Abstract
BackgroundSeveral 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.ResultsWe have developed a method that allows us to select cis-acting regulatory sequences that respond to diverse chemicals. The method is based on the β-lactamase gene containing a random sequence inserted into the beginning of the ORF. Several rounds of selection are used to isolate sequences that suppress β-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.ConclusionsSeveral 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.
Highlights
Several cis-acting regulatory sequences functioning at the level of mRNA or nascent peptide and influencing transcription or translation have been described
Nascent peptide:ribosome complexes respond to macrolide group antibiotics, chloramphenicol and tryptophan [3,4,5,6]
The sequences could affect gene expression by forming riboswitch mRNA structures, able to bind chemicals directly and regulate gene expression. They may be functional at the peptide level via specific interactions between the chemicals, peptides and the ribosome. To discriminate between these possibilities, we introduced synonymous mutations into one chloramphenicol and two erythromycin responsive sequences fused to b-galactosidase, changing the mRNA sequence but preserving the peptide sequence (Figure 1)
Summary
Several cis-acting regulatory sequences functioning at the level of mRNA or nascent peptide and influencing transcription or translation have been described. These regulatory elements often respond to specific chemicals. According to the classical scenario, trans factors, either protein or RNA, bind to the mRNA and modulate the level of translation. The nascent peptide can stall the ribosome, often in response to small molecules. This leads to the induction or repression of downstream genes [1,2]. The mRNA itself can recognize small molecules, leading to changes in its transcription
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