Genetic assay for the binding of the LtrA protein to the DIVa. (A) Plasmid pBDG-LtrA used in the genetic assay. The plasmid employs an L-arabinose-inducible promoter (PBAD) to express the wild-type LtrA protein, followed by a cassette consisting of DIVa with the Shine–Dalgarno (SD) sequence, ATG initiation codon, and next 14 codons of the LtrA ORF fused in-frame to codon 2 of GFP. In the genetic assay, the LtrA protein binds DIVa to down-regulate translation of GFP, leading to a GFP− phenotype, while mutations that impair binding of LtrA to DIVa lead to a GFP+ phenotype. The plasmid encodes the activator protein AraC, which binds L-arabinose to induce expression from the PBAD promoter. (camR) Chloramphenicol-resistance marker; (ori) plasmid replication origin. The region containing DIVa is expanded to emphasize structural features. (B) DIVa region of pBDG-LtrA and changes in DIVa mutants M1 and M2. The secondary structure of the LtrA-binding region of wild-type (WT) DIVa was deduced from in vitro selection experiments (Singh et al. 2002; Watanabe and Lambowitz 2004). Nucleotide residues found to be critical for LtrA binding in those experiments are boxed. The SD sequence is delineated, and the AUG initiation codon is circled. The insets at left and right show changes in DIVa mutants M1 and M2, with mutant nucleotide residues shown in lowercase letters. The UGA termination codon of the LtrA ORF located upstream of DIVa (see A) is boxed. The discontinuity of intron position numbers downstream from DIVa reflects deletion of the intron ORF. The locations of some restriction sites used for cloning are shown (see Materials and Methods).
