۱- Introduction

۲- Materials and methods

۳- Results

۴- Discussion

References

Abstract

Ribonuclease P (RNase P) is an RNA processing enzyme essential for production of functional tRNAs. Bacterial RNase P is a ribozyme, i.e., an RNA-based enzyme, which functions in all bacteria including those growing at high temperatures (≥۵۵ °C). We examined three bacterial RNase P ribozymes, one from a mesophilic bacterium and two from thermophilic bacteria, to understand the factor(s) providing efficient catalytic ability under conditions of high temperature. Thermophilic RNase P ribozymes show structural adaptations to allow correct folding at high temperature. The presence of a molecular crowder significantly enhanced the catalytic efficiency of thermophilic RNase P ribozyme reactions at 55 °C, while it modestly reduced the upper limit of the reaction temperature.

Introduction

Ribonuclease P (RNase P) is a class of RNA processing enzymes, the primary role of which is to remove 50 leader sequences from precursor tRNAs (Fig. 1A) [1]. Bacterial RNase P enzymes are commonly composed of one large RNA (typically ca. 300e400 nucleotides) and one small basic protein (ca. 120 amino acids). Bacterial RNase P is classed as a ribozyme because its catalytic ability originates from its RNA component, which can promote the sitespecific cleavage of pre-tRNAs in vitro without the protein component [1,2]. Bacterial RNase P ribozymes have a modular architecture consisting of two structural domains [1,3,4]: the Cdomain (catalytic domain) containing elements essential for catalysis, and the S-domain (specificity domain) that plays an important role in the specific recognition of pre-tRNA substrates (Fig. 1B) [5,6]. As cleavage of the 50 leader sequence is an essential step in production of functional tRNAs, RNase P ribozymes have to function in all bacterial species growing under various environmental conditions, including various growth temperatures. The RNase P ribozyme is also considered to have emerged at an early stage in the molecular evolution of life and to have functioned in the last universal common ancestor (LUCA) [7], which was proposed to have lived in hydrothermal vents [8]. Bacteria in several phylums, such as Aquificales, Firmicutes, Thermotogae, and DeinococcuseThermus, are known to grow under conditions of high temperature (65 Ce90 C). They also contain RNase P ribozymes [9e12], which need to be active at their growth temperature. The RNA components of these ribozymes may have adaptations for the high temperature growth conditions, and may require the assistance of external factors including their protein components, other intracellular macromolecules that provide molecular crowding conditions, and inorganic/organic cations other than Mg2þ. Thermophilic RNase P ribozymes from thermophilic bacteria are also interesting from the viewpoint of the RNA world hypothesis because it has been proposed that the RNA world may have emerged at hydrothermal vents [13,14].