Structural Biology of RNA and Atomic Coordinates
We wish to understand how RNA can adopt complex three-dimensional structures, how RNA can bind small-molecule ligands with high affinity and specificity, how small-molecule binding leads to allosteric conformational change of RNAs, and how all-RNA active sites can catalyze biochemical transformations. We are also interested in learning how proteins recognize RNA structure, and how proteins can facilitate RNA folding. We study three types of experimental systems: (1) catalytic RNAs, (2) riboswitches, and (3) protein enzymes and ribonucleoprotein complexes (RNPs) that modify RNA.
Catalytic RNA: the hairpin ribozyme The 2.4 Å-resolution crystal structure of a hairpin ribozyme in complex with an inhibitor that mimics the precursor to the cleavage reaction demonstrates that this catalytic RNA aligns the 2'-OH nucleophile and 5'-oxo leaving groups by splaying apart the nucleotides flanking the scissile phosphate.
The splayed substrate conformation is strikingly similar to that employed by ribonuclease A a protein enzyme that catalyzes the same reaction. Thus, we have the remarkable convergent evolution of a protein enzyme and a catalytic RNA.
Rupert, P.B. & Ferré-D'Amaré, A.R. Crystal structure of a hairpin ribozyme-inhibitor complex with implications for catalysis. Nature 410:780-786 (2001). PDB accession number 1M5K reprint supplementary info
We employed vanadate in order to mimic the transition state of the hairpin ribozyme (a 5'-Chloro RNA complex served as a control). Comparison of the transition state mimic structure with the ribozyme-inhibitor complex (that mimics the precursor structure) and a product complex structure suggests how the hairpin ribozyme employs preferential binding to the transition state as part or its catalytic mechanism.
Rupert, P.B. Massey, A. Sigurdsson, S.Th. & Ferré-D'Amaré, A.R. Transition state stabilization by a catalytic RNA Science 298:1421-1424 (2002). PDB accession numbers 1M5O 1M5V 1M5P reprint
Riboswitches: small molecule recognition and the regulation of gene expression by RNA The thi-box is an RNA domain that regulates gene expression in archaea, bacteria and eukaryotes in response to intracellular concentrations of thiamine pyrophosphate (TPP). Binding of TPP leads to a conformational change in the thi-box, which in turn leads to changes in gene expression at the transcriptional, translational or post-transcriptional levels. Remarkable for a polyanion, the thi-box binds TPP preferentially over the less negatively-charged thiamine monophosphate (TMP), or the positively charged thiamine. To understand the chemical basis of specificity, and to provide a residue-by-residue description of the conformational changes that accompany bindig of small molecules by the thi-box, we have solved its structure in complex with TPP, TMP, and the synthetic compounds benfotiamine and pyrithiamine. Comparison of our structures shows how the riboswitch adaptively recognizes different compounds, and how a disorder-to-order transition accompanies high-affinity binding.
Edwards, T.E. & Ferré-D'Amaré, A.R. Crystal structures of the thi-box riboswitch bound to thiamine pyrophosphate analogs reveal adaptive RNA-small molecule recognition. Structure 14:1459-1468 (2006). PDB accession numbers 2HOJ 2HOM 2HOO 2HOP
The glmS ribozyme-riboswitch: how RNA employs a coenzyme The glmS ribozyme-ribswich is a gene-regulatory RNA that is widespread among Gram-positive bacteria, such as Bacillus, Listeria, and Staphylococcus. This RNA is part of the 5'-untranslated region of the mRNA that encodes the protein responsible for synthesis of glucosamine-6-phosphate (GlcN6P), a key metabolic precursor to the bacterial cell wall. Binding of GlcN6P to the glmS ribozyme-riboswitch activates cleavage of the mRNA by this catalytic RNA. Cleavage results in reduced synthesis of the enzyme encoded by the mRNA, and thereby regulates production of GlcN6P through negative feedback.
Formally, GlcN6P could activate the glmS ribozyme allosterically, or by serving as a coenzyme. Structure determination of the glmS ribozyme in the precleavage and post-cleavage forms, as well as bound to the competitive inhibitor Glc6P demonstrates that this is not an allosteric RNA. Instead the activator functions as a coenzyme by providing a catalytically essential amine group to the pre-formed active site of the ribozyme.
Because the glmS ribozyme is highly conserved in Gram-positve bacteria but is absent from eukaryotes, because it has evolved to bind to a small molecule activator, and because it controls a key step in the biosynthesis of the bacterial cell wall, it constitutes an attractive target for the discovery and design of novel antibiotics.
Klein, D.J. & Ferré-D'Amaré, A.R. Structural basis of glmS ribozyme activation by glucosamine-6-phosphate. Science 313:1752-1756 (2006). PDB accession numbers 2GCS 2H0Z 2GCV
Klein, D.J. Wilkinson, S.R. Been, M.D. & Ferré-D'Amaré, A.R. Requirement of helix P2.2 and nucleotide G1 for positioning the cleavage site and cofactor of the glmS ribozyme. J. Mol. Biol. 373:178-189 (2007). PDB accession numbers 2Z74 2Z75
Klein, D.J. Been, M.D. & Ferré-D'Amaré, A.R. Essential role of an active-site guanine in glmS ribozyme catalysis. J. Am. Chem. Soc. 129:14858-14859 (2007). PDB accession numbers 3B4A 3B4B 3B4C
Protein-RNA recognition: pseudouridine synthases and the box H/ACA RNP The box H/ACA RNP is an RNA- protein complex conserved from archaea to human. It is responsible for pseudouridylation of ribosomal, spliceosomal and nucleolar RNAs. Pseudouridylation is the most common post-transcriptional modification of cellular RNAs. The box H/ACA RNP is also responsible for directing correct processing of pre-ribosmal RNA, and it is also an essential architectural component of vertebrate telomerases. The H/ACA RNP comprises a guide RNA and four proteins. We have reconstituted the particle in vitro from recombinant proteins, and have undertaken structural characterization of the intact RNP as well as of subcomplexes. Thus far, we have reported the structures of the K-turn binding protein L7Ae bound to its cognate site in an H/ACA RNA, and of the two core proteins Cbf5 and Nop10 associated with each other. The latter structure shows how Nop10 buttresses the catalytic site of the pseudouridine synthase Cbf5, and suggests that the Cbf5-Nop10 complex functions as a molecular bracket that organizes the multiple helices of the H/ACA and telomerase RNAs. We are collaborating with Gabriele Varani's laboratory (University of Washington) to analyze box H/ACA complex formation in solution by NMR.
Numerous pseudouridine synthases function as single polypeptides rather than being part of an elaborate RNP. We are studying several such "stand-alone" pseudouridine synthases to understand both how they recognize their RNA substrates and how they carry out catalysis. We determined the first structure of a pesudouridine synthase bound to RNA. The cocrystal structure of TruB bound to a substrate reveled that the enzyme recognizes the shape of the T-loop of tRNAs, and that it accesses its site of modification through base-flipping. The structure suggested how this enzyme could function as an RNA chaperone. In collaboration with Eugene Mueller's laboratory (University of Delaware) we are further characterizing both RNA recognition and catalysis by these ancient enzymes. We are also carrying out structural studies of highly divergent pseudouridyne synthases, such as TruD.
Hoang, C. & Ferré-D'Amaré, A.R. Cocrystal structure of a tRNA Psi55 pseudouridine synthase: nucleotide flipping by an RNA-modifying enzyme. Cell 107:929-939 (2001). PDB accession number 1K8W reprint
Hamma, T. & Ferré-D'Amaré, A.R. Structure of protein L7Ae bound to a K-turn derived from an archaeal box H/ACA sRNA at 1.8 Å resolution. Structure 12:893-903 (2004). PDB accession number 1SDS
Hoang, C. & Ferré-D'Amaré, A.R. Crystal structure of the highly divergent pseudouridine synthase TruD reveals a circular permutation of a conserved fold RNA 10:1026-1033 (2004). PDB accession number 1SB7
Hoang, C. Hamilton, C.S. Mueller, E.G. & Ferré-D'Amaré, A.R. Precursor complex structure of pseudouridine synthase TruB suggests coupling of active site perturbations to an RNA-sequestering peripheral protein domain Protein Sci. 14:2201-2206 (2005). PDB accession number 1ZL3
Hamma, T. Reichow, S.L. Varani, G. & Ferré-D'Amaré, A.R. The Cbf5-Nop10 complex is a molecular bracket that organizes box H/ACA RNPs Nature Struct. Mol. Biol. 12:1101-1107 (2005). PDB accession number 2APO
Hoang, C. Chen, J. Vizthum, C.A. Kandel, J.M. Hamilton, C.S. Mueller, E.G. & Ferré-D'Amaré, A.R. Crystal structure of pseudouridine synthase RluA: indirect sequence readout through protein-induced RNA structure Molecular Cell 24:535-545 (2006). PDB accession number 2I82
