Type of Document	Dissertation
Author	Mitchell, Michelle H.
Author's Email Address	mdh04@fsu.edu
URN	etd-07102009-145044
Title	Understanding Structural Mechanisms of Endolytic RNA Cleavage Enzymes
Degree	Doctor of Philosophy
Department	Molecular Biophysics, Institute of
Advisory Committee	Advisor Name Title Hong Li Committee Chair Branko Stefanovic Committee Member Brian Miller Committee Member W. Ross Ellington Committee Member Penny J. Gilmer Outside Committee Member
Keywords	RNA Cleavage tRNA Splicing Endonuclease RNA Processing
Date of Defense	2009-06-26
Availability	unrestricted
Abstract The RNA splicing and processing endonuclease from Nanoarchaeum equitans (NEQ) belongs to the recently identified (ab)2 family of splicing endonucleases that require two different subunits for splicing activity. N. Equitans splicing endonuclease consists of the catalytic subunit (NEQ205) and the structural subunit (NEQ261). Here we report the crystal structure of the functional NEQ enzyme at 2.1 Angstroms resolution containing both subunits, as well as that of the NEQ261 subunit alone at 2.2 Angstroms resolution. The functional enzyme resembles previously known a2 and a4 endonucleases but forms a heterotetramer; a dimer of two heterodimers of the catalytic subunit (NEQ205) and the structural subunit (NEQ261). Surprisingly, NEQ261 alone forms a homodimer, similar to the previously known homodimer of the catalytic subunit. The homodimers of isolated subunits are inhibitory to heterodimerization as illustrated by a covalently linked catalytic homodimer that had no RNA cleavage activity upon mixing with the structural subunit. Detailed structural comparison reveals a more favorable hetero- than homo-dimerization interface, thereby suggesting a possible regulation mechanism of enzyme assembly through available subunits. Finally, the uniquely flexible active site of the NEQ endonuclease provides a possible explanation for its broader substrate specificity.
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