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THE IRE RNA. |
Current
Hall Lab Projects
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Noncanonical
RRM structure and function. RNA binding selectivity and
protein dynamics (Caroline Maynard) Synopsis: I am interested
in using NMR techniques to probe the structure and dynamics of the
polyprimidine tract binding protein (PTB) in order to determine how the
interplay between structure and dynamics guides RNA binding selectivity. PTB: The human PTB
consists of 4 highly conserved RNA binding domains (RBDs) connected by
flexible linkers and is important in a variety of functions including mRNA
processing and stabilization, alternative splicing, and internal ribosomal
entry site (IRES)-mediated translation
initiation. All 4 domains
have the canonical βαββαβ sandwich fold, however, RBDs 2 and 3 have a
C-terminal extension consisting of a 5th beta strand antiparallel to β2,
extending the RNA binding surface, and which is connected to β4 by a long, flexible
linker that extends across the face of the β-sheet. In addition, RBDs 3 and 4 interact significantly, whereas
RBDs 1 and 2 appear to be independent.
Source: Conte, T. et al.;
EMBO J. 2000 19(12):3132-3141. PTB binds to different RNAs with different affinity,
further complicated by the fact that the affinities are dependent on which
RBDs are present. For example,
RBDs 1 and 2 have higher binding affinity for c-src RNA, which has a good
deal of secondary structure, than for the less structured RNA from the GABA
γ2 intron. Conversely, RBDs 3
and 4 bind the less structured RNA more tightly than the more structured RNA.
Protein Dynamics: The RNA
binding selectivities and protein-protein interactions of the RBDs of PTB
cannot be explained solely by structural differences. Thus, I hypothesize that protein
dynamics have evolved on a common structural template in order to facilitate
a variety of functions. In order
to test this, I aim to measure protein motions of RBDs 3 and 4 as well as a
truncated version of RBD3 which does not include the C-terminal extension
(protein db5), both as individual domains, and in concert, as well as with
and without RNA (both structured and unstructured) bound. Relaxation dispersion experiments can
be used to probe protein dynamics on the μs-ms timescale, which may be
relevant for PTB binding to RNA.
Correlation of the protein motions with equilibrium structure
differences and binding kinetics can then be used to implicate specific
protein motions in RNA binding. So far: We can show
that db5 can be overexpressed, purified, and has comparable secondary
structure and backbone amide chemical shifts to PTB RBD3 (see below). In addition, electrophoretic mobility
shift assays show that the binding affinities are PTB34 > PTB3 > db5. Currently, I am working on producing RBD 4 and determining
its structure and RNA binding affinity.
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Return to Washington University Biochemistry and Molecular Biophysics Homepage
Dr. Kathleen B. Hall (hall(at)biochem.wustl.edu)
Department of Biochemistry and
660
office: 314-362-4196
lab: 314-362-4197
or 314-747-8079
FAX: 314-362-7183
send email to kathleenhal(at)gmail.com
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