Study On The Proteins Interacting With PELF1,yELF1 And The Binding Structure Between PELF1 And Chromatin

y ELF1(elongation factor 1) is a DNA binding protein, which involved in meiosis chromosome and transcription elongation, evolutionary conserved in Eukaryote. However the role of y ELF1 in gene transcription elongation and packaged of chromosomes is unclear.PELF1 is an y ELF1-like protein. It was isolated from the c DNA library Physarum Polycephalum by yeast two-hybrid using P14-3-3 as bait.PELF1 c DNA encoding 100 amino acids, its molecule weight is 11 KDa and p I is 5.16.We observed that PELF1 mainly gathered in the nucleus by confocal laser scanning microscope.In order to study the Molecular function of PELF1 and y ELF1, we identified the proteins that interacting with PELF1 and y ELF1 by GST(Glutathione S-transferase)pull-down technique and identified interacting proteins using LC-MS. The results showed that most of them are ribosomal proteins, and the rest proteins are involved in gene transcription, protein translation, glycometabolism, signal transduction, stress response, phosphorylation / dephosphorylation and sustain cell structure.To confirm the role of PELF1 that involved in cell-cycle,we therefore produced two Py ELF1 monoclonal antibody by using the chip screening technology,respectively named m Ab P35 and m Ab P59. First, we performed westernblot assay with m Ab P35 to assess PELF1 expression level during the cell-cycle progress(S phase and G2 phase 1 h, 3 h, 5 h, M phase) in P. polycephalum.The reslusts demonstrated that PELF1 was up-regulated at the earlier G2 phase,then subsequently down regulated until the M phase arrived. Similar to the results as indicated before,the PELF1 m RNA expression was down-regulated until the later G2 phase, then upregulated throughout the M phase.In addition,by combining atomic force microscope with Immunogold labelling techique, we observed chromosomes structural transformation and interaction with theGST-y ELF1 during S phase, G2 phase and M phase in the P. polycephalum. The results demonstrated that over expression GST-y ELF1 leads to the tandem linear arrangement upon the chromatin under the natural conditions.And these were also in consistent with the structure of Py ELF1 binds to DNA observed in previous vitro study.During the cell-cycel progress(S phase and G2 phase 1 h, 3 h, 5 h, M phase),we found that Py ELF1 bind to chromatin in the S phase, early G2(G2 phase 1h) and mid G2(G2 phase 3h) while this phenomenon could be not observed during the later G2(G2 phase 5h) and M phase.In this study,we revealed the binding structure between PELF1 and chromatin during the cell cycle progress. And we aslo developed a technical route for better characterizated specific protein components in multiprotein complex by using our Immunogold labelling techique combined with AFM. Finally, we will get more insight into the function and mechanism that PELF1 interacted with DNA in vivo with these findings in our study.