Joseph Stackhouse

The ribonuclease from an extinct bovid ruminant

The sequence of the ribonuclease from the ancestor of swamp buffalo, river buffalo, and ox, corresponding approximately to Pachyportax latidens, an extinct ruminant known from the fossil record, has been reconstructed using the rule of ‘maximum parsimony’. This protein and two sequences that may have been intermediates in the evolution of modern ribonuclease have been constructed in the laboratory by site‐directed mutagenesis, and their properties examined.

Site-directed mutagenesis of bovine pancreatic ribonuclease: lysine-41 and aspartate-121.

Chemical modification studies suggest that two residues of bovine pancreatic ribonuclease A (RNase A), Lys‐41 and Asp‐121, are important for catalysis. Three mutants of RNase A have been prepared, two point mutants with Lys‐41 altered to Arg‐41 and Asp‐121 altered to Glu‐121, and a double mutant where both residues are altered. The Lys‐41 Arg mutant has ca. 2% the calatylic activity (kcalKm ) of the native protein, while the Asp‐121 Glu mutant has ca. 17% the catalytic activity of the native protein. The double mutant has catalytic activity comparable to the Lys‐41 Arg mutant.

Redesigning nucleic acids

A research program has applied the tools of synthetic organic chemistry to systematically modify the structure of DNA and RNA oligonucleotides to learn more about the chemical principles underlying their ability to store and transmit genetic information. Oligonucleotides (as opposed to nucleosides) have long been overlooked by synthetic organic chemists as targets for structural modification. Synthetic chemistry has now yielded oligonucleotides with 12 replicatable letters, modified backbones, and new insight into why Nature chose the oligonucleotide structures that she did.

7 – Evolutionary Reconstructions in the Ribonuclease Family

Publisher Summary This chapter provides an overview of the ribonuclease (RNases) family. RNases of the bovine pancreatic superfamily play a central role in the development of ideas and technologies to study protein structure and catalysis. This is because RNase is small, present in large amounts in the pancreas of ruminants, stable under a wide range of conditions, and therefore amenable to full chemical analysis. It was among the first proteins to be sequenced, the first protein to be examined by NMR spectroscopy, and the first to be unfolded and refolded in the laboratory. This chapter also discusses the use of protein engineering for understanding the evolution. It explains concepts related to experimental paleomolecular geobiology. It also describes collection of additional seminal RNase sequences from recently diverging artiodactyls. An overview of reconstructing evolution of biomolecular behavior in the RNase superfamily is also presented in this chapter. The chapter elaborates in detail about the repair of damaged pseudogenes by gene conversion. The chapter concludes with a discussion on physiological functions of seminal RNase.