Author: Anne Eliane ChiaramelloPublisher:
ISBN:
Category : DNA
Languages : en
Pages : 300
Book Description
The initiation of DNA synthesis at the chromosomal replication origin, oriC, in Escherichia coli involves an RNA polymerase-mediated step. The level of synthesis of transcripts moving counterclockwise toward oriC is controlled at two promoters, P1 (asnC) and P2 (mioC), and at two transcription terminator regions, T1 and T2. As shown by S1 mapping, termination at the T2 region occurs to the right of oriC at nucleotides 297-299 and 306-310, while major termination events in the T1 region occur in and near the mioC promoter. The majority of transcripts entering oriC originates from the mioC promoter. Transcription from the mioC promoter has been shown to enhance the frequency of initiation of DNA replication of oriC containing plasmids, and to stabilize these plasmids in the host cells. The mioC promoter, which is stringently controlled, is also growth rate regulated. The amount of mioC transcripts relative to the amount of total RNA was inversely correlated with growth rate. This transcript is characterized by a short half-life (1.5 min). The mioC promoter, which contains a DnaA protein binding site, was much less susceptible to repression by DnaA protein when located in the chromosome, than when located in a plasmid. Only a very high concentration of DnaA protein repressed the mioC promoter. The DnaA protein, which is required for initiation of DNA replication from oriC, is growth rate regulated. As shown by RNase protection, this regulation is exerted at the transcriptional level, affecting both promoters, dnaAp1 and dnaAp2. Transcription from these two promoters is also stringently controlled. The amount of DnaA protein in spoT mutants, which are deficient in ppGpp pyrophosphorylase activity, decreases as the severity in the mutation increases. Thus, the intracellular concentration of ppGpp influences the expression of the dnaA gene. In conclusion, the growth rate regulation and stringent control of the dnaA gene suggest that one way in which DNA replication is coordinated with the growth rate is via ppGpp synthesis at the ribosome.
