Robert T. Batey

Structure of a natural guanine-responsive riboswitch complexed with the metabolite hypoxanthine.

Riboswitches are genetic regulatory elements found in the 5′ untranslated region of messenger RNA that act in the absence of protein cofactors. They are broadly distributed across bacteria and account for the regulation of more than 2% of all genes in Bacillus subtilis, underscoring their importance in the control of cellular metabolism. The 5′ untranslated region of many mRNAs of genes involved in purine metabolism and transport contain a guanine-responsive riboswitch that directly binds guanine, hypoxanthine or xanthine to terminate transcription. Here we report the crystal structure at 1.95 Å resolution of the purine-binding domain of the guanine riboswitch from the xpt–pbuX operon of B. subtilis bound to hypoxanthine, a prevalent metabolite in the bacterial purine salvage pathway. This structure reveals a complex RNA fold involving several phylogenetically conserved nucleotides that create a binding pocket that almost completely envelops the ligand. Hypoxanthine functions to stabilize this structure and to promote the formation of a downstream transcriptional terminator element, thereby providing a mechanism for directly repressing gene expression in response to an increase in intracellular concentrations of metabolite.

Structure of the S-adenosylmethionine riboswitch regulatory mRNA element.

Riboswitches are cis-acting genetic regulatory elements found in the 5′-untranslated regions of messenger RNAs that control gene expression through their ability to bind small molecule metabolites directly. Regulation occurs through the interplay of two domains of the RNA: an aptamer domain that responds to intracellular metabolite concentrations and an expression platform that uses two mutually exclusive secondary structures to direct a decision-making process. In Gram-positive bacteria such as Bacillus species, riboswitches control the expression of more than 2% of all genes through their ability to respond to a diverse set of metabolites including amino acids, nucleobases and protein cofactors. Here we report the 2.9-Å resolution crystal structure of an S-adenosylmethionine (SAM)-responsive riboswitch from Thermoanaerobacter tengcongensis complexed with S-adenosylmethionine, an RNA element that controls the expression of several genes involved in sulphur and methionine metabolism. This RNA folds into a complex three-dimensional architecture that recognizes almost every functional group of the ligand through a combination of direct and indirect readout mechanisms. Ligand binding induces the formation of a series of tertiary interactions with one of the helices, serving as a communication link between the aptamer and expression platform domains.

Tertiary Motifs in RNA Structure and Folding

Specific tertiary structural motifs determine the complete architecture of RNA molecules (see picture for examples). Within the last few years a number of high-resolution crystal structures of complex RNAs have led to new insights into the mechanisms by which these complex folds are attained. In this review the structures of these tertiary motifs and how they influence the folding pathway of biological RNAs are discussed, as well as new developments in modeling RNA structure based upon these findings.

Riboswitches: Emerging Themes in RNA Structure and Function

Riboswitches are RNAs capable of binding cellular metabolites using a diverse array of secondary and tertiary structures to modulate gene expression. The recent determination of the three-dimensional structures of parts of six different riboswitches illuminates common features that allow riboswitches to be grouped into one of two types. Type I riboswitches, as exemplified by the purine riboswitch, are characterized by a single, localized binding pocket supported by a largely pre-established global fold. This arrangement limits ligand-induced conformational changes in the RNA to a small region. In contrast, Type II riboswitches, such as the thiamine pyrophosphate riboswitch, contain binding pockets split into at least two spatially distinct sites. As a result, binding induces both local changes to the binding pocket and global architecture. Similar organizational themes are found in other noncoding RNAs, making it possible to begin to build a hierarchical classification of RNA structure based on the spatial organization of their active sites and associated secondary structural elements.

A universal mode of helix packing in RNA.

RNA molecules fold into specific three-dimensional shapes to perform structural and catalytic functions. Large RNAs can form compact globular structures, but the chemical basis for close helical packing within these molecules has been unclear. Analysis of transfer, catalysis, in vitro-selected and ribosomal RNAs reveal that helical packing predominantly involves the interaction of single-stranded adenosines with a helix minor groove. Using the Tetrahymena thermophila group I ribozyme, we show here that the near-perfect shape complementarity between the adenine base and the minor groove allows for optimal van der Waals contacts, extensive hydrogen bonding and hydrophobic surface burial, creating a highly energetically favorable interaction. Adenosine is recognized in a chemically similar fashion by a combination of protein and RNA components in the ribonucleoprotein core of the signal recognition particle. These results provide a thermodynamic explanation for the noted abundance of conserved adenosines within the unpaired regions of RNA secondary structures.

Structure of the SAM-II riboswitch bound to S -adenosylmethionine

In bacteria, numerous genes harbor regulatory elements in the 5′ untranslated regions of their mRNA, termed riboswitches, which control gene expression by binding small-molecule metabolites. These sequences influence the secondary and tertiary structure of the RNA in a ligand-dependent manner, thereby directing its transcription or translation. The crystal structure of an S-adenosylmethionine–responsive riboswitch found predominantly in proteobacteria, SAM-II, has been solved to reveal a second means by which RNA interacts with this important cellular metabolite. Notably, this is the first structure of a complete riboswitch containing all sequences associated with both the ligand binding aptamer domain and the regulatory expression platform. Chemical probing of this RNA in the absence and presence of ligand shows how the structure changes in response to S-adenosylmethionine to sequester the ribosomal binding site and affect translational gene regulation.

Crystal Structure of the Lysine Riboswitch Regulatory mRNA Element

Riboswitches are metabolite-sensitive elements found in mRNAs that control gene expression through a regulatory secondary structural switch. Along with regulation of lysine biosynthetic genes, mutations within the lysine-responsive riboswitch (L-box) play a role in the acquisition of resistance to antimicrobial lysine analogs. To understand the structural basis for lysine binding, we have determined the 2.8Å resolution crystal structure of lysine bound to the Thermotoga maritima asd lysine riboswitch ligand-binding domain. The structure reveals a complex architecture scaffolding a binding pocket completely enveloping lysine. Mutations conferring antimicrobial resistance cluster around this site as well as highly conserved long range interactions, indicating that they disrupt lysine binding or proper folding of the RNA. Comparison of the free and bound forms by x-ray crystallography, small angle x-ray scattering, and chemical probing reveals almost identical structures, indicating that lysine induces only limited and local conformational changes upon binding.

[13] Preparation of isotopically enriched RNAs for heteronuclear NMR

Publisher Summary This chapter discusses a detailed procedure for the production of 13 C- and/or 15 N-labeled RNA from inexpensive and readily available sources of these isotopes. The ability to overexpress proteins, isotopically labeled with 13 C and 15 N, is a cornerstone of the current nuclear magnetic resonance (NMR) methodology used to solve solution structures of large proteins. Recently, similar heteronuclear NMR techniques have been applied to the RNA structures in solution. A number of laboratories have developed techniques for the synthesis of isotopically labeled ribonucleotide triphosphates as precursors for the preparation of any RNA of defined sequence and for the efficient assignment of labeled RNAs. Bacterial cells are grown in a minimal salts medium, containing isotopically labeled carbon and/or nitrogen substrates. E. coli are grown to produce 15 N-labeled nucleotides, whereas 13 C-labeled or 13 C/ 15 N nucleotides are best produced, by growing Methylophilus methylotrophus on 13 C-methanol that is an economical source of 13 C. The cells are harvested and lysed by detergent, the proteins are removed by phenol-chloroform extraction, and the total nucleic acids are precipitated with isopropanol. The boronate chromatography procedure allows one to quantitatively and reproducibly separate deoxyribonucleotides from ribonucleotides. This chromatography, however, must be performed carefully if one is to achieve these results.

B12 cofactors directly stabilize an mRNA regulatory switch

Structures of riboswitch receptor domains bound to their effector have shown how messenger RNAs recognize diverse small molecules, but mechanistic details linking the structures to the regulation of gene expression remain elusive. To address this, here we solve crystal structures of two different classes of cobalamin (vitamin B12)-binding riboswitches that include the structural switch of the downstream regulatory domain. These classes share a common cobalamin-binding core, but use distinct peripheral extensions to recognize different B12 derivatives. In each case, recognition is accomplished through shape complementarity between the RNA and cobalamin, with relatively few hydrogen bonding interactions that typically govern RNA–small molecule recognition. We show that a composite cobalamin–RNA scaffold stabilizes an unusual long-range intramolecular kissing-loop interaction that controls mRNA expression. This is the first, to our knowledge, riboswitch crystal structure detailing how the receptor and regulatory domains communicate in a ligand-dependent fashion to regulate mRNA expression.

The structure of a tetrahydrofolate sensing riboswitch reveals two ligand binding sites in a single aptamer

Transport and biosynthesis of folate and its derivatives are frequently controlled by the tetrahydrofolate (THF) riboswitch in Firmicutes. We have solved the crystal structure of the THF riboswitch aptamer in complex with folinic acid, a THF analog. Uniquely, this structure reveals two molecules of folinic acid binding to a single structured domain. These two sites interact with ligand in a similar fashion, primarily through recognition of the reduced pterin moiety. 7-deazaguanine, a soluble analog of guanine, binds the riboswitch with nearly the same affinity as its natural effector. However, 7-deazaguanine effects transcriptional termination to a substantially lesser degree than folinic acid, suggesting that the cellular guanine pool does not act upon the THF riboswitch. Under physiological conditions the ligands display strong cooperative binding, with one of the two sites playing a greater role in eliciting the regulatory response, which suggests that the second site may play another functional role.