The crystal structure of Escherichia coli glutaminyl-tRNA synthetase (GlnRS) complexed with its cognate glutaminyl transfer RNA (tRNA(Gln] and adenosine triphosphate (ATP) has been derived from a 2.8 angstrom resolution electron density map and the known protein and tRNA sequences. The 63.4-kilodalton monomeric enzyme consists of four domains arranged to give an elongated molecule with an axial ratio greater than 3 to 1. Its interactions with the tRNA extend from the anticodon to the acceptor stem along the entire inside of the L of the tRNA. The complexed tRNA retains the overall conformation of the yeast phenylalanine tRNA (tRNA(Phe] with two major differences: the 3' acceptor strand of tRNA(Gln) makes a hairpin turn toward the inside of the L, with the disruption of the final base pair of the acceptor stem, and the anticodon loop adopts a conformation not seen in any of the previously determined tRNA structures. Specific recognition elements identified so far include (i) enzyme contacts with the 2-amino groups of guanine via the tRNA minor groove in the acceptor stem at G2 and G3; (ii) interactions between the enzyme and the anticodon nucleotides; and (iii) the ability of the nucleotides G73 and U1.A72 of the cognate tRNA to assume a conformation stabilized by the protein at a lower free energy cost than noncognate sequences. The central domain of this synthetase binds ATP, glutamine, and the acceptor end of the tRNA as well as making specific interactions with the acceptor stem.2+t is

http://science.sciencemag.org/content/sci/246/4934/1135.full.pdf

Structure of E. coli glutaminyl-tRNA synthetase complexed with tRNA(Gln) and ATP at 2.8 A resolution

N(1)-Methyladenosine (m(1)A) is a prevalent post-transcriptional RNA modification, yet little is known about its abundance, topology and dynamics in mRNA. Here, we show that m(1)A is prevalent in Homo sapiens mRNA, which shows an m(1)A/A ratio of ∼0.02%. We develop the m(1)A-ID-seq technique, based on m(1)A immunoprecipitation and the inherent ability of m(1)A to stall reverse transcription, as a means for transcriptome-wide m(1)A profiling. m(1)A-ID-seq identifies 901 m(1)A peaks (from 600 genes) in mRNA and noncoding RNA and reveals a prominent feature, enrichment in the 5' untranslated region of mRNA transcripts, that is distinct from the pattern for N(6)-methyladenosine, the most abundant internal mammalian mRNA modification. Moreover, m(1)A in mRNA is reversible by ALKBH3, a known DNA/RNA demethylase. Lastly, we show that m(1)A methylation responds dynamically to stimuli, and we identify hundreds of stress-induced m(1)A sites. Collectively, our approaches allow comprehensive analysis of m(1)A modification and provide tools for functional studies of potential epigenetic regulation via the reversible and dynamic m(1)A methylation.

Transcriptome-wide mapping reveals reversible and dynamic N1-methyladenosine methylome

Crystallographic refinement of yeast aspartic acid transfer RNA.

In recent years, major breakthroughs in RNA-modification-mediated regulation of gene expression have been made, leading to the emerging field of epitranscriptomics.Our understanding of the distribution, regulation and function of these dynamic RNA modifications is based on sequencing technologies. In this Review, we focus on the major mRNA modifications in the transcriptome of eukaryotic cells: N6-methyladenosine, N6, 2'-O-dimethyladenosine, 5-methylcytidine, 5-hydroxylmethylcytidine, inosine, pseudouridine and N1-methyladenosine. We discuss the sequencing technologies used to profile these epitranscriptomic marks, including scale, resolution, quantitative feature, pre-enrichment capability and the corresponding bioinformatics tools. We also discuss the challenges of epitranscriptome profiling and highlight the prospect of future detection tools. We aim to guide the choice of different detection methods and inspire new ideas in RNA biology.

Epitranscriptome sequencing technologies: decoding RNA modifications

The structure of vitamin D-dependent calcium-binding protein from bovine intestine, determined crystallographically to 2.3 A, contains a pair of calcium-binding domains similar to those in parvalbumin, but one domain has a longer and rearranged calcium-binding loop. This result has implications for structure predictions of other calcium-binding proteins such as calmodulin and S-100.

Structure of vitamin D-dependent calcium-binding protein from bovine intestine.

OUR understanding of the molecular structure–function relationship in tRNA rests mainly on three types of information. First, on the common sequence patterns which have emerged from careful examination of many primary structures1–3; second, a wide variety of spectral and other physical and chemical results must be accounted for by the molecular structure4–6; and third, there is the detailed image of the yeast tRNAPhe molecule independently determined and refined from two different—albeit similar—crystal forms7–10. It is also clear, however, that the molecular model deduced from the yeast tRNAphe crystal structure cannot be easily reconciled with all structural requirements for function and is best considered a well-defined and stable canonical form of tRNA which is packed in an unusually well-ordered way in specific crystal lattices. Notwithstanding the enormous value of this canonical form in explaining the basic architectural features of tRNA, it is clearly important to image other crystalline tRNAs; particularly tRNAs that exhibit different functions (such as, initiators) or have significantly different covalent structures (for example, class III tRNAs)1 or those that crystallise in different solvent conditions. We report here the initial results of the crystal structure determination of a eukaryotic initiator tRNA crystallised from a highly polar aqueous solvent11,12. Its architecture is essentially the same as crystalline yeast tRNAphe, except for a small but significant difference in the position of the anticodon arm.

N. Krishnamachari

Papers

Crystal structure of a eukaryotic initiator tRNA