Introduction

RNA thermometers (RNATs) are RNA sequences that range from 40 to more than a 100 nucleotides commonly found in the 5' untranslated region of some genes and that regulate in cistheir translation without the need of other factors. These RNAT sequences show certain three dimensional structures, some of which interact with the ribosome binding site (RBS) of their regulated genes and hinders the proccessivity of the ribosome complex at certain temperatures. The dynamics of the formation of these structures is temperature dependent and is the basis of RNA thermoregulation.

Functional RNAT have been found in different organisms, mainly pathogenic bacteria, and many others have been predicted from a number of bioinformatic studies. They have been found to regulate the expression of virulence factors, heat and cold shock response factors and even proteins involved the development of some bacteriophages.

Their apparent widespread presence in living organisms has made RNATs attractive for some applications, specially the ones related to the replacement of chemical inducers and for the development of new drugs, since RNATs are usually found in pathogenic bacteria.

However, from the experience of those who have been working extensively with RNAT in the later years, the accurate bioinformatic prediction of functional RNAT has proven to be an exceptionally difficult task; the reasons for this are pointed to be the poor sequence conservation observed among RNATs and the gaps in our current understanding of the RNAT function and the effect of other regulatory sequences far from the RBS region.

The discovery of new RNATs has relied on a mixed approach that involves bioinformatics and experimental validation, as well as approaches that involve mutational libraries, synthetic constructions and directed evolution.

Nevertheless, even when the naturally found RNATs usually regulate the expression of transcription factors, the synthetic constructions made so far have focused mainly to characterize the effect of a given RNAT using a reporter protein (LacZ or a fluorescent protein) directly downstream of a RNAT. In our work, we intend to prove that RNATs can also be employed to effectively regulate the expression of transcription factors in synthetic circuits and point at possible applications for the circuit topologies that would be made feasible with this new kind of synthetic regulatory device.