K5280427

A RNA thermosensor, based on a stem-loop structure, exposes the double-stranded RNA cutting site of RNase III at low temperatures, causing transcript degradation and inhibiting downstream gene translation.

The design references the Cold-repressible RNA-based thermosensor (BBa_K2541201-BBa_K2541210) from iGEM18_Jilin_China and wants to make some improvements to it.

Usage and Biology

The RNA thermosensor is a non-coding RNA molecule that regulates gene expression sensitive to temperature and is found mainly in prokaryotes. In general, RNA thermosensors respond to temperature fluctuations through subtle changes in their secondary structures. Such structural transitions can expose or occlude important regions of RNA, such as ribosome binding sites, thereby affecting the rate of translation of nearby protein-coding genes.

Known naturally occurring RNA thermosensors, although abundant in nature, are difficult to implement in engineered systems because of the complexity of their mechanisms. The RNA thermosensor mechanism developed here is simple and easy to apply to synthetic biology. At low temperatures, the hairpin forms a cleavage site for the natural ribonuclease RNase III in the 5' untranslated region (UTR) of the target mRNA; at high temperatures, the hairpin unravels, the cleavage site disappears, and expression is turned on.

Figure 1. Mechanism of RNA thermosensors.

Design

The thermosensors are controlled by the constitutive Anderson promoter (BBa_J23106), express the red fluorescent protein mCherry (BBa_J428079), and use the double terminator BBa_B0015. Each thermosensor sequence was inserted downstream of the transcription start site and upstream of the RBS and constructed using Gibson Assembly. The strain E. coli DH5α was used. Thermosensor structures and parameters were estimated using the Mfold Web server (Zuker, 2003). RNA Folding Form V2.3 (RNA Folding Form V2.3) provided predicted secondary structures and ΔG values; melting temperature (Tm) was estimated using Hybridization of two Different Strands of DNA or RNA (DINAMelt) and simulated with its Absorbance Plot. This application predicts the unwinding temperature of two independent RNA strands. In this case, a thermosensor is considered to be two independent chains. This approximation ignores the effect of loop size.

The RNA thermosensor described here consists of a fluorescent reporter gene (i.e., mcherry), a universal RBS, and an RNase III cleavage site (RC), which cleaves double-stranded strands in stem loops. At low temperatures, the mRNA stem-loop is stabilized, exposing the RNase III cleavage site and the transcript will be degraded. At elevated temperatures, the stem-loop will unfold and translation will occur unimpeded. Thus, at low temperatures the expression will be “off” and at high temperatures it will be “on”.

RNase III was chosen because, first, it was necessary to choose an endoribonuclease so that it could cleave the transcript at an internal location. It was also important to choose an enzyme that could cut double-stranded RNA instead of single-stranded RNA so that the transcripts would degrade at low temperatures. Of course the non-specificity of RNase III is more problematic, but this conveniently adds to the diversity of the thermosensor.

The design of the RNA thermosensors started with the RNase III cutting site (RC) identified in the literature (Figure 2, Pertzev & Nicholson, 2006). This sequence does not appear elsewhere in the mCherry transcript, and the thermosensor sequence contains either one RC or two RC separated by 2 bp. Adjust the bases in the stem-loop to achieve the predicted melting temperature in the 25 - 35°C temperature range. The naming of the temperature sensors resulted in the first 13 thermosensor sequences being of the same length (30bp), the next four being 32bp, and the last five being duplicates of two stem-loop structures containing RC. To ensure that there were no potential downstream interactions that would prevent stem-loop formation, the secondary structure of the 5'UTR was also predicted and found to have no major deviation.

Figure 2. Effect of base pair sequence on the reactivity of RNase III to μR1.1 RNA cleavage. The diagram shows the sequence and proposed secondary structure of μR1.1 RNA. The arrow indicates the RNase III cleavage site, and the pb and db are also indicated. The numbers to the left of the two boxes refer to the specific subsites within each box. On the right are shown the base pair substitutions. The relative reactivity is provided below each substitution, and represents the average of three experiments, with a standard error of the mean of ±15%. (Pertzev & Nicholson, 2006)

Thermodynamic details of BBa_K5280410: ΔG = -7.6 kcal/mol ΔH = -68.0 kcal/mol ΔS = -199.9 Tm = 31.3°C

Figure 3. Absorbance Plot on the left and the secondary structure of BBa_K5280410 on the right.

Characterization

Due to our experimental schedule problems, we were not able to complete the measurement of BBa_K5280410 performance. Relevant data can be found in the predicted results above, but experiments are still needed to prove their reliability.

References

• Hoynes-O’Connor, A., Hinman, K., Kirchner, L., & Moon, T. S. (2015). De novo design of heat-repressible RNA thermosensors in E. coli. Nucleic Acids Research, 43(12), 6166–6179. https://doi.org/10.1093/nar/gkv499
• Pertzev, A. V., & Nicholson, A. W. (2006). Characterization of RNA sequence determinants and antideterminants of processing reactivity for a minimal substrate of Escherichia coli ribonuclease III. Nucleic Acids Research, 34(13), 3708–3721. https://doi.org/10.1093/nar/gkl459