Figure 10-2: Lysis phenotypes. A. Lysis of a lambda lysogen induced in exponential phase at t = 0. S+ R+ = triangles; 5 mM KCN added to S+ R+ = filled squares; S+ R- = open diamonds; S- R+ = circles; S- R+ with CHCl3 added, filled diamonds; S(a52g) R+ = open squares. Adapted from (112), with permission. B. Lysis phenotypes of single Cys substitution alleles. Exponential cells lysogenic for lambda-delta-(SR) and carrying a plasmid with the ??lysis cassette under pR’ control were induced at t = 0. All alleles except the null Sam7 are isogenic to the parental S105 allele (with codon 1, the initiation site for the S107 reading frame, inactivated). Stars indicate alleles with lysis times earlier than the S105 parental allele. Adapted from Gründling et al. (54), with permission. C. RNA structure control of the S105/S107 partition and lysis timing partition. Left: Western blot, using anti-S antibodies, of cytoplasmic membrane fractions from cells induced for various S alleles with mutations in the translational initiation region, including the dominant lysis defective sdi mutations that defined the upstream regulatory RNA stem-loop (see Fig. 10-1A inset). The first three lanes have (a) S+, (b) S107 and (c) S105; in the latter two alleles, the AUG start codons 3 and 1, respectively, are changed to CUG, eliminating the production of S105 and S107, respectively. Alleles in other lanes: d = sdi3 (G-8 to A); e = sdi1 (C-18 to U); f = G3 to A,; g = inversion of sdi stem-loop by swapping UCCCC and GGGGG sequences; h = UAAG (-6 to -3) replaced by GAGG; I = C33 to G ; j = G-3 to A and C33 to G, combined; k = U-24 to C; l = C36 to G and A48 to C; m = both codons 1 and 3 to CUG. Mutations that weaken the sdi (upstream) stem-loop, strengthen the downstream loop or damage the Shine-Dalgarno sequence for S105 (lanes (d), (e), (f) (l)) favor S107 production over S105. Conversely, strengthening the sdi structure, weakening the downstream loop, improving the S105 Shine-Dalgarno sequence, (lanes (h), (I), (k)) favor S105 production over S107. Inversion of the sdi structure separates the S107 reading frame from its Shine-Dalgarno sequence and abolishes S107 production, and changing both start codons to CUG (m) eliminates both S gene products. Adapted from Chang et al. (35), with permission.