Interaction of Chlamydia trachomatis transcriptional activator GrgA with RNA polymerase β' subunit
Objective To study the interaction between transcriptional activator GrgA and RNA polymerase(RNAP)β'sub-unit during the transcriptional regulation of Chlamydia trachomatis gene.Methods Preparation of chlamydia trachomatis RNA poly-merase(cRNAP)NH-β'(N-terminal with His tag)plasmid:Specific primers were designed according to gene sequences in GenBank,chlamydia genomic DNA(gene DNA,gDNA)was used as template,and the gene fragment of cRNAP β'subunit was amplified by Poly-merase chain reaction(PCR).The expression plasmid was constructed by homologous recombination,and β'subunits and deletion sub-units were prepared by the same method.Expresstion of cRNAP β'subunit protein(including subunits and missing subunits):The con-structed plasmid was transformed into ArcticExpress expressing bacteria,and the protein expression was identified by Coomasil bright blue staining method.Purification cRNAP β'subunits and their subunit proteins:The corresponding proteins were purified by im-mobilized metal affinity chromatography(IMAC).In vitro Pull-down protein binding assay(pull-down),polyacrylamide gel electro-phoresis(SDS-PAGE)and Western blot(WB)were used to identify a specific amino acid sequence of GrgA and cRNAP β'subunits.Results GrgA could bind to β'subunit of cRNAP.GrgA binds to the N-terminal(1-731)amino acid sequence of the β'subunit of cRNAP,but does not bind to the C-terminal(671-1396)amino acid sequence.GrgA binds to β'subunit(1-240)and(181-417)amino acid sequences,and there is no binding with β'(360-598),β'(533-731)amino acid sequence.GrgA is bound to β'△(1-180)and β'△(241-417),but not to β'△(1-417).Conclusion GrgA can bind to β'subunit of cRNAP.There are two binding sites between cRNAP's β'subunit and GrgA,which are located at positions 1-180 and 241-417 of the β'subunit N terminal respectively.
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