Transcript 1 Frameshift Stimulation by the ScYLV P1-P2 RNA

Structure and Folding of Ribosomal
Frameshift-stimulating mRNA Pseudoknots
David Giedroc
Department of Biochemistry and Biophysics
Texas A&M University
The Central Dogma….
DNA
transcription
REGULATION:
Transcriptional regulators; RNAi
Metal Homeostasis/Resistance
“A bioinorganic twist”
mRNA
translation
REGULATION:
Ribosomal Recoding, e.g.
-1 Ribosomal Frameshifting
Protein
Our interests: Molecular Determinants of Biological Regulation
Ribosomal Recoding
•Ribosomal or translational recoding: A programmed alteration in the usual triplet
decoding of the mRNA into protein by the elongating ribosome
•Documented to occur in all organisms (bacteria to mammals to plants)
•Translation recoding signals are embedded in the mRNA itself
•Recoding comes in several flavors:
1) Readthrough via stop codon (UAG) supression: fusion protein
2) Bypass or hopping (T4 gene 60)
3) Incorporation of nonstandard amino acids, including
selenocysteine (#21) and L-pyrrolysine (#22) via stop codon redefinition
4) Frameshifting: change in reading frame to create a fusion protein
+1 PRF: XXX YYY ZZ
XXX YYZ Z (antizyme: polyamine biosyn; E. coli RF2)
-1 PRF: X XXY YYZ
From Baranov et al. (2002) Gene
XXX YYY Z (animal/plant RNA viruses; E. coli dnaX)
Many RNA viruses employ -1 ribosomal frameshifting
fs = 5%
protease
P2
RdRP
antiviral target
Translational Recoding by -1 Frameshifting (e.g., PEMV-1)
[[
[[
-1:
0:
Frameshifting efficiencies typically range from 5-30% in RNA viruses
RNA Pseudoknot Folding Topology
Example: Phage T2 gene 32 translational operator
Du, Giedroc and Hoffman (1998) Biochemistry
Structural Diversity in RNA Pseudoknots
Giedroc et al. (2000) J Mol Biol
Modeling the FS signal on the bacterial 70S ribosome
L7/L12
Giedroc et al. (2000) J Mol Biol
(1JGO)
Plant et al. (2003) RNA
Overview of Translation
eEF2 (EF-G)-catalyzed
translocation perturbed by the
downstream pseudoknot
Pseudoknot kinetically enhances partitioning of the elongating ribosome into
the new -1 reading frame during translocation [Namy et al. (2006) Nature].
EM structure of a stalled 80S ribosome-pseudoknot complex
mRNA channel
Frameshift-stimulators are structurally diverse
Hypothesis:
Some general feature(s) of pseudoknot structure, stability and/or
kinetic lability are major determinants for frameshift stimulation
Our approach:
Systematically investigate the structures, stability determinants
of a group of closely related -1 PRF-stimulating pseudoknots
Objective:
Identify key molecular features that modulate frameshift stimulation,
independent of folding
Evaluate the functional importance of these interactions in detail
in a suitable mechanistic assay
Plant Luteoviruses
Mosaic yellow pattern on leafs
Enations
Typical luteovirus particles
Smith and Barker (1999) The Luteoviridae (CABI)
Symptoms of infection with pea enation mosaic virus (PEMV-1)
Proposed 2º structures of BWYV, PEMV-1 and ScYLV P1-P2 pseudoknots
Beet Western Yellows Virus
BWYV infection of escarole
Pea Enation Mosaic Virus (RNA-1)
Sugarcane Yellow Leaf Virus
1.6 Å structure of the BWYV pseudoknot
S2
5’
L2
A21
C22
U13 (L3)
A23
A24
S1
3’
C8
A25
L1
Su, Egli, Rich et al (1999) Nat Struct Biol
Sugarcane Yellow Leaf Virus P1-P2 RNA Pseudoknot as a Structural Target
Slip-site
NMR Spectroscopy as a Biomolecular Structural Tool
Texas A&M (500, 600 MHz:)
The Scripps Research Institute (900 MHz)
Large fixed Bo: Nuclear magnets (protons, etc.) align in the magnetic field,
and absorb radiofrequency energy; the decay of this excited state is a strong
function of structural environment of individual atoms.
The predicted ScYLV P1-P2 mRNA pseudoknot adopts a well-folded PK conformation
H5(C5C4N)H:
NH2 protons of C27 reside
in an unusual environment
Cornish, Hennig, Giedroc (2005) Proc Natl Acad Sci
Loop L2 adenosine amino protons are resolved and protected from solvent exchange
Cornish, Hennig, Giedroc (2005) Proc Natl Acad Sci
Solution structure of the ScYLV P1-P2 RNA pseudoknot
Cornish, Hennig, Giedroc (2005) Proc Natl Acad Sci
50±14º
Solution Structure of the ScYLV P1-P2 RNA Pseudoknot
L2
5’
S1
A13
C25
S2
3’
G9 (L1)
Cornish, Hennig, Giedroc (2005) Proc Natl Acad Sci
L2
S1
A13
C8+
S2
C25
G9 (L1)
Cornish, Hennig, Giedroc (2005) Proc Natl Acad Sci
ScYLV Pseudoknot: Five consecutive base triples
cis-Watson-Crick/sugar edge base pairing
Hoogsteen base pairing
*See also HCV IRES (Kieft et al., 2002)
*See also A riboswitch (Serganov et al., 2004);
G riboswith (Batey et al., 2004) [C•(U-A)]
Cornish, Hennig, Giedroc (2005) Proc Natl Acad Sci
CURVES analysis of BWYV, PEMV-1 and ScYLV pseudoknot topologies
BWYV
•Helical over-rotation: 89º
•Horiz. displacement: 5.5 Å
S1
S2
PEMV-1
A-form coaxial helices
S1
•Helical over-rotation: 98º
•Horiz. displacement: 5.0 Å
S2
ScYLV
S1
•Helical over-rotation: 103º
•Horiz. displacement: 7.6 Å
S2
-1 Frameshift Stimulation by the ScYLV P1-P2 RNA Pseudoknot
The ≈2.5-fold difference in FS stimulation
between ScYLV and BWYV pseudoknots
derives entirely with a 3’ C
A substitution
in loop L2
Cornish, Hennig, Giedroc (2005) Proc Natl Acad Sci
The structures of the C27A and WT ScYLV pseudoknots are essentially identical…
…despite easily measurable structural perturbations at the helical junction region
Cornish, Stammler & Giedroc (2006) RNA
The C27A ScYLV pseudoknot is destabilized relative to the WT RNA
Cornish, Hennig, Giedroc (2005) Proc Natl Acad Sci
Helical junction pairwise coupling free energies (d) of WT and C27A pseudoknots
Cornish & Giedroc (2006) Biochemistry
The mechanical model for stimulation of -1 PRF during translocation
Ian Brierley, Univ of London
Namy et al. (2006) Nature
Prediction: More stable pseudoknots would be more effective frameshift-stimulators,
generally consistent with our findings.
However,stabilizing interactions localized in the helical junction region appear far
more important.
Hypothesis: Helical junction interactions may function as GATEKEEPERS (kinetic
barrier) to ribosome-mediated pseudoknot unwinding.
Another perspective on ribosome-mediated unfolding during -1 PRF
So the moral of the story is…….
Embrace Physical Chemistry!!
NIH Predoctoral Training Programs in Biophysical Chemistry,
Chemistry-Biology Interface
Conclusions
Plant viral -1 frameshift-stimulating mRNA pseudoknots adopt unique triple
helical architectures characterized by numerous loop-stem (L1-S2 & L2-S1)
base triple (quadruple) interactions.
Despite significant differences in the helical junctions among all three luteoviral
pseudoknots, their global folds are remarkably similar.
A major determinant for modulating frameshifting efficiencies by luteoviral
pseudoknots is the 3’ nucleotide in loop L2. We propose that the helical junction
functions as a kinetic barrier to ribosome-mediated pseudoknot unfolding. Groundstate structure is a poor predictor of frameshift-stimulation.
These variant pseudoknots will be excellent tools with which to mechanistically
probe how pseudoknots stimulate frameshifting (laser-based optical tweezers).
Acknowledgments
Dr. Peter Cornish
Dr. Carla Theimer
Dr. Paul Nixon
Dr. Peter Cornish
Suzanne Stammler
Lichun Li
Saritha Suram
Dr. Raza Khan
Dr. Mirko Hennig
The Scripps Research Institute
Dr. David W. Hoffman
University of Texas at Austin
NIH
NSF
Texas Higher Education Coordinating Board