CONTROL OF INITIATION FREQUENCY OF COLE2 REPLICATION

点击翻译按钮获取中文摘要

The ColE2 Rep protein is unique among many essential initiator proteins in that it is a plasmid-specified primase specific for initiation of DNA replication in the origin by DNA polymerase I.Expression of the Rep protein is kept constant through negative regulation by a small antisense RNA (RNAI). RNAl is entirely complementary to the 5' nontranslated region of the Rep mRNA.Since the Rep protein is trans-acting, it is not so obvious how the initiation frequency can be kept constant.Analyses of various derivatives of the ColE2 origin carrying deletions or single-base substitutions showed that the region may be divided into three subregions : one important for stable binding of the Rep protein, another important for binding and for replication and another important for replication but apparently not for binding. Transformation frequency of the autonomously replicating plasmids carrying deletions in the first region is lower and nevertheless the copy numbers of them in hostbacteria are higher as compared with the wild-type plasmid. The Rep protein might inhibit over-replication or re-replication of newly replicated daughter molecules by stable binding to the origin. This might be important to keep the initiation frequency at a constant level.The 5' nontranslated region of the Rep mRNA far upstream of the initiation codon and another region in the coding region near the initiation codon are important for efficient expression of the Rep protein. Partial digestion with various RNases in the presence or absence of RNAI suggested that binding of RNAI to the Rep mRNA causes changes in the secondary structure of the region containing the initiation codon. Mutant plasmids carrying base substitutions in the upstream and downstream regions of the initiation codon of the Rep mRNA have been isolated. Some of the mutations abolished or decreased expression of the Rep protein.