Fig. 1.

The toxT 5′ UTR modulates temperature-dependent translation of LacZ. (A) Schematic of the predicted structure of the toxT 5′ UTR. The secondary structure was derived from Mfold (16). Shown are the relevant nucleotides within the putative fourU thermometer and the SD sequence; the complete nucleotide sequence and structure is shown in Fig. S1. The start codon and the U5 and U7 residues are indicated. (B) Effect of temperature on LacZ translation from toxT 5′ UTR. The toxT 5′UTR-lacZ (WT) construct, and the U5C, U7C, and U5C U7C (U5, 7C) mutant toxT 5′ UTR-lacZ constructs were expressed from the pBAD promoter in plasmids pBO1296, pBO1826, pBO1829, and pKEK1807, respectively. Plasmids were transformed into V. cholerae O1 strains O395 (Classical), C6706 (El Tor), and E. coli strain DH5α, and strains assayed for β-galactosidase activity during midlog growth (OD₆₀₀ of 0.4–0.5) at the temperatures indicated in the presence of 0.1% arabinose. Translation from the WT UTR was significantly greater at 37 °C than at 25 °C (P < 0.01), and translation from WT UTR was significantly greater than from U5C and U5,7C UTRs at 37 °C (P < 0.001); Student’s two-tailed t test.