A key element of control of ColE1-type plasmids in vivo is the rate of degradation of the antisense repressor RNAI. Endonucleolytic removal of 5 bp from the 5' end of RNAI by RNaseE relieves repression converting RNAI to pRNAI-5' a rapidly decaying product. An artificially constructed 5'triphosphate-terminated homolog of pRNAI-5' pppRNAI-5' is degraded slowly and consequently inhibits replication. The decay of pppRNAI-5' which lacks an RNaseE cleavage site, is markedly affected by the rate of cell growth. When bacterial growth is slowed, the cellular concentration of pppRNAI-5 increases, plasmid DNA replication is inhibited and plasmid copy number decreases.
The aim of this project is to identify the growth-rate controlled enzyme (GRCE) that degrades the 5'triphosphate terminus of this RNAI variant. Since pRNAI-5 and pppRNAI-5 are identical except for the mono or triphosphate 5' terminus, it is likely that the difference in decay rate results from a 5'-exonucleolytic activity. Mutations that increase the activity or expression of the GRCE will be isolated selecting for an increase of plasmid copy number in conditions of slowed cell growth. After cloning the gene encoding this enzyme, regulatory regions will be identified and response to cell growth at the level of gene expression will be tested. The long-term objective is to investigate how changes in cell growth rate can affect the degradative pathway of an RNA.
