Cloning and expression of the redox-sensing transcriptional repressor Rex and in vitro DNA-binding assay of the Rex and rex operator in Streptomyces rimosus M4018

Abstract:

Abstract:［Objective］The aim is to explore the self-regulation mechanism of the rex in Streptomyces rimosus M4018．［Methods］We cloned the rex of S． rimosus M4018 (Sr-rex) based on its homologoussequence in Streptomyces coelicolor A3(2) and its upstream rex operator (ROP) fragment using PCR and genome walking． An electrophoretic mobility shift assay (EMSA) was applied to analyze the regulation of rex to ROP in vitro．［Results］Sr-rex is 846 bp in length and has a 84% identity with the one in S．coelicolor A3 (2) in amino acid sequence． It was deposited in Genbank under the accession number GQ849479． The expressed Sr-Rex by E．coli was mainly composed of alpha-helixes and beta-sheets，which was in compliance with the prediction． An Electrophoretic Mobility Shift Assay (EMSA) confirmed the specific binding activity of Sr-Rex with ROP． Meanwhile，we synthesized a 22 bp DNA fragment (ROP1) based on the minimal binding site of ROP． The maximal binding ratio of this fragment to Sr-Rex was 5∶1(molar)．NADH negatively affected the binding activity，however，NAD + had no impact on it．［Conclusion］In S． rimosus M4018，the Rex regulated the gene expression of ROP via sensing the intracellular level of NAD (H) ．