The Role Of Proteins Interacting With The Escherichia Coli RNA Polymerase ? Subunit In Transcription

In the complex process of gene transcription,the ? subunit of the prokaryotic RNA polymerase recognizes the promoter of genes and induces the RNA polymerase assembling on the promoter.When transcription elongation begins,the ? subunit dissociates from the promoter and induces the assembly of next transcription division.However,the mechanism of the dynamic cycle of the ? subunit(Rpo D)is still unclear.(1)Through the GFP-Trap immunoprecipitation technology,we found that chaperone proteins such as ClpB,HtpG,DnaK and YhgF,protein of unknow function,interact with Rpo D.The analysis results of the biomacromolecule interaction instrument showed that Rpo D does interact with ClpB,HtpG,DnaK and YhgF proteins in vitro.Bacterial two-hybrid assay also proved the interaction of these proteins with Rpo D in vivo.(2)The fluorescence localization experiment of Rpo D-GFP shows that the fluorescence localization of Rpo D in wild-type and ?clp B,?htp G,?dnaK and ?yhg F mutant cells will not be affected by ClpB,HtpG,DnaK or YhgF.It is found that YhgF-GFP colocalize with the chromosome,indicating that YhgF may be involved in transcription initiation mediated by Rpo D.(3)Through RNA-seq analysis,we found that there is an extensive change in expression of many genes in ?clp B,?htp G,?dnaK and ?yhg F mutants cells,indicating that ClpB,HtpG,DnaK and YhgF proteins affect transcription.The differentially expressed genes caused by the deletion of ClpB are mainly concentrated in DNA recombination and electronic transport;when YhgF is deficient,the differentially expressed genes are mainly concentrated in basic biological processes such as cell localization;when HtpG is deficient,the differentially expressed genes are mainly concentrated in cell components and electronic transport;Deficiency of DnaK has a greater impact on gene expression,and differentially expressed genes are mainly concentrated in basic biological processes and cellular components.It is worth emphasizing that compared with other chaperone proteins,deletion of DnaK affects gene expression more widely.(4)The absence of ClpB,HtpG,DnaK and YhgF does not affect cell cycle parameters,but the absence of HtpG and DnaK weakens cell motility,while the absence of ClpB and YhgF does not significantly affect cell motility.The results indicate that these proteins may regulate the expression of flagellar gene.(5)The lack of QseB/Qse C signaling system affects cellular motility,which is regulated by the interaction between QseB and DnaK.(6)The QseB/Qse C system affects the initiation of chromosome replication by regulating the expression of dna A gene.In summary,the molecular chaperone proteins ClpB,HtpG,DnaK and YhgF,protein of unknow function,interact with RNA polymerase ? subunit(Rpo D);the deletion of ClpB,YhgF,HtpG and DnaK affects the level of genome transcription,and HtpG and DnaK affects cell motility,while DnaK interacts with QseB to regulate cell motility,which influences expression of dna A gene and regulates the initiation of replication.