Study On DNA Binding Function And Structure Of LmbU The Pathway-Specific Regulators Of Lincomycin Biosynthesis Pathway

Lincomycin is a lincosamide antibiotic and widely used in clinical treatment.In the past decade,the research on lincomycin biosynthesis has progressed rapidly,and the synthetic pathway has been elucidated.In the regulatory network of lincomycin synthesis,several global regulators such as AdpA,BldA and SLCG₂919 have been identified.Besides,the clustersituated regulators LmbU has been shown to regulate transcription of clustered gene and positively regulate lincomycin biosynthesis.And LmbU was named as a novel regulatory factor family for its sequence is no homologous to other known regulators.Based on the DNA binding function of LmbU,this paper attempts to analyze the structure of LmbU,LmbU and DNA complex by X-ray diffraction.For the study on DNA binding function of LmbU,site-directed mutated DNA binding region of LmbU were obtainded.And the binding abilities of these mutant proteins was analyzed by EMSA assay.Experiments showed that most of the amino acids in LmbU DBD region are related to DNA binding,while the side amino acids are not involved with DNA binding.With bioinformatic analysis,we found that homologoes of LmbU are numerous and widely distributed.But no structure information was reported.So related research on LmbU structure was carried out in this work.Firstly,LmbUc12GC63 was constructed and purified,and crystals were obtained by crystallization optimization,and the diffraction data were successfully obtained.Further,we tried to resolved the crystal structure using selenium protein derivatives.We successfully constructed and purified four protein variants with a single point mutation of methionine.Finally,the DNA binding abilities of the truncated proteins of LmbU were analyzed by EMSA.And it was found that N-terminus of LmbU inhibits LmbU binding activity.So LmbU40-225 was expressed and purified.LmbU40-225 and various DNA substrates are screened to obtain diverse LmbU40-225-DNA complex crystallization conditions.These work,especially the attempt of crystallization laid a foundation for the subsequent analysis of LmbU structure.