Figure 2.

Identification of single-nucleotide suppressor substitutions in the hok/Sok locus. (A) The hok/Sok locus consists of two overlapping CDSs, the leader peptide mok and the toxic protein hok, and of the Sok noncoding RNA. The mok_hok mRNA (or hok mRNA) is transcribed as an untranslatable FL mRNA that is processed at the 3′ end to form a translatable, truncated mRNA (Tr-hok). Here, the HS, HΔS and ΔHΔS constructs were amplified by PCR to induce mutations and individually introduced into the E. coli chromosome. The expected expression of each of the three constructs designed for FASTBAC-Seq is indicated. HS: The Sok antitoxin inhibits the Tr-hok mRNA translation by inducing degradation of the toxin/antitoxin RNA duplex by RNase III. HΔS: In the absence of the Sok antitoxin, Tr-hok mRNA can be translated and is toxic for the host cell. ΔHΔS: The locus with the promoters of both hok and Sok inactivated was used as a control; hok and Sok are not transcribed. (B) FASTBAC-Seq results. The differential distribution of single-nucleotide substitutions was statistically analyzed between the hok*/ΔpSok and the controls (ctrl) hok/Sok and Δhok/ΔpSok. We obtained loss-of-function substitutions (occurring statistically more in hok*/ΔpSok) well distributed over the hok/Sok locus. The arrow represents the promoter, and the ‘stem-loop’ represents the terminator. The CDSs of mok and hok are shown in orange colors. The zoomed gray box shows the location substitution leading to stop codons in mok or hok CDS.