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Polymers and Self Assembly: From Biology to Nanomaterials Poster Session I
22-POS
Board 22
RNA Structural Isomerization in the Peptide Bond Formation Site: Molecular Mechanism
and Physiological Function of DbpA.
Jared J. Childs,
Eda Koculi
.
University of Central Florida , Orlanod , USA.
DbpA is a DEAD-box RNA helicase. Like all the members of this family, DbpA has two RecA-
like domains, which use the energy of ATP binding and hydrolysis to unwind short double helix
substrates. Different from other members of the DEAD-box family of enzymes, DbpA has a
structured C-terminal domain, which imparts the DbpA protein’s specificity for helix 92. Helix
92 is located in the peptide bond formation site; therefore, DbpA performs RNA structural
isomerization in a region of the ribosome that is crucial for cell survival. Using a number of
DbpA constructs, and RNA-DNA and RNA-PEG chimeras we show that the DbpA protein
unwinds the double helix substrates by directly loading on them and that the residues outside the
double-helix region are mechanistically unimportant for the DbpA catalytic activity. Moreover,
our data indicates that the interaction of the DbpA C-terminal domain with the double helix
regions near helix 92 determine the double helix substrates that are accessible by the DbpA
catalytic core. Hence, during ribosome assembly the role of the DbpA C-terminal domain could
be both to tether DbpA to its site of action and to prevent the DbpA catalytic core from
unwinding correctly annealed double helices. In vivo pulse-labeling experiments indicate that
DbpA acts in two pathways of large subunit assembly. When helicase inactive DbpA, R331A, is
expressed in the cell one of the pathways of the large subunit assembly never goes to completion
and a dead-end large subunit intermediate accumulates. We are in the process of investigating
both protein compositions and RNA structures of various intermediates on large subunit
assembly in the presence of R331A DbpA. These experiments will determine the precise RNA
structural isomerization that DbpA performs during ribosome assembly.