STRUCTURAL CHARACTERIZATION OF LIGAND RECOGNITION AND ACTIVATION OF A

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STRUCTURAL CHARACTERIZATION OF LIGAND RECOGNITION
AND ACTIVATION OF A METABOLITE-RESPONSIVE
RIBOZYME Introduction to riboswitches and
ribozymes Glucosamine 6-phosphate
riboswitch/ribozyme Results - Ligand recognition
and pH dependence Conclusions and future
structural analyses
Dr. Julie Soukup Nebraska INBRE
Associate Creighton University
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Genetic Control Mechanisms
Tom Cech, Nature (2004) 428 263-264.
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Riboswitches contain an aptamer domain
expression platform
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J.K. Soukup and G.A. Soukup, Curr. Opin. Struct.
Biol. (2004) 14 344-349.
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Riboswitches
J.K. Soukup and G.A. Soukup, Curr. Opin. Struct.
Biol. (2004) 14 344-349.
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Glucosamine 6-phosphate riboswitch
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A conserved RNA structure precedes the glmS gene
in Gram-positive organisms
Kobayashi et al., Proc. Natl. Acad. Sci. USA
(2003) 10 4678-4683.
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Glucosamine 6-phosphate ribozyme
self-cleavage RNA transesterification
Might glucosamine 6-phosphate serve as the
general acid-base (coenzyme) for self-cleavage?
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Ribozyme exhibits self-cleavage activity in TRIS
buffer in the absence of ligand
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Ligand specificity - importance of amine
functionality
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Ribozyme-coenzyme activity depends on pH
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pKa of the amine functionality of GlcN6P affects
catalysis more so than binding
GlcN
serinol
pH 8.5
pH 7.5
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CONCLUSIONS GlcN6P ribozyme self-cleavage both
requires and is dependent upon the pKa of the
amine functionality of GlcN6P GlcN6P plays a
role in general acid-base catalysis Novel
coenzyme role for GlcN6P in ribozyme
self-cleavage Binding is largely uneffected by
protonation state of the ligand FUTURE In order
to design effective antibiotics Determine role
of the phosphate functionality of
GlcN6P Determine role of divalent metal ion
interactions with GlcN6P
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ACKNOWLEDGEMENTS Tom McCarthy, Creighton
University Melissa Plog, Creighton
University Joshua Jansen, Creighton University
INBRE Scholar Dr. Garrett Soukup, Creighton
University INBRE Mentor Dr. Ron Breaker, Yale
University SUPPORT NIH INBRE/BRIN grant Clare
Boothe Luce Program of the Henry Luce Foundation
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glmS riboswitch mechanism of gene regulation RNA
processing
Role of glucosamine 6-phosphate?
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Nucleotide Analog Interference Mapping (NAIM)
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Nucleotide Analogs
Adenosine (A?S)
Cytosine (C?S)
Guanosine (G?S)
Uridine (U?S)
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