The discovery of a catalytic RNA within RNase P and its legacy.

Sidney Altman's discovery of the processing of one RNA by another RNA that acts like an enzyme was revolutionary in biology and the basis for his sharing the 1989 Nobel Prize in Chemistry with Thomas Cech. These breakthrough findings support the key role of RNA in molecular evolution, where replicating RNAs (and similar chemical derivatives) either with or without peptides functioned in protocells during the early stages of life on Earth, an era referred to as the RNA world. Here, we cover the historical background highlighting the work of Altman and his colleagues and the subsequent efforts of other researchers to understand the biological function of RNase P and its catalytic RNA subunit and to employ it as a tool to downregulate gene expression. We primarily discuss bacterial RNase P-related studies but acknowledge that many groups have significantly contributed to our understanding of archaeal and eukaryotic RNase P, as reviewed in this special issue and elsewhere.

Trials, travails and triumphs: an account of RNA catalysis in RNase P.

Last December marked the 20th anniversary of the Nobel Prize in Chemistry to Sidney Altman and Thomas Cech for their discovery of RNA catalysts in bacterial ribonuclease P (an enzyme catalyzing 5' maturation of tRNAs) and a self-splicing rRNA of Tetrahymena, respectively. Coinciding with the publication of a treatise on RNase P, this review provides a historical narrative, a brief report on our current knowledge, and a discussion of some research prospects on RNase P.

Surprising contribution to aminoacylation and translation of non-Watson-Crick pairs in tRNA.

Molecules of transfer RNA (tRNA) typically contain four stems composed of Watson-Crick (W-C) base pairs and infrequent mispairs such as G-U and A-C. The latter mispairs are fundamental units of RNA secondary structure found in nearly every class of RNA and are nearly isomorphic to W-C pairs. Therefore, they often substitute for G-C or A-U base pairs. The mispairs also have unique chemical, structural, and dynamic conformational properties, which can only be partially mimicked by W-C base pairs. Here, I characterize the identities and tasks of six mutant G-U and A-C mispairs in Escherichia coli tRNA(Gly) using genetic and bioinformatic tools and show that mispairs are significantly more important for aminoacylation and translation than previously realized. Mispairs boost aminoacylation and translation primarily because they activate tRNA by means of their conformational flexibility. The statistical preservation of the six mutant mispair sites across tRNA(Gly) in many organisms points to a fundamental structure-function signature within tRNA(Gly) with possible analogous missions in other RNAs.

Structure-function analysis of tRNA(Gln) in an Escherichia coli knockout strain.

The diverse and highly specific interaction between RNAs and proteins plays an essential role in many important biological processes. In the glutamine aminoacylation system, crystal structures of the free and ligated macromolecules have provided a description of the tRNA-protein interactions at the molecular level. This data lays the foundation for genetic, biochemical, and structural analyses to delineate the set of key interactions that governs the structure-function relationships of the two macromolecules. To this end the chromosomal tRNA(Gln) genes were disrupted in Escherichia coli to produce a tRNA(Gln) knockout strain that depends upon expression of a functional tRNA(Gln) from a plasmid for cell viability. Mutants of an inactive tester tRNA derived from tRNA(Ala) were generated by hydroxylamine mutagenesis, and the active derivatives were selected by their ability to support knockout cell growth. Two of the mutants contained substitutions in the first base pair of the acceptor stem that likely facilitate the formation of a hairpin loop that places A76 in the active site. The third mutation was located at position 13 in the D loop region of the tRNA, and suggests that an interaction with residue 13 contributes to a specific conformational change in unliganded GlnRS, which helps configure the enzyme active site in its catalytically proficient form. This work demonstrates the efficacy of an integrated approach that combines genetic selections and biochemical analyses with the physical data from crystal structures to reveal molecular steps that control the specificity of RNA-protein interactions.

Aptamer redesigned tRNA is nonfunctional and degraded in cells.

An RNA aptamer derived from tRNA(Gln) isolated in vitro and a rationally redesigned tRNA(Gln) were used to address the relationship between structure and function of tRNA(Gln) aminoacylation in Escherichia coli. Two mutant tRNA(Gln) sequences were studied: an aptamer that binds 26-fold tighter to glutaminyl-tRNA synthetase than wild-type tRNA(Gln) in vitro, redesigned in the variable loop, and a mutant with near-normal aminoacylation kinetics for glutamine, redesigned to contain a long variable arm. Both mutants were tested in a tRNA(Gln) knockout strain of E. coli, but neither supported knockout cell growth. It was later found that both mutant tRNAs were present in very low amounts in the cell. These results reveal the difference between in vitro and in vivo studies, demonstrating the complexities of in vivo systems that have not been replicated in vitro.

Recognition of acceptor-stem structure of tRNA(Asp) by Escherichia coli aspartyl-tRNA synthetase.

Protein-RNA recognition between aminoacyl-tRNA synthetases and tRNA is highly specific and essential for cell viability. We investigated the structure-function relationships involved in the interaction of the Escherichia coli tRNA(Asp) acceptor stem with aspartyl-tRNA synthetase. The goal was to isolate functionally active mutants and interpret them in terms of the crystal structure of the synthetase-tRNA(Asp) complex. Mutants were derived from Saccharomyces cerevisiae tRNA(Asp), which is inactive with E. coli aspartyl-tRNA synthetase, allowing a genetic selection of active tRNAs in a tRNA(Asp) knockout strain of E. coli. The mutants were obtained by directed mutagenesis or library selections that targeted the acceptor stem of the yeast tRNA(Asp) gene. The mutants provide a rich source of tRNA(Asp) sequences, which show that the sequence of the acceptor stem can be extensively altered while allowing the tRNA to retain substantial aminoacylation and cell-growth functions. The predominance of tRNA backbone-mediated interactions observed between the synthetase and the acceptor stem of the tRNA in the crystal and the mutability of the acceptor stem suggest that many of the corresponding wild-type bases are replaceable by alternative sequences, so long as they preserve the initial backbone structure of the tRNA. Backbone interactions emerge as an important functional component of the tRNA-synthetase interaction.

Genetic perturbations of RNA reveal structure-based recognition in protein-RNA interaction.

Protein-RNA recognition is an essential foundation of cellular processes, yet much remains unknown about these important interactions. The recognition between aminoacyl-tRNA synthetases and their cognate tRNA substrates is highly specific and essential for cell viability, due to the necessity for accurate translation of the genetic code into protein sequences. We selected an active tRNA that is highly mutated in the recognition nucleotides of the acceptor stem region in the alanine system. The functional properties of this mutant and its secondary derivatives demonstrate that recognition cannot be reduced to isolated structural elements, but rather the amino acid acceptor stem is being recognized as a unit.