Michael E. Harris

Native mRNA editing complexes from Trypanosoma brucei mitochondria.

The aim of this study was to identify multicomponent complexes involved in kinetoplastid mitochondrial mRNA editing. Mitochondrial extracts from Trypanosoma brucei were fractionated on 10–30% glycerol gradients and assayed for RNAs and activities potentially involved in editing, including pre‐edited mRNA, guide RNA (gRNA), endonuclease, terminal uridylyltransferase (TUTase), RNA ligase and gRNA‐mRNA chimera‐forming activities. These experiments suggest that two distinct editing complexes exist. Complex I (19S) consists of gRNA, TUTase, RNA ligase and chimera‐forming activity. Complex II (35–40S) is composed of gRNA, preedited mRNA, RNA ligase and chimera‐forming activity. These studies provide the first evidence that editing occurs in a multicomponent complex. The possible roles of complex I, complex II and RNA ligase in editing are discussed.

Use of photoaffinity crosslinking and molecular modeling to analyze the global architecture of ribonuclease P RNA.

Bacterial ribonuclease P (RNase P), an endonuclease involved in tRNA maturation, is a ribonucleoprotein containing a catalytic RNA. The secondary structure of this ribozyme is well established, but comparatively little is understood about its 3‐D structure. In this analysis, orientation and distance constraints between elements within the Escherichia coli RNase P RNA‐pre‐tRNA complex were determined by intra‐ and intermolecular crosslinking experiments. A molecular mechanics‐based RNA structure refinement protocol was used to incorporate the distance constraints indicated by crosslinking, along with the known secondary structure of RNase P RNA and the tertiary structure of tRNA, into molecular models. Seven different structures that satisfy the constraints equally well were generated and compared by superposition to estimate helix positions and orientations. Manual refinement within the range of conformations indicated by the molecular mechanics analysis was used to derive a model of RNase P RNA with bound substrate pre‐tRNA that is consistent with the crosslinking results and the available phylogenetic comparisons.

Comparative photocross-linking analysis of the tertiary structures of Escherichia coli and Bacillus subtilis RNase P RNAs.

Bacterial ribonuclease P contains a catalytic RNA subunit that cleaves precursor sequences from the 5′ ends of pre‐tRNAs. The RNase P RNAs from Bacillus subtilis and Escherichia coli each contain several unique secondary structural elements not present in the other. To understand better how these phylogenetically variable elements affect the global architecture of the ribozyme, photoaffinity cross‐linking studies were carried out. Photolysis of photoagents attached at homologous sites in the two RNAs results in nearly identical cross‐linking patterns, consistent with the homology of the RNAs and indicating that these RNAs contain a common, core tertiary structure. Distance constraints were used to derive tertiary structure models using a molecular mechanics‐based modeling protocol. The resulting superimposition of large sets of equivalent models provides a low resolution (5–10 Å) structure for each RNA. Comparison of these structure models shows that the conserved core helices occupy similar positions in space. Variably present helical elements that may play a role in global structural stability are found at the periphery of the core structure. The P5.1 and P15.1 helical elements, unique to the B.subtilis RNase P RNA, and the P6/16/17 helices, unique to the E.coli RNA, occupy similar positions in the structure models and, therefore, may have analogous structural function.

RNA editing in kinetoplastid mitochondria.

RNA editing in the mitochondrion of kinetoplastid protozoa results in the posttranscriptional addition and deletion of uridine residues in mRNAs. Editing of mRNAs can lead to the formation of initiation codons for mitochondrial translation, the correction of frame‐shifted genes at the RNA level, and in extensively edited mRNAs, the formation of complete reading frames. Kinetoplastid RNA editing requires that genetic information from two or more separately transcribed genes be brought together to form the mature, edited mRNA. The information necessary for the proper insertion or deletion of uridines in the mRNA is present in small mitochondrial transcripts termed guide RNAs (gRNAs). Editing of mRNAs appears to be associated with a high molecular weight complex, called the editosome, containing specific gRNAs, unedited mRNAs, and proteins. Editing is likely a two‐step process involving first the breakage of a phosphodiester bond at the editing site and formation of a chimeric molecule with a gRNA covalently joined to the 5′ end of the 3′ portion of an mRNA. The chimera is resolved by the rejoining of the 5′ end of the mRNA to the 3′ portion of the mRNA with the addition or deletion of a uridine at the junction point. Two models are proposed for the biochemical mechanism of RNA editing. The first is an enzymatic cascade of cleavage and ligation while the other supports successive rounds of transesterification. The obvious functional necessity for editing in kinetoplastid mitochondria is the formation of translatable mRNAs. Far less clear is the evolutionary origin of editing and the role editing plays in regulating mitochondrial gene expression.— Hajduk, S. L., Harris, M. E., and Pollard, V. W. RNA editing in kinetoplastid mitochondria. FASEB J. 7: 54‐63; 1993.

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.

Evidence that substrate-specific effects of C5 protein lead to uniformity in binding and catalysis by RNase P

The ribonucleoprotein enzyme RNase P processes all pre‐tRNAs, yet some substrates apparently lack consensus elements for recognition. Here, we compare binding affinities and cleavage rates of Escherichia coli pre‐tRNAs that exhibit the largest variation from consensus recognition sequences. These results reveal that the affinities of both consensus and nonconsensus substrates for the RNase P holoenzyme are essentially uniform. Comparative analyses of pre‐tRNA and tRNA binding to the RNase P holoenzyme and P RNA alone reveal differential contributions of the protein subunit to 5′ leader and tRNA affinity. Additionally, these studies reveal that uniform binding results from variations in the energetic contribution of the 5′ leader, which serve to compensate for weaker tRNA interactions. Furthermore, kinetic analyses reveal uniformity in the rates of substrate cleavage that result from dramatic (>900‐fold) contributions of the protein subunit to catalysis for some nonconsensus pre‐tRNAs. Together, these data suggest that an important biological function of RNase P protein is to offset differences in pre‐tRNA structure such that binding and catalysis are uniform.

Practical Access to 2-Alkylsuccinates through Asymmetric Catalytic Hydrogenation of Stobbe-Derived Itaconates

Enantiomerically pure 2-alkylsuccinates are obtained on a 500-g scale after hydrogenation with the cationic rhodium complexes with tetraalkyl-substituted 1,2-bis(phospholanyl)ethane or -benzene ligands [R′-DuPHOS; Eq. (a)]. The catalysts allow smooth hydrogenation of mixtures of the prochiral E and Z isomers of itaconate derivatives with very high enantioselectivities and catalytic efficiencies; even tetrasubstituted itaconates were hydrogenated with 96% ee.

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.

Experimental and computational analysis of the transition state for ribonuclease A-catalyzed RNA 2′-O-transphosphorylation

Enzymes function by stabilizing reaction transition states; therefore, comparison of the transition states of enzymatic and nonenzymatic model reactions can provide insight into biological catalysis. Catalysis of RNA 2′-O-transphosphorylation by ribonuclease A is proposed to involve electrostatic stabilization and acid/base catalysis, although the structure of the rate-limiting transition state is uncertain. Here, we describe coordinated kinetic isotope effect (KIE) analyses, molecular dynamics simulations, and quantum mechanical calculations to model the transition state and mechanism of RNase A. Comparison of the 18O KIEs on the 2′O nucleophile, 5′O leaving group, and nonbridging phosphoryl oxygens for RNase A to values observed for hydronium- or hydroxide-catalyzed reactions indicate a late anionic transition state. Molecular dynamics simulations using an anionic phosphorane transition state mimic suggest that H-bonding by protonated His12 and Lys41 stabilizes the transition state by neutralizing the negative charge on the nonbridging phosphoryl oxygens. Quantum mechanical calculations consistent with the experimental KIEs indicate that expulsion of the 5′O remains an integral feature of the rate-limiting step both on and off the enzyme. Electrostatic interactions with positively charged amino acid site chains (His12/Lys41), together with proton transfer from His119, render departure of the 5′O less advanced compared with the solution reaction and stabilize charge buildup in the transition state. The ability to obtain a chemically detailed description of 2′-O-transphosphorylation transition states provides an opportunity to advance our understanding of biological catalysis significantly by determining how the catalytic modes and active site environments of phosphoryl transferases influence transition state structure.