Figure 1.

Influence of cytidylate kinase on the phosphorylation state and decay rate of A-initiated transcripts in E. coli. (A) Effect of gene deletions on the expression of a yeiP-GFP reporter. Reporter gene expression was calculated from the ratio of cellular GFP fluorescence to cell density and normalized to reporter expression in wild-type cells (WT). Each value is the average of three biological replicates. Error bars correspond to standard deviations. (B) Effect of gene deletions on the 5′ phosphorylation state of yeiP mRNA. The 5′ phosphorylation state of yeiP mRNA was analyzed by using a splinted ligation assay (PABLO) to determine the percentage of 5′ ends that were monophosphorylated (MonoP) in an isogenic set of E. coli strains. This percentage was calculated by normalizing the ligation yield to that of fully monophosphorylated yeiP mRNA generated by exhaustive pretreatment of cellular RNA with an excess of purified E. coli RppH (PPase), as the ligation of monophosphorylated 5′ ends generally is incomplete. The bent arrow beside the blot leads from the RNA substrate to its ligation product. A representative experiment is shown. (C) Effect of a cmk gene deletion on the 5′ phosphorylation state of A-initiated mRNAs in E. coli. The percentage of monophosphorylated 5′ ends for each cellular transcript was determined by PABLO, as in (B). Each value is the average of three biological replicates. Error bars correspond to standard deviations. (D) Effect of gene deletions on the decay rate of yeiP mRNA. The decay of yeiP mRNA in an isogenic set of E. coli strains was monitored as a function of time after inhibiting transcription with rifampicin. Representative experiments are shown.