Agris Lab


AGRIS LABORATORY RESEARCHERS
FRANCK A. P. VENDEIX RESEARCH Characterization and determination of the structure, dynamics and interactions of Nucleic acids (DNA/RNA) and their complexes with small molecules and proteins by using solution state NMR and biophysical techniques. RESEARCH IN PROGRESS Project 1: The decoding of genome – ASL tRNA (anticodon)-mRNA (codon) interactions Transfer RNAs (tRNAs) play a crucial role in decoding the genome. This decoding process is critical to the accuracy and efficiency of protein synthesis. Accurate protein synthesis on the ribosome depends on the correct base pairing between the many tRNA anticodons and the 61 degenerate, amino acid codons in messenger RNA (mRNA) codon. Because there are far fewer tRNAs than codons, many tRNAs recognize multiple codons. A few tRNAs are restricted to their recognition of the cognate or canonical codon, whereas others decode 1, 2 and or even 3 codons besides the cognate codon. In many cases, proper recognition of the wobble codon by the corresponding anticodon requires the presence of naturally occurring modified nucleosides found at specific positions in the anticodon stem-loop (ASL) of the tRNA. Our objectives are to gain a detailed physicochemical understanding of tRNA’s anticodon modifications from which general principles for codon recognition in all organisms can be formulated. The solution structures of unmodified ASLVal3UAC and ASLVal3UAC-cmo5U34;m6A37 have been successfully determined. ASLVal3UAC-cmo5U34;m6A37 has an A-form RNA stem, and a canonical "U-turn" at U33. Considerable stacking in the anticodon loop structures the modified anticodon relative the anticodon of the unmodified ASL. The stacking continues up the 3‘ side of the loop and into the 3'side of the stem. This clearly demonstrates that modifications pre-structure the anticodon loop prior to A-site codon binding. Additional, subtle conformational changes accompany anticodon binding to the codon on the ribosome. cmo5U34 Loop cmo5U34 Stem A37 U34 m6A37 ASLVal3UAC-cmo5U34;m6A37 ASLVal3UAC Project 2: HIV RNA/tRNA primer interactions Human tRNALys3 is recruited by HIV to prime reverse transcription. The U-rich anticodon of tRNALys3 is annealed to the A-rich Loop 1 region of the HIV genome and thus, could be similar to anticodon/codon binding on the ribosome. However, the anticodon domain conformation is "melted" in vivo by HIV nucleocapsid protein and then, the tRNA annealed to a similarly denatured HIV Loop 1. Using NMR derived distance and angle constraints and refined molecular dynamics, we determined the resulting structure of this interaction to RMSD of ~1.5 Å. The tRNA's anticodon's "U-turn" conformation is lost. Its modifications (s2U34, Ψ39) enhance the affinity of the interaction. The structure is stabilized by two non-canonical base pairs: Ψ39-U, and A38-G. We conclude that though the nucleoside base pairs of anticodon to codon are the same as that of ribosome-mediated codon interaction, and that modifications stabilize the binding, the final structure is quite different than that of the anticodon loop binding to codon in the A-site. PUBLICATIONS Agris, P.F., Vendeix, F.A.P. and Graham, W.D. (2007) tRNA's Wobble Decoding of the Genome: 40 Years of Modification, J. Mol. Biol. 366, 1-13. Weixlbaumer, A., Murphy, F.,Vendeix, F., Graham, W.D., Dziergowska, A., Gustilo, E., Sproat, B., Ramakrishnan, V., and Agris, P.F. (2007) The structure of four codon decoding by a modified uridine wobble base. Nature Struct. Mol. Biol., in press F. A. Vendeix, R. Banks, Y.Z. Xu and V. Ramesh (2006) NMR structural characterisation of mismatch DNAs and their interaction with E. coli MutS repair enzyme; Nucleic acids Research :under review. Publications in preparation: F. A. Vendeix, W. D. Graham, A. Dziergowska, B. Sproat, A. Malkiewicz, and P. F. Agris (2006) Wobble-Position Modifications Pre-structure tRNA's Anticodon for Ribosome-Mediated Codon Binding. F. A. Vendeix, E. DeRose, R. E. London, R. Guenther, A. Malkiewicz, and P. F. Agris (2006) HIV RNA/tRNA Primer Anticodon Interactions: Enhancement by Modified Nucleosides Reduced Dynamics. F. A. Vendeix, R. Banks, Y.Z. Xu and V. Ramesh (2006) NMR studies of O4meT and O6meT modified DNA base pairs and their interactions with E. coli MutS repair enzyme. Franck A. Vendeix, William D. Graham and Paul F. Agris (2007) Predictive Modified Anticodon-Codon Base Pair Formation in silico.

AGRIS LABORATORY RESEARCHERS

FRANCK A. P. VENDEIX

RESEARCH

Characterization and determination of the structure, dynamics and interactions of Nucleic acids (DNA/RNA) and their complexes with small molecules and proteins by using solution state NMR and biophysical techniques.

RESEARCH IN PROGRESS

Project 1: The decoding of genome – ASL tRNA (anticodon)-mRNA (codon) interactions

Transfer RNAs (tRNAs) play a crucial role in decoding the genome. This decoding process is critical to the accuracy and efficiency of protein synthesis. Accurate protein synthesis on the ribosome depends on the correct base pairing between the many tRNA anticodons and the 61 degenerate, amino acid codons in messenger RNA (mRNA) codon. Because there are far fewer tRNAs than codons, many tRNAs recognize multiple codons. A few tRNAs are restricted to their recognition of the cognate or canonical codon, whereas others decode 1, 2 and or even 3 codons besides the cognate codon. In many cases, proper recognition of the wobble codon by the corresponding anticodon requires the presence of naturally occurring modified nucleosides found at specific positions in the anticodon stem-loop (ASL) of the tRNA.

Our objectives are to gain a detailed physicochemical understanding of tRNA’s anticodon modifications from which general principles for codon recognition in all organisms can be formulated.

The solution structures of unmodified ASLVal3UAC and ASLVal3UAC-cmo5U34;m6A37 have been successfully determined. ASLVal3UAC-cmo5U34;m6A37 has an A-form RNA stem, and a canonical "U-turn" at U33. Considerable stacking in the anticodon loop structures the modified anticodon relative the anticodon of the unmodified ASL. The stacking continues up the 3‘ side of the loop and into the 3'side of the stem. This clearly demonstrates that modifications pre-structure the anticodon loop prior to A-site codon binding. Additional, subtle conformational changes accompany anticodon binding to the codon on the ribosome.

cmo5U34
Loop
cmo5U34 Stem A37 U34 m6A37 ASLVal3UAC-cmo5U34;m6A37 ASLVal3UAC
Project 2: HIV RNA/tRNA primer interactions
Human tRNALys3 is recruited by HIV to prime reverse transcription. The U-rich anticodon of tRNALys3 is annealed to the A-rich Loop 1 region of the HIV genome and thus, could be similar to anticodon/codon binding on the ribosome. However, the anticodon domain conformation is "melted" in vivo by HIV nucleocapsid protein and then, the tRNA annealed to a similarly denatured HIV Loop 1.
Using NMR derived distance and angle constraints and refined molecular dynamics, we determined the resulting structure of this interaction to RMSD of ~1.5 Å. The tRNA's anticodon's "U-turn" conformation is lost. Its modifications (s2U34, Ψ39) enhance the affinity of the interaction. The structure is stabilized by two non-canonical base pairs: Ψ39-U, and A38-G. We conclude that though the nucleoside base pairs of anticodon to codon are the same as that of ribosome-mediated codon interaction, and that modifications stabilize the binding, the final structure is quite different than that of the anticodon loop binding to codon in the A-site.

PUBLICATIONS

Agris, P.F., Vendeix, F.A.P. and Graham, W.D. (2007) tRNA's Wobble Decoding of the Genome: 40 Years of Modification, J. Mol. Biol. 366, 1-13.
Weixlbaumer, A., Murphy, F.,Vendeix, F., Graham, W.D., Dziergowska, A., Gustilo, E., Sproat, B., Ramakrishnan, V., and Agris, P.F. (2007) The structure of four codon decoding by a modified uridine wobble base. Nature Struct. Mol. Biol., in press
F. A. Vendeix, R. Banks, Y.Z. Xu and V. Ramesh (2006) NMR structural characterisation of mismatch DNAs and their interaction with E. coli MutS repair enzyme; Nucleic acids Research :under review.
Publications in preparation:
F. A. Vendeix, W. D. Graham, A. Dziergowska, B. Sproat, A. Malkiewicz, and P. F. Agris (2006) Wobble-Position Modifications Pre-structure tRNA's Anticodon for Ribosome-Mediated Codon Binding.
F. A. Vendeix, E. DeRose, R. E. London, R. Guenther, A. Malkiewicz, and P. F. Agris (2006) HIV RNA/tRNA Primer Anticodon Interactions: Enhancement by Modified Nucleosides Reduced Dynamics.
F. A. Vendeix, R. Banks, Y.Z. Xu and V. Ramesh (2006) NMR studies of O4meT and O6meT modified DNA base pairs and their interactions with E. coli MutS repair enzyme.
Franck A. Vendeix, William D. Graham and Paul F. Agris (2007) Predictive Modified Anticodon-Codon Base Pair Formation in silico.