A method for selecting cis-acting regulatory sequences that respond to small molecule effectors.

BACKGROUND

Several cis-acting regulatory sequences functioning at the level of mRNA or nascent peptide and specifically influencing transcription or translation have been described. These regulatory elements often respond to specific chemicals.

RESULTS

We have developed a method that allows us to select cis-acting regulatory sequences that respond to diverse chemicals. The method is based on the beta-lactamase gene containing a random sequence inserted into the beginning of the ORF. Several rounds of selection are used to isolate sequences that suppress beta-lactamase expression in response to the compound under study. We have isolated sequences that respond to erythromycin, troleandomycin, chloramphenicol, meta-toluate and homoserine lactone. By introducing synonymous and non-synonymous mutations we have shown that at least in the case of erythromycin the sequences act at the peptide level. We have also tested the cross-activities of the constructs and found that in most cases the sequences respond most strongly to the compound on which they were isolated.

CONCLUSIONS

Several selected peptides showed ligand-specific changes in amino acid frequencies, but no consensus motif could be identified. This is consistent with previous observations on natural cis-acting peptides, showing that it is often impossible to demonstrate a consensus. Applying the currently developed method on a larger scale, by selecting and comparing an extended set of sequences, might allow the sequence rules underlying the activity of cis-acting regulatory peptides to be identified.