Part:BBa_K5280428

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

This design is inspired by the cold-repressible RNA-based thermosensor (BBa_K2541201-BBa_K2541210) developed by iGEM18_Jilin_China, with the aim of enhancing its functionality.

Usage and Biology

The RNA thermosensor is a non-coding RNA molecule that regulates gene expression in response to temperature changes, primarily observed in prokaryotes. Generally, RNA thermosensors respond to temperature fluctuations through subtle changes in their secondary structures. Such structural transitions can either expose or occlude some important regions of the RNA, such as ribosome binding sites, thereby influencing the translation rate of nearby protein-coding genes.

While naturally occurring RNA thermosensors are prevalent in nature, their implementation in engineered systems is often complicated by the complexity of their mechanisms. The RNA thermosensor mechanism developed here is straightforward and readily applicable to synthetic biology. At low temperatures, the hairpin structure creates a cleavage site for the natural ribonuclease RNase III within the 5' untranslated region (UTR) of the target mRNA; conversely, at elevated temperatures, the hairpin unwinds, the cleavage site disappears, and expression is turned on.

Figure 1. Mechanism of RNA thermosensors. (Created with biorender.com).

Design

The design of the RNA thermosensors started with the RNase III recognition site (distal box, db) and cleavage site (proximal box, pb) identified in the literature. Adjust other bases in the stem-loop to achieve the predicted melting temperature (Tm) within the range of 25–35°C. Changes in nucleotide sequence also affect the catalytic efficiency of RNase III. Furthermore, increasing the length of the stem can optimize the RNA thermosensors for higher temperatures, while reducing the stem length has the opposite effect.

Following the design phase, the structural and thermodynamic parameters of the thermosensors were evaluated using the Mfold web server. 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 on DINAMelt and simulated with its Absorbance Plot. This application predicts the unwinding temperature of two independent RNA strands, treating the thermosensor as two separate chains. It is important to note that this approximation does not account for the influence of loop size.

The thermosensors ranging from BBa_K5280427 to BBa_K5280431 all contain two stem loops derived from existing thermosensors with the aim of enhancing their reaction efficiency with RNase III. The part BBa_K5280428 replicates and doubles the stem loop structure of BBa_K5280412.

Figure 2. Effect of base pair sequence on the catalytic efficiency of RNase III. pb and db are shown in the figure. Arrows indicate the cutting sites of RNase III. Base pair substitutions are shown on the right. Relative reactivity is labeled below each substitution, and the standard error of the mean is ±15%. (Pertzev and Nicholson, 2006).

Figure 3. The secondary structure of BBa_K5280428.

Characterization

We conducted a preliminary characterization of several thermosensors to assess their ability to regulate the expression of downstream genes.

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. We used Abs700 to represent cell density, as mCherry has significant absorption around 600 nm.

Figure 4. The values of Fluorescence/OD700 of BBa_K5280428 with mCherry incubated at 25°C (purple), 29°C (blue), 33°C (green), and 37°C (orange).

Divide the fluorescence by the OD700 to provide an approximate “per cell” fluorescence measurement. Figure 4 demonstrates the change in fluorescence intensity of BBa_K5280428 with temperature.

Reference

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

Zuker, M. (2003). Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Research, 31(13), 3406–3415. https://doi.org/10.1093/nar/gkg595

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]