Mutagenic and structural studies of the active center of oligoribonuclease, an essential enzyme for Escherichia coli viability

Date of Award




Degree Name

Doctor of Philosophy (Ph.D.)


Biochemistry and Molecular Biology

First Committee Member

Arun Malhotra - Committee Chair


Maturation and degradation of RNA molecules are essential processes in both prokaryotic and eukaryotic cells. Oligoribonuclease (ORN) is the only exoribonuclease able to efficiently degrade small oligonucleotides. Upon loss of oligoribonuclease activity, E. coli cells accumulate short oligonucleotides and lose viability.ORN is a DEDDh type exoribonuclease. The importance of the conserved histidine in ORN activity is investigated using mutants of the enzyme. Kinetic analyses of these mutants show that histidine is essential and cannot be replaced by either tyrosine or asparagine.ORN activity is inhibited by zinc like RNase T, another DEDDh exoribonuclease. To investigate the role of His157 in attenuation of ORN activity by zinc, this residue was mutated to alanine. Activity of H157A mutant compared to that of wild type enzyme shows that His157 residue is responsible for the sensitivity of ORN to zinc, and its removal abolishes zinc inhibition. A metal preference of DEDDh versus DEDDy exonucleases is hypothesized.ORN structure reveals a basic patch near the active center, possibly involved in binding and orientation of the substrate at the active center. Two of the basic residues located in this patch, Arg129 and Lys153, were analyzed in detail using mutagenesis. Both residues were determined to be important for activity of the enzyme and productive binding of the substrate. The Arg129 residue is probably involved in correctly orienting the substrate at the active center, likely through charge interactions. A model of substrate binding by ORN, with special emphasis on Arg129, is proposed.X-ray crystal structures of two ORN mutants, H157A and H157Y, were solved. The overall folds of these mutants are similar to that of the wild type ORN. An approximate shift of 2 A was observed in the H157A structure at position 157 which provides further evidence for the flexible nature of this region and suggests that the ORN active center may be dynamic in nature.These studies advance our understanding of ORN active center and how ORN selects and binds substrates. This research has direct relevance to understanding the molecular mechanism of RNA processing and metabolism.


Biology, Molecular; Chemistry, Biochemistry

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