Two-gene operons have been identified in bacteria and eukaryotes in which the nascent peptide product of the upstream gene interferes with a function(s) of its translating ribosome. This can activate or abolish translation of the downstream coding sequence depending on the gene system. In translation attenuation regulation of inducible chloramphenicol resistance genes in bacteria, ribosome stalling at a specific codon in the leader coding sequence (the upstream gene) destabilizes an adjacent region of mRNA secondary structure that sequesters the ribosome binding site for the downstream coding sequence, which specifies the antibiotic resistance protein. thus, ribosome stalling in the upstream coding sequence activates translation of the downstream coding sequence. We propose the pivotal first two steps of a three step model for translation attenuation depend on cis-interaction of the nascent leader peptide with its translating ribosome. Mutations in 23S rRNA that prevent Step 1 (the binding of peptide to rRNA) will be tested for induction in vivo and for their effect on peptide inhibition of peptidyltransferase. Ribosome pausing in the leader due to Step 1 is converted to ribosome stalling (Step 2) in the presence of inducer. We suggest that the nascent peptide alters the response of the target ribosome to chloramphenicol. This will be tested by identifying the ribosomal components altered during the combined addition of peptide and inducer, and by in vivo analysis of translational fusions of the leader to lacZ in bacteria and yeast. Step 3 is proposed to result from an interaction of the rRNA with mRNA nucleotides in the leader proximal region of secondary structure. This will be examined by experiments that swap expressed 23S rRNA's and by modifications in the sequence of the secondary structures that regulate translation attenuation. The amino acid sequence of the leader peptides dictates 23S rRNA binding activity. We have identified a simple method that should allow determination of the critical amino acids for binding. Further, we will refine the identification of the rRNA site for peptide binding by high resolution footprinting.
| Lodato, Patricia B; Rogers, Elizabeth J; Lovett, Paul S (2006) A variation of the translation attenuation model can explain the inducible regulation of the pBC16 tetracycline resistance gene in Bacillus subtilis. J Bacteriol 188:4749-58 |
| Rogers, Elizabeth J; Rahman, M Sayeedur; Hill, Russell T et al. (2002) The chloramphenicol-inducible catB gene in Agrobacterium tumefaciens is regulated by translation attenuation. J Bacteriol 184:4296-300 |
