Figure 1. Generation of stable 5′ ends in wild type and 2.4.1rne^{E. coli(ts)} strains of R. sphaeroides.

(A) Internal cleavage by RNase E. The ends of primary transcripts are protected from degradation by a triphosphate at their 5′ end and by secondary structures (often terminators) at their 3′ end. Internal cleavage of RNase E generates an unprotected 3′ end, which allows rapid degradation by 3′–5′ exoribonucleases. The new monophosphorylated 5′ end is a new target for RNase E. The 5′ end stemming from RNase E cleavage is accumulated in the wild type at the nonpermissive temperature. (B) 5′ end–dependent degradation by RNase E. RNase E can bind to monophosphorylated 5′ ends if a stretch of unpaired nt is present and will subsequently introduce cleavages in an overall 5′–3′ direction. The 5′ monophosphate ends can stem from previous RNase E cleavage, from cleavage by other endoribonucleases, or by the action of a pyrophosphohydrolase. The 5′ monophosphate end is enriched in the rne mutant strain compared with the wild type at the nonpermissive temperature. Global analysis of 5′ end profiles at a permissive (32°C) (C) and a nonpermissive temperature (42°C) (D). The plots show average first-base-in-read-coverage level in wild-type samples compared with 2.4.1rne^{E. coli(ts)} samples and the relative log₂ fold change. The x axis (base mean) represents the average, library-size normalized coverage values, whereas the y axis (fold change) represents the ratio of the normalized coverage values of the mutant in comparison with the wild type (both base-means and fold changes were calculated by DESeq2). The green dots represent sites with a significant (i.e., Benjamini–Hochberg adjusted, P < 0.05) enrichment in the wild type, whereas the brown dots represent sites with a significant enrichment in the mutant. Black dots represent sites without a significant enrichment.