Revision as of 14:46, 12 October 2018

Heat-inducible RNA-based thermosensor-1

A RNA-based thermosensor that can be used for temperature sensitive post-transcriptional regulation which is based on the change of RNA sencondary structure. The heat-inducible RNA-based thermosensors can initiate translation of downstream genes at high temperatures.

1. Usage and Biology

Heat-inducible RNA-based thermosensors are RNA genetic control systems that sense temperature changes. At low temperatures, the mRNA adopts a stem-loop conformation that masks the Shine–Dalgarno (SD) sequence within the 5’-untranslated region (5’-UTR) and, in this way, prevents ribosome binding and translation. At elevated temperatures, the RNA secondary structure melts locally, thereby giving the ribosomes access to the SD sequence to initiate translation (Figure 1). Whereas natural RNA-based thermosensors have a relatively complicated secondary structure with multiple stems, hairpin loops and bulges. The highly complex RNA secondary structures into which most naturally occurring RNA-based thermosensors can be folded has led to the hypothesis that RNA-based thermosensors may not function as simple on/off switches. Our team designed synthetic heat-inducible RNA-based thermosensors that are considerably simpler than naturally occurring thermosensors and can be exploited as convenient on/off switches of gene expression.

2. Design

The temperature response of these thermosensors was designed on the basis of the melting temperature of the minimum free energy structure. The 5’-UTR contained an ASD (anti-SD sequence) and the downstream consensus SD sequence (5’-AAGGAG-3’) was followed by 8-nt spacer derived from----the bacteriophage T7 gene 10 leader sequence. To optimize the thermosensors for the desired melting temperature, intensity and sensitivity, a number of structural parameters come into consideration: stem length, loop size and mismatches or bulges in the stem. Stem length is determined by ASD sequence because the SD sequence is conserved. Adding stem length can optimize heat inducible RNA-based thermosensors to more high temperature, while decreasing stem length has the opposite effect. The stem length is 4 base parings in K2541001. Loop size can moderate thermosensors melting temperature to a suitable temperature. In K2541001, the loop sequence is AAUAA.

Figure 2. Design of synthetic heat inducible RNA-based thermosensor. (A) The RNA secondary structure is predictred by mFOLD. (B) Thermosensor sequence, ASD sequence, loop sequence, the site of mismatch or bulge in the stem and △G are in the table.

Characterization

The thermosensor is constructed on the pSB1C3 vector by GoldenGate assembly. As shown below, the measurement device is composed of Anderson promotor (BBa_J23104), thermosensor (BBa_K2541001) and sfGFP(BBa_K2541400). We measured the sfGFP expression to get the actual melting temperature of the heat-inducible RNA thermosensor.

As shown in the figure, the thermosensor melting temperature range is [ ]. Our data show that efficient RNA thermosensors can be built from a single small RNA stem-loop structure masking the ribosome binding site, thus providing useful RNA-based toolkit for the regulation of gene expression.

Sequence and Features