Eric L. Christian

Helix P4 is a divalent metal ion binding site in the conserved core of the ribonuclease P ribozyme.

The ribonuclease P ribozyme (RNase P RNA), like other large ribozymes, requires magnesium ions for folding and catalytic function; however, specific sites of metal ion coordination in RNase P RNA are not well defined. To identify and characterize individual nucleotide functional groups in the RNase P ribozyme that participate in catalytic function, we employed self-cleaving ribozyme-substrate conjugates that facilitate measurement of the effects of individual functional group modifications. The self-cleavage rates and pH dependence of two different ribozyme-substrate conjugates were determined and found to be similar to the single turnover kinetics of the native ribozyme. Using site-specific phosphorothioate substitutions, we provide evidence for metal ion coordination at the pro-Rp phosphate oxygen of A67, in the highly conserved helix P4, that was previously suggested by modification-interference experiments. In addition, we detect a new metal ion coordination site at the pro-Sp phosphate oxygen of A67. These findings, in combination with the proximity of A67 to the pre-tRNA cleavage site, support the conclusion that an important role of helix P4 in the RNase P ribozyme is to position divalent metal ions that are required for catalysis.

Recent insights into the structure and function of the ribonucleoprotein enzyme ribonuclease P

In bacteria, the tRNA-processing endonuclease ribonuclease P is composed of a large ( approximately 400 nucleotide) catalytic RNA and a smaller ( approximately 100 amino acid) protein subunit that is essential for substrate recognition. Current biochemical and biophysical investigations are providing fresh insights into the modular architecture of the ribozyme, the mechanisms of substrate specificity and the role of essential metal ions in catalysis. Together with recent high-resolution structures of portions of the ribozyme, these findings are beginning to reveal how the functions of RNA and protein are coordinated in this ribonucleoprotein enzyme.

Evidence for a polynuclear metal ion binding site in the catalytic domain of ribonuclease P RNA

Interactions with divalent metal ions are essential for the folding and function of the catalytic RNA component of the tRNA processing enzyme ribonuclease P (RNase P RNA). However, the number and location of specific metal ion interactions in this large, highly structured RNA are poorly understood. Using atomic mutagenesis and quantitative analysis of thiophilic metal ion rescue we provide evidence for metal ion interactions at the pro‐RP and pro‐SP non‐bridging phosphate oxygens at nucleotide A67 in the universally conserved helix P4. Moreover, second‐site modifications within helix P4 and the adjacent single stranded region (J3/4) provide the first evidence for metal ion interactions with nucleotide base functional groups in RNase P RNA and reveal the presence of an additional metal ion important for catalytic function. Together, these data are consistent with a cluster of metal ion interactions in the P1–P4 multi‐helix junction that defines the catalytic core of the RNase P ribozyme.

Differential chemical probing of a group II self-splicing intron identifies bases involved in tertiary interactions and supports an alternative secondary structure model of domain V.

Dimethyl sulfate modification was used to probe for tertiary structural elements in the group II intron PI.LSU/2 from the mitochondrial pre-ribosomal RNA of the brown alga Pylaiella littoralis. Modification of the lariat form of the intron under conditions that allow both native folding and conformational homogeneity is found to be generally consistent with secondary and tertiary structural features identified previously for group II ribozymes. A comparison of chemical probing at temperatures just below and above the first melting transition illustrates the cooperative unfolding of tertiary structure and identifies novel candidates for tertiary interactions in addition to defining elements of secondary structure. Substitution of the GAAA terminal loop of domain V is shown to be compatible with retention of conformational homogeneity (despite the loss of an important tertiary interaction), but produces a concise methylation footprint in domain I at the site previously shown to harbor the receptor for that loop. The analysis also identified two nucleotide positions in domain V with novel secondary and potential tertiary structural roles. The proposed refinement of domain V secondary structure is supported by an expanded comparative analysis of group II sequences and bears increased resemblance to U2:U6 snRNA pairing in the spliceosome.

Conservation of Helical Structure Contributes to Functional Metal Ion Interactions in the Catalytic Domain of Ribonuclease P RNA

Like protein enzymes, catalytic RNAs contain conserved structure motifs important for function. A universal feature of the catalytic domain of ribonuclease P RNA is a bulged-helix motif within the P1-P4 helix junction. Here, we show that changes in bulged nucleotide identity and position within helix P4 affect both catalysis and substrate binding, while a subset of the mutations resulted only in catalytic defects. We find that the proximity of the bulge to sites of metal ion coordination in P4 is important for catalysis; moving the bulge distal to these sites and deleting it had similarly large effects, while moving it proximal to these sites had only a moderate effect on catalysis. To test whether the effects of the mutations are linked to metal ion interactions, we used terbium-dependent cleavage of the phosphate backbone to probe metal ion-binding sites in the wild-type and mutant ribozymes. We detect cleavages at specific sites within the catalytic domain, including helix P4 and J3/4, which have previously been shown to participate directly in metal ion interactions. Mutations introduced into P4 cause local changes in the terbium cleavage pattern due to alternate metal ion-binding configurations with the helix. In addition, a bulge deletion mutation results in a 100-fold decrease in the single turnover cleavage rate constant at saturating magnesium levels, and a reduced affinity for magnesium ions important for catalysis. In light of the alternate terbium cleavage pattern in P4 caused by bulge deletion, this decreased ability to utilize magnesium ions for catalysis appears to be due to localized structural changes in the ribozymes catalytic core that weaken metal ion interactions in P4 and J3/4. The information reported here, therefore, provides evidence that the universal conservation of the P4 structure is based in part on optimization of metal ion interactions important for catalysis.

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.

RNA Crosslinking Methods

RNA-RNA crosslinking provides a rapid means of obtaining evidence for the proximity of functional groups in structurally complex RNAs and ribonucleoproteins. Such evidence can be used to provide a physical context for interpreting structural information from other biochemical and biophysical methods and for the design of further experiments. The identification of crosslinks that accurately reflect the native conformation of the RNA of interest is strongly dependent on the position of the crosslinking agent, the conditions of the crosslinking reaction, and the method for mapping the crosslink position. Here, we provide an overview of protocols and experimental considerations for RNA-RNA crosslinking with the most commonly used long- and short-range photoaffinity reagents. Specifically, we describe the merits and strategies for random and site-specific incorporation of these reagents into RNA, the crosslinking reaction and isolation of crosslinked products, the mapping crosslinked sites, and assessment of the crosslinking data.

A quantitative Raman spectroscopic signal for metal-phosphodiester interactions in solution.

Accurate identification and quantification of metal ion-phosphodiester interactions are essential for understanding the role of metal ions as determinants of three-dimensional folding of large RNAs and as cofactors in the active sites of both RNA and protein phosphodiesterases. Accomplishing this goal is difficult due to the dynamic and complex mixture of direct and indirect interactions formed with nucleic acids and other phosphodiesters in solution. To address this issue, Raman spectroscopy has been used to measure changes in bond vibrational energies due to metal interactions. However, the contributions of inner-sphere, H-bonding, and electrostatic interactions to the Raman spectrum of phosphoryl oxygens have not been analyzed quantitatively. Here, we report that all three forms of metal ion interaction result in attenuation of the Raman signal for the symmetric vibration of the nonbridging phosphate oxygens (nu(s)PO(2)(-)), while only inner-sphere coordination gives rise to an apparent shift of nu(s)PO(2)(-) to higher wavenumbers (nu(s)PO(2)(-)M) in solution. Formation of nu(s)PO(2)(-)M is shown to be both dependent on metal ion identity and an accurate measure of site-specific metal ion binding. In addition, the spectroscopic parameter reflecting the energetic difference between nu(s)PO(2)(-) and nu(s)PO(2)(-)M (DeltanuM) is largely insensitive to changes in phosphodiester structure but strongly dependent on the absolute electronegativity and hardness of the interacting metal ion. Together, these studies provide strong experimental support for the use of nu(s)PO(2)(-)M and DeltanuM as general spectroscopic features for the quantitative analysis of metal binding affinity and the identification of metal ions associated with phosphodiesters in solution.

Deconvolution of Raman spectroscopic signals for electrostatic, H-bonding, and inner-sphere interactions between ions and dimethyl phosphate in solution

Quantitative analysis of metal ion-phosphodiester interactions is a significant experimental challenge due to the complexities introduced by inner-sphere, outer-sphere (H-bonding with coordinated water), and electrostatic interactions that are difficult to isolate in solution studies. Here, we provide evidence that inner-sphere, H-bonding and electrostatic interactions between ions and dimethyl phosphate can be deconvoluted through peak fitting in the region of the Raman spectrum for the symmetric stretch of non-bridging phosphate oxygen (ν(s)PO(2)(-)). An approximation of the change in vibrational spectra due to different interaction modes is achieved using ions capable of all or a subset of the three forms of metal ion interaction. Contribution of electrostatic interactions to ion-induced changes to the Raman ν(s)PO(2)(-) signal could be modeled by monitoring attenuation of ν(s)PO(2)(-) in the presence of tetramethylammonium, while contribution of H-bonding and inner-sphere coordination could be approximated from the intensities of altered ν(s)PO(2)(-) vibrational modes created by an interaction with ammonia, monovalent or divalent ions. A model is proposed in which discrete spectroscopic signals for inner-sphere, H-bonding, and electrostatic interactions are sufficient to account for the total observed change in ν(s)PO(2)(-) signal due to interaction with a specific ion capable of all three modes of interaction. Importantly, the quantitative results are consistent with relative levels of coordination predicted from absolute electronegativity and absolute hardness of alkali and alkaline earth metals.

Understanding the Role of Metal Ions in RNA Folding and Function: Lessons from RNase P, a Ribonucleoprotein Enzyme

There is a large and rapidly growing literature relating RNA function to metal ion identity and concentration; however, due to the complexity and large number of interactions it remains a significant experimental challenge to tie the interactions of individual ions to specific aspects of RNA function. Investigation of the ribonculeopro- tein enzyme RNase P function has assisted in defining characteristics of RNA-metal ion interactions and provided a useful model system for understanding RNA catalysis and ribonucleoprotein assembly. The goal of this chapter is to review progress in under- standing the physical basis of functional metal ion interactions with P RNA and relate this progress to the development of our understanding of RNA metal ion interactions in general. The research results reviewed here encompass: (1) Determination of the contribution of divalent metal ion binding to specific aspects of enzyme function, (2) Identification of individual metal ion binding sites in P RNA and their contribution to function, and (3) The effect of protein binding on RNA-metal ion affinity.