Molecular Mechanism Of SRP Mediating The Translocation Of Proteins In Streptomyces Coelicolor

In this thesis the biochemical characteristics of Ffh and FtsY proteins from S.coelicolor were investigated to reveal the mechanism of protein translocation in this bacterium. The localizting manner of SRP receptor FtsY was illustrated by the method of fluorescent protein labelling. In addition the regulating function of FtsY on the cell differentation and secondary metabolism was discovered through the gene-deletion and the molecular hybriding techniques. In all, the molecular mechanism of protein translocation mediated by SRP pathway in S.coelicolor was researched, which would contibute to deeply understand the conservation and diversity of SRP pathway.In first chapter, ffh and ftsY genes and their encoding proteins were analyzed by bioinformatic method. Both Ffh and FtsY were single unit protein, and their molecular weight was 59.1kDa and 43.6kDa, respectively. SMART analysis indicated that both proteins contained a greatly conserved NG domain which was responsible for binding and hydrolyzing GTP. Ffh has an M domain for binding RNA, while FtsY has a significant transmembrane N-terminal domain with high hydrophobic. Multi-sequences alignment showed that Ffh and FtsY were conserved among lives from E.coli to mammalian cell. During evolutionary history, S.coelicolor emerged later than B.subtilis and E.coli predicted by phylogenesis.In the next chapter, the relationship between structure and enzymic activity of Ffh and FtsY were analyzed. The whole protein FtsY, NG domain and G domain were expressed and purified to assay their GTPase activities. The same procedure was performed for Ffh protein and its domains. The results revealed that both S.coelicolor Ffh and FtsY had GTPase activities. Moreover, their NG domain had similar GTPase activity to whole protein. The parameter Km of FtsY and NG were 2.056μmol·l^-1 and 1.143μmol·l^-1 orderly, while Km of its G domains was 9.807μmol·l^-1, which indicated that the whole protein and NG domain had higher affinity for GTP than that of G domain. As for Ffh and its domains, the affinity for substrate of G domain was the lowest even than that of FtsY G domain. Similarly, Ffh and its NG domain had similar GTPase activity. In addition, the data of cross-linking also supported the results above. Moreover, the difference in binding substrate might be caused by their protein structure. The data of proteinase K digestion indicated that after binding GMPPCP the FtsY , Ffh and their NG domains could resist to delay the destroy, and while G domains could fail to do so.Furthermore, to determine the activity sits in the core NG domain of FtsY and Ffh site-directed mutagenesis was performed. The results revealed that a conserved box GXXGXGK was essential for them to bind and catalyse GTP, and lys¹⁴⁷ in Ffh and Lys²²⁵ played an important role in binding GTP. In addition, Gly²¹⁹ in FtsY also affected the GTPase activity of NG domain, and while mutations in Gly¹⁴¹ and Ala¹⁴⁵ of Ffh and in Thr²²³ of FtsY had no meaning. Therefore, Lys residues in GXXGXGK of Ffh and FtsY were necessary. Moreover, a structure model was constructed to explain the significance of Lysine residue during the interaction between NG domins of Ffh and FtsY. In this model the two key Lysine residues could form two hydrogen bonds to bind GMPPCP.In the fourth chapter the GFP reporter was introduced to study the localizaion of FtsYs from S.coelicolor, B.subtilis and E.coli and reveal the localization mechanism of SRP receptor. The ftsY,ffh genes and their domains were fused to gfp gene in the parent plasmid pSG1729 to make six kinds of recombination plasmids. Sequently, six kinds of mutant were constructed by double cross-over recombination. Under the induction of xylose, the gfp fusion proteins expressed successfully. The localization of ten kinds of fusion proteins were analyzed by the laser confocal fluorescent microscopy. The results indicated that both the E.coli FtsY and its NG domain/B.subtilis FtsY-NG could bind the membrane, while only the S.coelicolor FtsY also was located to the membrane, and its NG domain lost the ability of binding. Therefore, the N terminal domain was necessary for S.coelicolor FtsY to bind the membrane. Moreover, the mechanism of localizaiton for SRP receptor FtsY in bacteria was not conserved as shown in this study, which also revealed the diversification of SRP pathway.In the fifth chapter the E.coli mutants, whose ffh and ftsY genes were disrupted, were constructed to investigate the conservation of SRP pathway in bacteria. The properties of the E.coli mutants were consistent to that the previous research reported. They were sensitive to the higher temperature condition and showed longer cell shape. Specially, the membrane of the fts Y-disrupted mutant became roughness possible due to the lack of membrane structure proteins which was proved by SDS-PAGE analysis for membrane fraction. Interestingly, S.coelicolor ftsY, which were controlled to express in the corresponding mutant, could restore the defects of mutant. But its NG domain could do nothing.In the last study, the ffh and ftsY gene in chromosome of S.coelicolor were deleted to evaluate their functions in vivo, respectively. The two mutants were titled as ff5 and ft2. The results indicated that the ft2 mutant could not develop into spore and was inhibited in growth. Moreover, the production of antibiotic Act decreased greatly. However, the production of antibiotic Red took no change. As for the ff5 mutant, no changes in growth and physiology were observed. To determine the function of ffh and ftsY, a series of recombination plasmids were constructed which contained ffh, ftsY, ffh-NG, ftsY-NG fragments. The complemental experiments showed that the whole gene ftsY could restore the defects of mutant ft2, while other three complementations made no effect. Therefore, FtsY in S.coelicolor could not only take part in the recognition and translocation of membrane protein but also be involved in cell differentation. In addition, the NG domain of FtsY could not substitute for the function of the whole protein FtsY in vivo. In our work, the possible mechanism of FtsY regulating sprulation was identified. the ftsY was predicted to be involved in the synthesis of glycine betaine which could effectively adjust the osmosis. So, the lower level of glycine betaine in mutant ft12 might be caused by the abnormal translocation of membrane protein glycine betaine synthase. To reveal the possible mechanism, we analyzed the transcription level of prox, whiG, whiB and whiH. The results indicated that the transcription level of prox and whiG decreased in mutant ft12, and the transcription of whiH could not take place. Once when the ftsY gene was placed in genome again, the transcription of whiH could resume.