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. 2021 Oct 13;49(19):11038–11049. doi: 10.1093/nar/gkab884

Figure 3.

Figure 3.

Mechanism by which cytidylate kinase influences the degradation of A-initiated transcripts in E. coli. (A) Increased percentage of yeiP 5′ ends that are diphosphorylated in cells lacking cytidylate kinase activity. The 5′ phosphorylation state of yeiP mRNA was analyzed by using a capping assay (PACO) to determine the percentage of yeiP 5′ ends that were diphosphorylated in an isogenic set of E. coli strains (bottom) versus a set of triphosphorylated (TriP), diphosphorylated (DiP) and monophosphorylated (MonoP) yeiP standards synthesized by in vitro transcription (top). Capping by the guanylyltransferase Pce1 protects the 5′-terminal phosphates of diphosphorylated RNA (and to a lesser extent triphosphorylated RNA) from removal by alkaline phosphatase; subsequent decapping by the pyrophosphatase RppH generates a monophosphorylated 5′ end able to undergo splinted ligation to an oligonucleotide (15). A bent arrow leads from each RNA to its ligation product. The calculated percentage of diphosphorylated (DiP) yeiP 5′ ends in each strain, as determined by comparison to the yeiP standards (Supplementary Tables S3A–D), is indicated below the lane in which the cellular RNA underwent a complete series of four consecutive enzyme treatments. Representative experiments are shown. (B) Invariant concentration of RppH and DapF in cells lacking cytidylate kinase activity. Equal amounts of total cellular protein extracted from an isogenic set of E. coli strains were analyzed by immunoblotting. RppH and DapF were detected via a chromosomally encoded epitope tag added to the carboxyl end of the protein (FLAG-hexahistidine (FH) or FLAG, respectively). RNase E served as an internal standard. ✻, untagged E. coli protein bound nonspecifically by the anti-FLAG antibodies. Representative experiments are shown. (C) Epistatic effect of cytidylate kinase and DapF on the phosphorylation state of yeiP and ydfG mRNA. The percentage of monophosphorylated yeiP and ydfG 5′ ends in an isogenic set of E. coli strains was determined by PABLO, as in Figure 1. Each value is the average of three biological replicates. Error bars correspond to standard deviations.