Dramatic Improvement of Crystals of Large RNAs by Cation Replacement and Dehydration

SummaryCompared to globular proteins, RNAs with complex 3D folds are characterized by poorly differentiated molecular surfaces dominated by backbone phosphates, sparse tertiary contacts stabilizing global architecture, and conformational flexibility. The resulting generally poor order of crystals of large RNAs and their complexes frequently hampers crystallographic structure determination. We describe and rationalize a postcrystallization treatment strategy that exploits the importance of solvation and counterions for RNA folding. Replacement of Li+ and Mg2+ needed for growth of crystals of a tRNA-riboswitch-protein complex with Sr2+, coupled with dehydration, dramatically improved the resolution limit (8.5–3.2 Å) and data quality, enabling structure determination. The soft Sr2+ ion forms numerous stabilizing intermolecular contacts. Comparison of pre- and posttreatment structures reveals how RNA assemblies redistribute as quasi-rigid bodies to yield improved crystal packing. Cation exchange complements previously reported postcrystallization dehydration of protein crystals and represents a potentially general strategy for improving crystals of large RNAs

The Structure and Function of Small Nucleolar Ribonucleoproteins

Eukaryotes and archaea use two sets of specialized ribonucleoproteins (RNPs) to carry out sequencespecific methylation and pseudouridylation of RNA, the two most abundant types of modifications of cellular RNAs. In eukaryotes, these protein–RNA complexes localize to the nucleolus and are called small nucleolar RNPs (snoRNPs), while in archaea they are known as small RNPs (sRNP). The C/D class of sno(s)RNPs carries out ribose- 20-O-methylation, while the H/ACA class is responsible for pseudouridylation of their RNA targets. Here, we review the recent advances in the structure, assembly and function of the conserved C/D and H/ACA sno(s)RNPs. Structures of each of the core archaeal sRNP proteins have been determined and their assembly pathways delineated. Furthermore, the recent structure of an H/ACA complex has revealed the organization of a complete sRNP. Combined with current biochemical data, these structures offer insight into the highly homologous eukaryotic snoRNPs

Structure and functional reselection of the Mango-III fluorogenic RNA aptamer

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Crystal structure of a DNA containing the planar, phenoxazine-derived bi-functional spectroscopic probe Ç

Previously, we developed the deoxycytosine analog Ç (C-spin) as a bi-functional spectroscopic probe for the study of nucleic acid structure and dynamics using electron paramagnetic resonance (EPR) and fluorescence spectroscopy. To understand the effect of Ç on nucleic acid structure, we undertook a detailed crystallographic analysis. A 1.7 Å resolution crystal structure of Ç within a decamer duplex A-form DNA confirmed that Ç forms a non-perturbing base pair with deoxyguanosine, as designed. In the context of double-stranded DNA Ç adopted a planar conformation. In contrast, a crystal structure of the free spin-labeled base ç displayed a ∼20° bend at the oxazine linkage. Density function theory calculations revealed that the bent and planar conformations are close in energy and exhibit the same frequency for bending. These results indicate a small degree of flexibility around the oxazine linkage, which may be a consequence of the antiaromaticity of a 16-π electron ring system. Within DNA, the amplitude of the bending motion is restricted, presumably due to base-stacking interactions. This structural analysis shows that the Ç forms a planar, structurally non-perturbing base pair with G indicating it can be used with high confidence in EPR- or fluorescence-based structural and dynamics studies

Modulating RNA structure and catalysis: lessons from small cleaving ribozymes

RNA is a key molecule in life, and comprehending its structure/function relationships is a crucial step towards a more complete understanding of molecular biology. Even though most of the information required for their correct folding is contained in their primary sequences, we are as yet unable to accurately predict both the folding pathways and active tertiary structures of RNA species. Ribozymes are interesting molecules to study when addressing these questions because any modifications in their structures are often reflected in their catalytic properties. The recent progress in the study of the structures, the folding pathways and the modulation of the small ribozymes derived from natural, self-cleaving, RNA motifs have significantly contributed to today’s knowledge in the field

Many Activities, One Structure: Functional Plasticity of Ribozyme Folds

Catalytic RNAs, or ribozymes, are involved in a number of essential biological processes, such as replication of RNA genomes and mobile genetic elements, RNA splicing, translation, and RNA degradation. The function of ribozymes requires the formation of active sites decorated with RNA functional groups within defined three-dimensional (3D) structures. The genotype (sequence) of RNAs ultimately determines what 3D structures they adopt (as a function of their environmental conditions). These 3D structures, in turn, give rise to biochemical activity, which can further elaborate them by catalytic rearrangements or association with other molecules. The fitness landscape of a non-periodic linear polymer, such as RNA, relates its primary structure to a phenotype. Two major challenges in the analysis of ribozymes is to map all possible genotypes to their corresponding catalytic activity (that is, to determine their fitness landscape experimentally), and to understand whether their genotypes and three-dimensional structures can support multiple different catalytic functions. Recently, the combined results of experiments that employ in vitro evolution methods, high-throughput sequencing and crystallographic structure determination have hinted at answers to these two questions: while the fitness landscape of ribozymes is rugged, meaning that their catalytic activity cannot be optimized by a smooth trajectory in sequence space, once an RNA achieves a stable three-dimensional fold, it can be endowed with distinctly different biochemical activities through small changes in genotype. This functional plasticity of highly structured RNAs may be particularly advantageous for the adaptation of organisms to drastic changes in selective pressure, or for the development of new biotechnological tools

The tRNA Elbow in Structure, Recognition and Evolution

Prominent in the L-shaped three-dimensional structure of tRNAs is the “elbow” where their two orthogonal helical stacks meet. It has a conserved structure arising from the interaction of the terminal loops of the D- and T-stem-loops, and presents to solution a flat face of a tertiary base pair between the D- and T-loops. In addition to the ribosome, which interacts with the elbow in all three of its tRNA binding sites, several cellular RNAs and many proteins are known to recognize the elbow. At least three classes of non-coding RNAs, namely 23S rRNA, ribonuclease P, and the T-box riboswitches, recognize the tRNA elbow employing an identical structural motif consisting of two interdigitated T-loops. In contrast, structural solutions to tRNA-elbow recognition by proteins are varied. Some enzymes responsible for post-transcriptional tRNA modification even disrupt the elbow structure in order to access their substrate nucleotides. The evolutionary origin of the elbow is mysterious, but, because it does not explicitly participate in the flow of genetic information, it has been proposed to be a late innovation. Regardless, it is biologically essential. Even some viruses that hijack the cellular machinery using tRNA decoys have convergently evolved near-perfect mimics of the tRNA elbow