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Team:UANL Mty-Mexico/Project
From 2013.igem.org
Functional RNAT have been found in different organisms, mainly pathogenic bacteria, and many others have been predicted in almost everyfrom 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.
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, their structural diversity and the effect of other regulatory sequences far from the RBS region [Kortmann and Narberhaus, (2012); Waldminghaus, et al., (2007)].
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.
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.
Although RNATs show almost no sequence similarity among them, a number of structural features can be used to classify them. Here we enlist the most described RNATs structural groups described to date:
- ROSE.- ROSE stands for "Regulation Of heat Shock Expression". ROSE elements are 60 to >100 nucleotide sequences found upstream of heat shock proteins. They have been found to be conserved in alpha and gamma-proteobacteria. Among the structural features of the ROSE element family are: a) their folding in 2 to 4 stemloop structures; b) a short conserved sequence (UU/CGCU) near the Shine-Dalgarno sequence; and c) the presence of a number of non-cannonical base interactions (the G83-G94 pair; a triple bair among U96-C80-C81; the U79-U97; and the interaction of the AUG codon and C71, G72 and U73. Functional ROSE elements have been found in E. coli (rpoH and ibpA) and B. japonicum (hspA).
- FourU elements.- these elements are characterized by a short motif composed of four uridines that pair with the Shine-Dalgarno region and is embedded in a hairpin that shows temperature-induced conformational changes. FourU elements have only one A-G non-cannonical base interaction. Among the structural features that characterize FourU elements are a) the A-G pair and b) the G34-C46 pair that regulates melting. Functional FourU elements have been described in Salmonella (agsA) and Yersina pseudotuberculosis (lcrF).
- Synechocystis hsp17 element.- with a length of 46 nucleotides, this is the shortest RNAT described so far. The distinctive structural features essential for the function of this element are a) the pairing of a UCCU sequence with the AGGA in the Shine-Dalgarno sequence and b) the presence of two loops in its stems.
- Coding region spanning RNATs.- RNATs are not exclusively found in the 5'UTR of genes; they can also span into the coding region and even be intergenic.
- Cold shock RNATs.- cold shock RNATs also depend on the dynamics of the folding of different loops, but in contrast to heat shock RNATs, the conformation that prevents the binding of the ribosome is found at high temperatures, while at low temperatures, the RNAT folds into a conformation that allows for the ribosome to proceed.
