Study On The RNA-binding Property Of The N-terminal Tandem RRM Domain Of Human RBM45 And Preliminary X-ray Analysis Of Mycobacterium Tuberculosis LexA

RNA-binding proteins（RBPs）regulate RNA metabolism,including splicing,maturation,localization,translation and degradation.RBPs play a key role in many diseases,including neurodegenerative diseases,cardiovascular diseases,genetic diseases,developmental disorders and cancers.RBM45 is an RNA-binding protein involved in neural development,whose aggregation is associated with neurodegenerative diseases,such as amyotrophic lateral sclerosis（ALS）and frontotemporal lobar dementia（FTLD）.This process requires the participation of RNA.It is crucial for the diagnosis and treatment of the disease to determine its specific recognition sequence.This study explores the binding property of RBM45 with the Fluorescence polarization（FP）analysis.The results show that RRM1 and RRM2 of RBM45 can recognize the GAC sequence of RNA.And neither RRM has selectivity for RNA methylation.According to the structure information of the complex,the key amino acids were mutated to obtain the mutant protein,and the interaction with the RNA containing the GAC sequence was detected.The results showed that two aromatic residues and an arginine residue in each RRM were critical for RNA-binding.These results will be helpful in the identification of physiologic targets of RBM45 and provide evidence for understanding the physiologic and pathologic functions of RBM45.Tuberculosis（TB）is one of the leading causes of death in the world.Mycobacterium tuberculosis（Mtb）is the bacterium that causes tuberculosis.It infects 23–32%population of the world.In the course of infection,DNA damage response plays a critical role in the survival of the bacterium in host cells.The transcriptional repressor LexA is a key component of the SOS response,the main mechanism for the regulation of DNA repair genes in many bacteria.However,gene induction of DNA damage is carried out by two mechanisms in mycobacterium:relatively a few genes are thought to be regulated by LexA,and more genes by an alternate,independent mechanism.This study attempts to parse the structure of MtLexA and MtLexA^NTD protein in the mycobacterium tuberculosis,and analyze their function,through methods of gene synthesis and molecular cloning to construct recombinant plasmid,heterologous expression in Escherichia coli and purified protein.Crystals were grown by the vapor phase diffusion dropping method.We change the length of ds DNA by increasing the length of the flanking sequence.And the crystal of complex with MtLexA^NTD was grown.The crystal of complex was obtained.The crystal structure of MtLexA^NTD-DNA complexes will lay the foundation for understanding the molecular mechanism of SOS response in Mycobacterium tuberculosis.