(A) Relative fitness of strains carrying single point mutations introduced into the relevant ancestral background competed against the respective ancestor. The mutations correspond to those listed in Supplementary files 2, 3 for the respective genes. Initial screening for fitness defects involved two biological replicates (grey diamonds). For the two mutations where the initial screen suggested a measurable fitness deficit, lamB and rplSsyn, all competitions were carried out in quadruplicate. Bar heights and error bars are as described in Figure 1. **p < 0.01, *p < 0.05 (one-sample t-test). OE: overexpression; EP: empty plasmid. Additional results for competitions terminated in mid-exponential phase (after 2 hr) are shown in Figure 3—figure supplements 1, 2. (B) Linear Feynman graph of the lamB region that harbours the mutation in the evolved ΔmutH strain (highlighted in grey). We predicted RNA secondary structure for the entire malK-lamB-malM transcription unit (RegulonDB identifier: ECK120009315) and its mutated counterpart using RNAfold (Lorenz et al., 2011). The malK-lamB-malM operon contains a repetitive extragenic palindromic (REP) element downstream of lamB, which prevents premature degradation of the lamB cistron following cleavage from malM. Resolution of this REP element as part of regulated degradation was previously shown to require RhlB (Khemici and Carpousis, 2004). However, comparison of predicted minimum free energy structures between wild type and mutant malK-lamB-malM transcripts suggested structural changes that do not interfere with REP element formation but rather lead to decreased positional entropy at the local level, as highlighted here.