XRay Crystallographic Studies of RNAProtein Interactions in Translation

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X-Ray Crystallographic Studies of RNA-Protein
Interactions in Translation

• Mark Dunstan
• Conn Lab G19

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Key areas of interest

• Regulation of PKR functionVA RNA and IFN-? 5
  UTR mRNA
• L11-rRNA Ribosomal Domain thiostrepton binding
• Structural Basis for Thiostrepton Antibiotic
  Resistance (TRMT)

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Co-evolution of protein and RNA structures within
a highly conserved ribosomal domain

• G1076-C1062 is replaced with U-A
• in the RNA and Ser is replaced by Asn
  at position 69 in L11.
• 2. Illustrates natural co-evolution of protein
  and RNA structures to maintain key contacts.

Dunstan, M.S. et al Chemistry biology, vol 12,
1-6 February, 2005
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Key areas of interest

• Regulation of PKR functionVA RNA and IFN-? 5
  UTR mRNA
• L11-rRNA Ribosomal Domain thiostrepton binding
• Structural Basis for Thiostrepton Antibiotic
  Resistance (TRMT)

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Thiostrepton Resistance Methyltransferase.

• 30KDa protein Found in streptomyces azeus.
• Methylates RNA base A1067 making the organism
  resistant to thiostrepton.
• Numerous Crystal targets in the enzyme cycle.

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TRMT the 58 nt rRNA

• Several crystal hits form Hampton and JB
  screens.
• Crystals obtained for both TRMT only and
  TRMT58nt rRNA.
• SDS and acrylamide gel analysis of one crystal
  shows presence of both protein and RNA.
• Crystals diffract to 2.8A

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Future goals

• Solve the structure of TRMT in various stages to
  fully characterize its enzymatic function.
• X-ray crystal structure of Thiostrepton bound
  RNA-protein complex.

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Intermission
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Investigating ligand binding in sweet taste G
protein-coupled receptors
Jeanette Hobbs Conn Lab - G19
T1R2
T1R3
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(No Transcript)
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Expression strategies for T1R NTDs
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A change in fluorescence intensity indicates
ligand interactions with MBP-T1R3 NTD
Normalised fluorescence intensity (AU)
Wavelength (nm)
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SRCD can differentiate between types of secondary
structure
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Glucose induces a structural change in T1R3
Glucose-dependent structural changes are
T1R3-specific
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Sugars induce a structural changes in T1R2 NTD
that are distinct from those seen in T1R3
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X-ray crystallography
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Sweet proteins likely interact with both T1R2 and
T1R3 NTDs
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X-ray structures of MNEI and G16A MNEI
GLY16
MNEI 1.15 Å
G16A MNEI 2.0 Å
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G16A mutation (red) causes local change in MNEI
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In summary..
Our data suggest a novel model for T1R2-T1R3
sweet receptor activation. -Distinct binding
affinities for each domain. -Different
conformational changes.