencing results showed that PAF1 complex not only is involved in gene transcription elongation but also participates in regulating the VSG ES silencing, which were also verified with the results of the real-time PCR. Moreover, we confirmed that PAF1 complex interacts with ISWI directly by the yeast two hybrids. Therefore, we speculated that PAF1 complex might be involved in VSG ES silencing via ISWI, but the specific mechanism remains to be further studied.