The Effects Of Dsra-srna And Oric Transcription On Initiation Of DNA Replication In Escherichia Coli

sRNAs(small noncoding RNAs)are widely involved in bacterial life events including gene expression,metabolism and stress response,but systematic studies of sRNAs on initiation of DNA replication have not been found.Analysis of 11 sRNA-deletion mutants showed that the Escherichia coli DsrA-sRNA was as a negative regulator for initiation of DNA replication.DsrA-sRNA may interact with dnaA-mRNA,but this interaction was not shown to affect the DnaA expression level.However,qPCR experiment showed that overexpression of DsrA-sRNA increased rpoS,dps,hchA,cbpA and yiaG gene expression.Deletion of the dsrA gene decreased expression of the dps gene,subsequently Dps inhibited initiation of DNA replication.In addition,bacterial two-hybrid experiment confirmed that HchA interacted with the Dps protein,but HchA was not found to interact with the DnaA protein.Interestingly,overexpression of DsrA-sRNA in the Adps cells promoted initiation frequency of DNA replication,which is not in agreement with the effect of overexpressed DsrA-sRNA in wild-type cells.The overexpression of DsrA-sRNA in AhchA cells still inhibits initiation of replication.In summary,the inhibitory effect of DsrA-sRNA on initiation of replication is achieved by Dps.However,in the absence of Dps,DsrA-sRNA may affect initiation of replication in an unknown pathway.The gene transcription through the origin for replication(oriC)is necessary for initiation of replication.In order to investigate the control mechanism of oriC transcription on initiation of replication,the PBAD promoter was inserted into the right side of the oriC region where the oriC region is able to transcribe through oriC,resulting in construction of the WT-PBAD,?AdksA-PBAD and ?mfd-PBAD strains.Flow cytometry found that addition of arabinose did not affect initiation of replication in WT-PBAD cells;the arabinose-induced effect on initiation of replication in AdksA-PBAD cells was shown to be dependent on nutrition.The initiation frequency of replication in ?dksA-PBAD cells grown in LB medium was significantly higher than that of AdksA cells,and addition of arabinose further increased initiation frequency of replication.Thus,oriC transcription promotes initiation of chromosomal replication,but its effect is regulated by DksA activity.