Identification And Analysis Of The Transcriptional Regulation Of EsrE In Escherichia Coli

In Escherichia coli,gentic locus yigP is recently identified as gene ubiJ which is responsible for the biosynthesis of ubiquinone.Promoter of upstream gene ubiE controls the transcription of gene ubiJ.In addition,there is a promoter within coding region of ubiJ(promoter of esrE),which transcripts a 252nt sRNA participated in the transcriptional regulation of operon sdhCDAB associated with the biosynthesis of succinodehydrogenase.Evidence shows that region P42,which is upstream of esrE promoter,is detected as a negative transcriptional regulatory element.However,we have no idea about the proteins interacted with esrE promoter and its regulatory region yet.In this study,we use promoter probe vector to confirm there are several regulatory regions in upstream of esrE promoter collaborating its transcription,which leads to active or negative effect.Base mutation experiment of cis-acting element P42 further confirms that this region plays an important role in the activity of esrE promoter and the key motif is"GGCGAT" There are interactions between the key motif and two palindromic sequences,which attract proteins with different functions to participate in transcriptional regulation of esrE promoter together.Later,we use DNA pull down coupled with mass spectrometry to separate and identify the DNA binding proteins interacted with fragment SIV3(probe P-PRO,including esrE promoter and its regulatory regions).Software is used to retrieve E.coli K-12 database and get 105 groups of proteins.Next,dual-plasmid system and electrophoresis mobility shift assay(EMSA)are applied to analyse the interaction between candicate proteins(RdgC/SeqA/RpoH)and regulatory regions.Experience shows that protein RdgC and the DNA-binding domain of protein SeqA attach to esrE promoter region nonspecifically,which don't transcribethe expression of esrE promoter.On the other hand,RpoH has an active effect on the activity of esrE promoter,which has already been proved as a DNA-binding protein(RNA polymerase subunit ?32).As a result,we come to a conclusion that the transcription of esrE promoter is initated by RpoH,which encodes ?32 factor.