Roopa Biswas

Molecular Modeling of the Three-dimensional Structure of the Bacterial RNase P Holoenzyme

Bacterial ribonuclease P (RNase P), an enzyme involved in tRNA maturation, consists of a catalytic RNA subunit and a protein cofactor. Comparative phylogenetic analysis and molecular modeling have been employed to derive secondary and tertiary structure models of the RNA subunits from Escherichia coli (type A) and Bacillus subtilis (type B) RNase P. The tertiary structure of the protein subunit of B.subtilis and Staphylococcus aureus RNase P has recently been determined. However, an understanding of the structure of the RNase P holoenzyme (i.e. the ribonucleoprotein complex) is lacking. We have now used an EDTA-Fe-based footprinting approach to generate information about RNA-protein contact sites in E.coli RNase P. The footprinting data, together with results from other biochemical and biophysical studies, have furnished distance constraints, which in turn have enabled us to build three-dimensional models of both type A and B versions of the bacterial RNase P holoenzyme in the absence and presence of its precursor tRNA substrate. These models are consistent with results from previous studies and provide both structural and mechanistic insights into the functioning of this unique catalytic RNP complex.

Inhibition of bacterial RNase P by aminoglycoside-arginine conjugates

The potential of RNAs and RNA–protein (RNP) complexes as drug targets is currently being explored in various investigations. For example, a hexa‐arginine derivative of neomycin (NeoR) and a tri‐arginine derivative of gentamicin (R3G) were recently shown to disrupt essential RNP interactions between the trans‐activator protein (Tat) and the Tat‐responsive RNA (trans‐activating region) in the human immunodeficiency virus (HIV) and also inhibit HIV replication in cell culture. Based on certain structural similarities, we postulated that NeoR and R3G might also be effective in disrupting RNP interactions and thereby inhibiting bacterial RNase P, an essential RNP complex involved in tRNA maturation. Our results indicate that indeed both NeoR and R3G inhibit RNase P activity from evolutionarily divergent pathogenic bacteria and do so more effectively than they inhibit partially purified human RNase P activity.

Use of EPR spectroscopy to study macromolecular structure and function.

Electron paramagnetic resonance (EPR) spectroscopy is now part of the armory available to probe the structural aspects of proteins, nucleic acids and protein–nucleic acid complexes. Since the mobility of a spin label covalently attached to a macromolecule is influenced by its microenvironment, analysis of the EPR spectra of site-specifically incorporated spin labels (probes) provides a powerful tool for investigating structure–function correlates in biological macromolecules. This technique has become readily amenable to address various problems in biology in large measure due to the advent of techniques like site-directed mutagenesis, which enables site-specific substitution of cysteine residues in proteins, and the commercial availability of thiol-specific spin-labeling reagents (Figure 1)1. In addition to the underlying principle and the experimental strategy, several recent applications are discussed in this review.

Structures of the catalytic site mutants D99A and H48Q and the calcium-loop mutant D49E of phospholipase A2

Crystal structures of the active-site mutants D99A and H48Q and the calcium-loop mutant D49E of bovine phospholipase A2 have been determined at around 1.9 A resolution. The D99A mutant is isomorphous to the orthorhombic recombinant enzyme, space group P212121. The H48Q and the calcium-loop mutant D49E are isomorphous to the trigonal recombinant enzyme, space group P3121. The two active-site mutants show no major structural perturbations. The structural water is absent in D99A and, therefore, the hydrogen-bonding scheme is changed. In H48Q, the catalytic water is present and hydrogen bonded to Gln48 N, but the second water found in native His48 is absent. In the calcium-loop mutant D49E, the two water molecules forming the pentagonal bipyramid around calcium are absent and only one O atom of the Glu49 carboxylate group is coordinated to calcium, resulting in only four ligands.

CORRELATION OF HYDROPHOBICITY AND PACKING IN A-DNA OLIGONUCLEOTIDES

A-DNA oligomers pack in a slanted fashion with the terminal base pairs abutting into the minor groove of neighboring molecules unlike the other forms of DNA which pack by vertically stacking one over the other into helical columns. To explain the differences in packing we have advanced a hypothesis that the orientation of the sugar-phosphate backbone is different in A-DNA from that in the other forms of DNA, mainly due to the differences in the sugar puckering.

Crystal Structure of an RNA Duplex [r(gugcaca)dC]2 with 3′-Dinucleoside Overhangs Forming a Superhelix

Abstract Crystal structure of the RNA octamer duplex, [r(gugcaca)dC] 2, with space group I212121 and the cell constants a=24.29, b=45.25 and c=73.68Å, has been determined and refined. The structural and packing architecture of the molecule consist of a highly bent six base paired duplex forming a right-handed superhelix stacked in tandem compared to an infinite pseudo- continuous column as is usually present in RNA crystal structures. The super helix could be formed by the head-to-head stacking (g1 over g1 and g9 over g9), the large bend and the twists at the junctions may also be responsible. The sugar-phosphate backbones of the 3′-end dinucleoside overhangs snuggly fit into the minor grooves of adjacent double helical stacks. The 3′-terminal deoxycytidines form antiparallel hemiprotonated trans (C·C)+ pairs with symmetry related deoxycytidines, while the penultimate adenines form base triples (a*g·c) with the capping g·c base pairs of the hexamer duplex with the adenine (a7) at one end being syn and at the other anti. These triple interactions are the same as those found in the tetrahymena ribozyme and group I intron.