Ligand-dependent tRNA processing by a rationally designed RNase P riboswitch

Abstract

We describe a synthetic riboswitch element that implements a regulatory principle which directly addresses an essential tRNA maturation step. Constructed using a rational in silico design approach, this riboswitch regulates RNase P-catalyzed tRNA 5′-processing by either sequestering or exposing the single-stranded 5′-leader region of the tRNA precursor in response to a ligand. A single base pair in the 5′-leader defines the regulatory potential of the riboswitch both in vitro and in vivo. Our data provide proof for prior postulates on the importance of the structure of the leader region for tRNA maturation. We demonstrate that computational predictions of ligand-dependent structural rearrangements can address individual maturation steps of stable non-coding RNAs, thus making them amenable as promising target for regulatory devices that can be used as functional building blocks in synthetic biology.

Synthetic riboswitches for the analysis of tRNA processing by eukaryotic RNase P enzymes

“…In a previous study, we successfully generated RNase P riboswitch constructs through computational predictions (Ender et al 2021). These riboswitches consist of the theophylline-dependent TCT8 aptamer (Jenison et al 1994) overlapping with a designed expression platform (Fig.…”

“…For the latter ones, computational folding predictions were combined with experimental characterization by Wachsmuth et al (2013Wachsmuth et al ( , 2015. Based on the most efficient transcription regulating riboswitch from that work (RS10), new synthetic riboswitches were generated that control RNase P-dependent 5 ′ -maturation of a tRNA (Ender et al 2021).…”

“…While several studies focus on the role of the tRNA 5 ′ -leader sequence composition (Zahler et al 2003(Zahler et al , 2005Sun et al 2006;Niland et al 2017), only very little information is available on the impact of its structural conformation. Several lines of experiments indicate that bacterial RNase P enzymes have an increased substrate affinity to unstructured 5 ′ -leader regions (Lin et al 2016;Niland et al 2017;Ender et al 2021). For other types of RNase P, the structural status of the leader region is postulated to be important as well (Lee et al 1997;Ziehler et al 2000;Brillante et al 2016).…”

“…The recently designed RNase P riboswitches (Ender et al 2021) provide a tool to address this question. These riboswitches are based on the theophylline-binding aptamer (Jenison et al 1994), where its 3 ′ -part forms a sequester hairpin through interaction with a computationally designed sequence in the OFF state.…”

Cell-free riboswitches

“…While many synthetic riboswitches mimicking natural mechanisms have been designed in bacteria, synthetic riboswitches that function in other cells and organisms, and riboswitches based on mechanisms not found in nature have also been developed. 9,[12][13][14][15][16][17][18][19] Bottom-up or cell-free synthetic biology is emerging as a frontier in synthetic biology whose goal is to design and build biochemical or genetic networks (cell-free systems) that display complex functions without using living cells. [20][21][22] Building such cell-free systems can improve our understanding of the design principles of biological systems.…”