The conserved AU dinucleotide at the 5' end of nascent U1 snRNA is optimized for the interaction with nuclear cap-binding-complex.

Splicing is initiated by a productive interaction between the pre-mRNA and the U1 snRNP, in which a short RNA duplex is established between the 5' splice site of a pre-mRNA and the 5' end of the U1 snRNA. A long-standing puzzle has been why the AU dincucleotide at the 5'-end of the U1 snRNA is highly conserved, despite the absence of an apparent role in the formation of the duplex. To explore this conundrum, we varied this AU dinucleotide into all possible permutations and analyzed the resulting molecular consequences. This led to the unexpected findings that the AU dinucleotide dictates the optimal binding of cap-binding complex (CBC) to the 5' end of the nascent U1 snRNA, which ultimately influences the utilization of U1 snRNP in splicing. Our data also provide a structural interpretation as to why the AU dinucleotide is conserved during evolution.

Structure and function of phosphonoacetaldehyde dehydrogenase: the missing link in phosphonoacetate formation.

Phosphonates (C-PO3²â») have applications as antibiotics, herbicides, and detergents. In some environments, these molecules represent the predominant source of phosphorus, and several microbes have evolved dedicated enzymatic machineries for phosphonate degradation. For example, most common naturally occurring phosphonates can be catabolized to either phosphonoacetaldehyde or phosphonoacetate, which can then be hydrolyzed to generate inorganic phosphate and acetaldehyde or acetate, respectively. The phosphonoacetaldehyde oxidase gene (phnY) links these two hydrolytic processes and provides a previously unknown catabolic mechanism for phosphonoacetate production in the microbial metabolome. Here, we present biochemical characterization of PhnY and high-resolution crystal structures of the apo state, as well as complexes with substrate, cofactor, and product. Kinetic analysis of active site mutants demonstrates how a highly conserved aldehyde dehydrogenase active site has been modified in nature to generate activity with a phosphonate substrate.

Functions of the DExD/H-box proteins in nuclear pre-mRNA splicing.

In eukaryotes, many genes are transcribed as precursor messenger RNAs (pre-mRNAs) that contain exons and introns, the latter of which must be removed and exons ligated to form the mature mRNAs. This process is called pre-mRNA splicing, which occurs in the nucleus. Although the chemistry of pre-mRNA splicing is identical to that of the self-splicing Group II introns, hundreds of proteins and five small nuclear RNAs (snRNAs), U1, U2, U4, U5, and U6, are essential for executing pre-mRNA splicing. Spliceosome, arguably the most complex cellular machine made up of all those proteins and snRNAs, is responsible for carrying out pre-mRNA splicing. In contrast to the transcription and the translation machineries, spliceosome is formed anew onto each pre-mRNA and undergoes a series of highly coordinated reconfigurations to form the catalytic center. This amazing process is orchestrated by a number of DExD/H-proteins that are the focus of this article, which aims to review the field in general and to project the exciting challenges and opportunities ahead. This article is part of a Special Issue entitled: The Biology of RNA helicases - Modulation for life.

Structures of the PelD cyclic diguanylate effector involved in pellicle formation in Pseudomonas aeruginosa PAO1.

The second messenger bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) plays a vital role in the global regulation in bacteria. Here, we describe structural and biochemical characterization of a novel c-di-GMP effector PelD that is critical to the formation of pellicles by Pseudomonas aeruginosa. We present high-resolution structures of a cytosolic fragment of PelD in apo form and its complex with c-di-GMP. The structure contains a bi-domain architecture composed of a GAF domain (commonly found in cyclic nucleotide receptors) and a GGDEF domain (found in c-di-GMP synthesizing enzymes), with the latter binding to one molecule of c-di-GMP. The GGDEF domain has a degenerate active site but a conserved allosteric site (I-site), which we show binds c-di-GMP with a K(d) of 0.5 µm. We identified a series of residues that are crucial for c-di-GMP binding, and confirmed the roles of these residues through biochemical characterization of site-specific variants. The structures of PelD represent a novel class of c-di-GMP effector and expand the knowledge of scaffolds that mediate c-di-GMP recognition.

Metal binding motif in the active site of the HDV ribozyme binds divalent and monovalent ions.

The hepatitis delta virus (HDV) ribozyme uses both metal ion and nucleobase catalysis in its cleavage mechanism. A reverse G·U wobble was observed in a recent crystal structure of the precleaved state. This unusual base pair positions a Mg(2+) ion to participate in catalysis. Herein, we used molecular dynamics (MD) and X-ray crystallography to characterize the conformation and metal binding characteristics of this base pair in product and precleaved forms. Beginning with a crystal structure of the product form, we observed formation of the reverse G·U wobble during MD trajectories. We also demonstrated that this base pair is compatible with the diffraction data for the product-bound state. During MD trajectories of the product form, Na(+) ions interacted with the reverse G·U wobble in the RNA active site, and a Mg(2+) ion, introduced in certain trajectories, remained bound at this site. Beginning with a crystal structure of the precleaved form, the reverse G·U wobble with bound Mg(2+) remained intact during MD simulations. When we removed Mg(2+) from the starting precleaved structure, Na(+) ions interacted with the reverse G·U wobble. In support of the computational results, we observed competition between Na(+) and Mg(2+) in the precleaved ribozyme crystallographically. Nonlinear Poisson-Boltzmann calculations revealed a negatively charged patch near the reverse G·U wobble. This anionic pocket likely serves to bind metal ions and to help shift the pK(a) of the catalytic nucleobase, C75. Thus, the reverse G·U wobble motif serves to organize two catalytic elements, a metal ion and catalytic nucleobase, within the active site of the HDV ribozyme.

Evolution of I-SceI homing endonucleases with increased DNA recognition site specificity.

Elucidating how homing endonucleases undergo changes in recognition site specificity will facilitate efforts to engineer proteins for gene therapy applications. I-SceI is a monomeric homing endonuclease that recognizes and cleaves within an 18-bp target. It tolerates limited degeneracy in its target sequence, including substitution of a C:G(+4) base pair for the wild-type A:T(+4) base pair. Libraries encoding randomized amino acids at I-SceI residue positions that contact or are proximal to A:T(+4) were used in conjunction with a bacterial one-hybrid system to select I-SceI derivatives that bind to recognition sites containing either the A:T(+4) or the C:G(+4) base pairs. As expected, isolates encoding wild-type residues at the randomized positions were selected using either target sequence. All I-SceI proteins isolated using the C:G(+4) recognition site included small side-chain substitutions at G100 and either contained (K86R/G100T, K86R/G100S and K86R/G100C) or lacked (G100A, G100T) a K86R substitution. Interestingly, the binding affinities of the selected variants for the wild-type A:T(+4) target are 4- to 11-fold lower than that of wild-type I-SceI, whereas those for the C:G(+4) target are similar. The increased specificity of the mutant proteins is also evident in binding experiments in vivo. These differences in binding affinities account for the observed ∼36-fold difference in target preference between the K86R/G100T and wild-type proteins in DNA cleavage assays. An X-ray crystal structure of the K86R/G100T mutant protein bound to a DNA duplex containing the C:G(+4) substitution suggests how sequence specificity of a homing enzyme can increase. This biochemical and structural analysis defines one pathway by which site specificity is augmented for a homing endonuclease.

A 1.9 A crystal structure of the HDV ribozyme precleavage suggests both Lewis acid and general acid mechanisms contribute to phosphodiester cleavage.

The hepatitis delta virus (HDV) ribozyme and HDV-like ribozymes are self-cleaving RNAs found throughout all kingdoms of life. These RNAs fold into a double-nested pseudoknot structure and cleave RNA, yielding 2',3'-cyclic phosphate and 5'-hydroxyl termini. The active site nucleotide C75 has a pK(a) shifted >2 pH units toward neutrality and has been implicated as a general acid/base in the cleavage reaction. An active site Mg(2+) ion that helps activate the 2'-hydroxyl for nucleophilic attack has been characterized biochemically; however, this ion has not been visualized in any previous structures. To create a snapshot of the ribozyme in a state poised for catalysis, we have crystallized and determined the structure of the HDV ribozyme bound to an inhibitor RNA containing a deoxynucleotide at the cleavage site. This structure includes the wild-type C75 nucleotide and Mg(2+) ions, both of which are required for maximal ribozyme activity. This structure suggests that the position of C75 does not change during the cleavage reaction. A partially hydrated Mg(2+) ion is also found within the active site where it interacts with a newly resolved G.U reverse wobble. Although the inhibitor exhibits crystallographic disorder, we modeled the ribozyme-substrate complex using the conformation of the inhibitor strand observed in the hammerhead ribozyme. This model suggests that the pro-R(P) oxygen of the scissile phosphate and the 2'-hydroxyl nucleophile are inner-sphere ligands to the active site Mg(2+) ion. Thus, the HDV ribozyme may use a combination of metal ion Lewis acid and nucleobase general acid strategies to effect RNA cleavage.

Competition between Co(NH(3)(6)3+ and inner sphere Mg2+ ions in the HDV ribozyme.

Divalent cations play critical structural and functional roles in many RNAs. While the hepatitis delta virus (HDV) ribozyme can undergo self-cleavage in the presence of molar concentrations of monovalent cations, divalent cations such as Mg(2+) are required for efficient catalysis under physiological conditions. Moreover, the cleavage reaction can be inhibited with Co(NH(3))(6)(3+), an analogue of Mg(H(2)O)(6)(2+). Here, the binding of Mg(2+) and Co(NH(3))(6)(3+) to the HDV ribozyme is studied by Raman microscopic analysis of crystals. Raman difference spectra acquired at different metal ion conditions reveal changes in the ribozyme. When Mg(2+) alone is introduced to the ribozyme, inner sphere coordination of Mg(H(2)O)(x)(2+) (x

Raman crystallography of RNA.

Raman crystallography is the application of Raman spectroscopy to single crystals. This technique has been applied to a variety of protein molecules where it has provided unique information about biopolymer folding, substrate binding, and catalysis. Here, we describe the application of Raman crystallography to functional RNA molecules. RNA represents unique opportunities and challenges for Raman crystallography. One issue that confounds studies of RNA is its tendency to adopt multiple non-functional folds. Raman crystallography has the advantage that it isolates a single state of the RNA within the crystal and can evaluate its fold, metal ion binding properties (ligand identity, stoichiometry, and affinity), proton binding properties (identity, stoichiometry, and affinity), and catalytic potential. In particular, base-specific stretches can be identified and then associated with the binding of metal ions and protons. Because measurements are carried out in the hanging drop at ambient, rather than cryo, conditions and because RNA crystals tend to be approximately 70% solvent, RNA dynamics and conformational changes become experimentally accessible. This review focuses on experimental setup and procedures, acquisition and interpretation of Raman data, and determination of physicochemical properties of the RNA. Raman crystallographic and solution biochemical experiments on the HDV RNA enzyme are summarized and found to be in excellent agreement. Remarkably, characterization of the crystalline state has proven to help rather than hinder functional characterization of functional RNA, most likely because the tendency of RNA to fold heterogeneously is limited in a crystalline environment. Future applications of Raman crystallography to RNA are briefly discussed.

A catalytic metal ion interacts with the cleavage Site G.U wobble in the HDV ribozyme.

The HDV ribozyme self-cleaves by a chemical mechanism involving general acid-base catalysis to generate 2',3'-cyclic phosphate and 5'-hydroxyl termini. Biochemical studies from several laboratories have implicated C75 as the general acid and hydrated magnesium as the general base. We have previously shown that C75 has a pK(a) shifted >2 pH units toward neutrality [Gong, B., Chen, J. H., Chase, E., Chadalavada, D. M., Yajima, R., Golden, B. L., Bevilacqua, P. C., and Carey, P. R. (2007) J. Am. Chem. Soc. 129, 13335-13342], while in crystal structures, it is well-positioned for proton transfer. However, no evidence for a hydrated magnesium poised to serve as a general base in the reaction has been observed in high-resolution crystal structures of various reaction states and mutants. Herein, we use solution kinetic experiments and parallel Raman crystallographic studies to examine the effects of pH on the rate and Mg(2+) binding properties of wild-type and 7-deazaguanosine mutants of the HDV ribozyme. These data suggest that a previously unobserved hydrated magnesium ion interacts with N7 of the cleavage site G.U wobble base pair. Integrating this metal ion binding site with the available crystal structures provides a new three-dimensional model for the active site of the ribozyme that accommodates all available biochemical data and appears competent for catalysis. The position of this metal is consistent with a role of a magnesium-bound hydroxide as a general base as dictated by biochemical data.

Detection of innersphere interactions between magnesium hydrate and the phosphate backbone of the HDV ribozyme using Raman crystallography.

A Raman microscope and Raman difference spectroscopy are used to detect the vibrational signature of RNA-bound magnesium hydrate in crystals of hepatitis delta virus (HDV) ribozyme and to follow the effects of magnesium hydrate binding to the nonbridging phosphate oxygens in the phosphodiester backbone. There is a correlation between the Raman intensity of the innersphere magnesium hydrate signature peak, near 322 cm-1, and the intensity of the PO2- symmetric stretch, near 1100 cm-1, perturbed by magnesium binding, demonstrating direct observation of -PO2-...Mg2+(H2O)x innersphere complexes. The complexes may be pentahydrates (x = 5) and tetrahydrates (x = 4). The assignment of the Raman feature near 322 cm-1 to a magnesium hydrate species is confirmed by isotope shifts observed in D2O and H218O that are semiquantitatively reproduced by calculations. The standardized intensity changes in the 1100 cm-1 PO2- feature seen upon magnesium hydrate binding indicates that there are approximately 5 innersphere Mg2+...-O2P contacts per HDV molecule when the crystal is exposed to a solution containing 20 mM magnesium.

Structure of a tyrosyl-tRNA synthetase splicing factor bound to a group I intron RNA.

The 'RNA world' hypothesis holds that during evolution the structural and enzymatic functions initially served by RNA were assumed by proteins, leading to the latter's domination of biological catalysis. This progression can still be seen in modern biology, where ribozymes, such as the ribosome and RNase P, have evolved into protein-dependent RNA catalysts ('RNPzymes'). Similarly, group I introns use RNA-catalysed splicing reactions, but many function as RNPzymes bound to proteins that stabilize their catalytically active RNA structure. One such protein, the Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (TyrRS; CYT-18), is bifunctional and both aminoacylates mitochondrial tRNA(Tyr) and promotes the splicing of mitochondrial group I introns. Here we determine a 4.5-A co-crystal structure of the Twort orf142-I2 group I intron ribozyme bound to splicing-active, carboxy-terminally truncated CYT-18. The structure shows that the group I intron binds across the two subunits of the homodimeric protein with a newly evolved RNA-binding surface distinct from that which binds tRNA(Tyr). This RNA binding surface provides an extended scaffold for the phosphodiester backbone of the conserved catalytic core of the intron RNA, allowing the protein to promote the splicing of a wide variety of group I introns. The group I intron-binding surface includes three small insertions and additional structural adaptations relative to non-splicing bacterial TyrRSs, indicating a multistep adaptation for splicing function. The co-crystal structure provides insight into how CYT-18 promotes group I intron splicing, how it evolved to have this function, and how proteins could have incrementally replaced RNA structures during the transition from an RNA world to an RNP world.

Direct measurement of a pK(a) near neutrality for the catalytic cytosine in the genomic HDV ribozyme using Raman crystallography.

The hepatitis delta virus (HDV) ribozyme uses a cytosine to facilitate general acid-base catalysis. Biochemical studies suggest that C75 has a pKa perturbed to near neutrality. To measure this pKa directly, Raman spectra were recorded on single ribozyme crystals using a Raman microscope. A spectral feature arising from a single neutral cytosine was identified at 1528 cm(-1). At low pH, this mode was replaced with a new spectral feature. Monitoring these features as a function of pH revealed pKa values for the cytosine that couple anticooperatively with Mg2+ binding, with values of 6.15 and 6.40 in the presence of 20 and 2 mM Mg2+, respectively. These pKa values agree well with those obtained from ribozyme activity experiments in solution. To correlate the observed pKa with a specific nucleotide, crystals of C75U, which is catalytically inactive, were examined. The Raman difference spectra show that this mutation does not affect the conformation of the ribozyme. However, crystals of C75U did not produce a signal from a protonatable cytosine, providing strong evidence that protonation of C75 is being monitored in the wild-type ribozyme. These studies provide the first direct physical measurement of a pKa near neutrality for a catalytic residue in a ribozyme and show that ribozymes, like their protein enzyme counterparts, can optimize the pKa of their side chains for proton transfer.