Structure and Folding of RibosomalFrameshift-stimulating mRNA Pseudoknots

David GiedrocDepartment of Biochemistry and Biophysics

Texas A&M University

The Central Dogma….

DNA

mRNA

Protein

transcription

translation

REGULATION:Transcriptional regulators; RNAi

Metal Homeostasis/Resistance“A bioinorganic twist”

REGULATION:Ribosomal Recoding, e.g.

-1 Ribosomal Frameshifting

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, includingselenocysteine (#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 XXX YYY Z (animal/plant RNA viruses; E. coli dnaX)

From Baranov et al. (2002) Gene

Many RNA viruses employ -1 ribosomal frameshifting

P2

protease

RdRP

fs = 5% antiviral target

0:-1:

Translational Recoding by -1 Frameshifting (e.g., PEMV-1)

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

Giedroc et al. (2000) J Mol Biol

Structural Diversity in RNA Pseudoknots

L7/L12

Plant et al. (2003) RNA

Giedroc et al. (2000) J Mol Biol

Modeling the FS signal on the bacterial 70S ribosome

(1JGO)

Overview of Translation

Pseudoknot kinetically enhances partitioning of the elongating ribosome intothe new -1 reading frame during translocation translocation [Namy et al. (2006) Nature][Namy et al. (2006) Nature].

eEF2 (EF-G)-catalyzedtranslocation perturbed by thedownstream pseudoknot

EM structure of a stalled 80S ribosome-pseudoknot complex

mRNA channel

Frameshift-stimulators are structurally diverse

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

Some general feature(s) of pseudoknot structure, stability and/orkinetic lability are major determinants for frameshift stimulation

Our approach:

Systematically investigate the structures, stability determinantsof a group of closely related -1 PRF-stimulating pseudoknots

Hypothesis:

Objective:

Smith and Barker (1999) The Luteoviridae (CABI)

Typical luteovirus particlesSymptoms of infection with pea enation mosaic virus (PEMV-1)

Mosaic yellow pattern on leafs

Enations

Plant Luteoviruses

Beet Western Yellows Virus Pea Enation Mosaic Virus (RNA-1) Sugarcane Yellow Leaf Virus

BWYV infection of escarole

Proposed 2º structures of BWYV, PEMV-1 and ScYLV P1-P2 pseudoknots

Su, Egli, Rich et al (1999) Nat Struct Biol

1.6 Å structure of the BWYV pseudoknot

L2

S1

S2

L1

A23

A24

A25

5’

3’

C8

U13 (L3)C22

A21

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 strongfunction of structural environment of individual atoms.

The predicted ScYLV P1-P2 mRNA pseudoknot adopts a well-folded PK conformation

NH2 protons of C27 reside in an unusual environment

H5(C5C4N)H:

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

50±14º

Solution structure of the ScYLV P1-P2 RNA pseudoknot

Cornish, Hennig, Giedroc (2005) Proc Natl Acad Sci

Solution Structure of the ScYLV P1-P2 RNA Pseudoknot

S1

S2

L2

5’

3’

C25

G9 (L1)

A13

Cornish, Hennig, Giedroc (2005) Proc Natl Acad Sci

S1

S2

L2

C25

G9 (L1)

A13

C8+

Cornish, Hennig, Giedroc (2005) Proc Natl Acad Sci

ScYLV Pseudoknot: Five consecutive base triples

*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)]

cis-Watson-Crick/sugar edge base pairing

Hoogsteen base pairing

Cornish, Hennig, Giedroc (2005) Proc Natl Acad Sci

•Helical over-rotation: 98º•Horiz. displacement: 5.0 Å

•Helical over-rotation: 89º•Horiz. displacement: 5.5 Å

•Helical over-rotation: 103º•Horiz. displacement: 7.6 Å

CURVES analysis of BWYV, PEMV-1 and ScYLV pseudoknot topologies

BWYV

PEMV-1

ScYLV

S1

S1

S1

S2

S2

S2

A-form coaxial helices

The ≈2.5-fold difference in FS stimulationbetween ScYLV and BWYV pseudoknots

derives entirely with a 3’ C A substitution

in loop L2

-1 Frameshift Stimulation by the ScYLV P1-P2 RNA Pseudoknot

Cornish, Hennig, Giedroc (2005) Proc Natl Acad Sci

The structures of the C27A and WT ScYLV pseudoknots are essentially identical…

Cornish, Stammler & Giedroc (2006) RNA

…despite easily measurable structural perturbations at the helical junction region

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 () of WT and C27A pseudoknots

Cornish & Giedroc (2006) Biochemistry

The mechanical model for stimulation of -1 PRF during translocation

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.

Ian Brierley, Univ of London

Namy et al. (2006) Nature

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. Ground- state 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. Carla TheimerDr. Paul Nixon

Dr. Peter CornishSuzanne Stammler

Lichun LiSaritha SuramDr. Raza Khan

Dr. Mirko HennigThe Scripps Research Institute

Dr. David W. HoffmanUniversity of Texas at Austin

NIHNSF

Texas Higher Education Coordinating Board

Dr. Peter Cornish