The choreography of biological processes
Structural Biology Revolution
Driven by technological improvements
Structures in the protein database (pdb)
Mechanism is the key challenge in
biology
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Genomic Data Molecular Structure
MECHANISM
mRNA
50S
30S
250 Å
200 Å
1.6 MDa 2 RNAs 34 proteins
0.9 MDa 1 RNA 21 proteins
P E A
The ribosome
5’
3’
translocation
milliseconds - seconds
µM
1-20 aa/s in vivo, 1-3 aa/s in vitro
Nascent chain folding
Time evolution of biological systems
Biological systems evolve temporally
• Conformation • Composition • Chemical • Spatial
FRET probes conformational dynamics
Low FRET High FRET
Low FRET High FRET
Time Flu
ore
scence I
nte
nsity
Low FRET
High FRET
Single-Molecule FRET
time
inte
nsi
ty
time in
ten
sity
averaging
Solution Averaging Destroys Signal
time
inte
nsi
ty
time in
ten
sity
averaging
Solution Averaging Destroys Signal
Obverve translation
directly by single
ribosomes
Why?
•Translation is endpoint of gene expression
• highly regulated, controlled
initiation/start site selection
termination
microRNAs and RNA-protein interactions
• linked to protein folding, processing
elongation rates?
• misregulation in many diseases
cancer, viral infection, genetic disorders
How?
• Fluorescently-label tRNA, mRNA, ribosomes and
factors (bacteria, yeast, human)
• Track single-ribosomes composition and
conformation in real time.
• First understand each step in translation:
initiation, elongation, termination
• Then understand dynamic regulation on
specific mRNAs
• Sort through pathways of translation
Monitor composition in real time with fluorescent
ligands
5’
3’
µM
Observing signal-molecule binding kinetics
Solution
Glass
Observing signal molecule binding kinetics using TIRF
on
off
Time Flu
ore
scence I
nte
nsity
Statistically analyze the distributions to obtain kinetics
Arrival time Lifetime
Time Flu
ore
scence I
nte
nsity
Extending to multiple colors tracking multiple factors
Time
Flu
ore
scence I
nte
nsity
TIRF is limited to 50 nM of labeled factors in solution
Limited to 50 nM
Most physiological Kd are in µM range
Overcoming the concentration barrier with the zero-
mode waveguide (ZMW)
Chen, Jin, Dalal, Ravi, et al. PNAS 111.2 (2014)
~150 nm
Up to 5 µM
High-throughput multicolor detection with the ZMW
Signal Signal
Signal
Signal
Al
SiO2
Chen, Jin, Dalal, Ravi, et al. PNAS 111.2 (2014)
High-throughput multicolor single-molecule dynamics
on the PacBio RS
Chen, Jin, Dalal, Ravi, et al. PNAS 111.2 (2014)
Obverve translation
directly by single
ribosomes
Compositional dynamics of translation in real time
Real time observation of translation
Tracking conformation with FRET
210 Å
45 Å
Inte
nsity
Wavelength
Intersubunit FRET follows ribosome
conformational dynamics
Time Flu
ore
scence I
nte
nsity
Low FRET High FRET
Following the substeps of elongation
Following ribosome conformation in real time
tRNA arrival
Peptide bond formation GTP hydrolysis
Translocation
Non-ROTATED State
High FRET
ROTATED State
Low FRET
Aitken et al. Nat. Struct & Mol. Biol. 2010
Peptide bond formation
Non-rotated
Subunit rotation
Non-rotated
Subunit rotation
6°
Rotated
EF-
G(GTP)
Preparation for translocation
Rotated
Chen, Jin, et al. Nature structural & molecular biology 20.6 (2013)
Non-rotated Rotated Non-rotated
Phe
Correlating conformation and tRNA occupancy
Chen, Jin, et al. Nature structural & molecular biology 20.6 (2013)
Non-rotated Rotated Non-rotated Rotated
Phe Lys
Correlating conformation and tRNA occupancy
Chen, Jin, et al. Nature structural & molecular biology 20.6 (2013)
Non-rotated Rotated Non-rotated Rotated Non-rotated
Phe Lys
Correlating conformation and tRNA occupancy
Experiments to map composition and
conformation of biological systems
Ligand binding multiple ligand binding conformational changes Ligand binding-conformational
changes
Flu
ore
scence inte
nsity
Time (s)
TC waiting time
EF-G waiting time
1 2 3 4 5 6 7 8 9 10 1
1 12 13 14 15 16 17 18 19
Tracking translation codon by codon
Does translation unfurl smoothly?
mRNA structure and folding protein structure and folding
Translation rates at each codon in an mRNA
Codon in A-site
No
n-r
ota
ted
sta
te (
s)
Ro
tate
d s
tate
(s)
Translation rate
Codon in A-site
Spectinomycin causes a 3-4x decrease in
translocation rate N
on
-ro
tate
d s
tate
(s)
Ro
tate
d s
tate
(s)
Translation rate
Recoding
ORF1
Frameshifting: a shift in reading frame
Recoding
ORF1
Frameshifting: a shift in reading frame
Recoding
ORF1
ORF2
-1 frame
Frameshifting: a shift in reading frame
Recoding
Bypassing: re-starting translation downstream
Recoding
Bypassing: re-starting translation downstream
Recoding involves multiple pathways
75% Pathway A Protein
A
Recoding involves multiple pathways
75%
25%
75% Pathway A
Pathway B
Protein A
Protein B
Multiple pathways
Recoding involves multiple pathways
mRNA sequence and structure might change
translational dynamics?
Recoding—frameshifting and bypassing
Frameshifting requires breaking of tRNA-mRNA
interactions and slippage
Slip back by one nucleotide
mRNA
codon-anticodon interactions and reading frame
P
A
mRNA features stimulate frameshifting
The P site is positioned 3 nucleotides upstream
of the slippery sequence
A P E
6-7 nts
6 nts
Barriers created by SD and hairpin set the stage
for frame shifting
The long rotated state pause is characteristic of
frameshifted ribosomes
75% 25%
Codon Lys7
Codon in A site Codon in A site
Branchpoint of pathways in -1 frameshifting
Normal translation
No stall
+
No frameshift
Stall
+
Frameshift
25%
75%
Normal translation
decision to frameshift is made prior to pause
Uncoupled translocation leaves the ribosome in
an unconventional rotated state
Unconventional rotated state
with peptidyl-tRNA in P site and
exposed A-site
A/P, P/E hybrid P/E hybrid P classical
rotated rotated non-rotated
EF-Tu-tRNA
substrate EF-G
substrate
frustrated,
non-canonical
state
Uncoupled translocation leaves the ribosome in
an unconventional rotated state
tRNA binds the ribosome multiple times during
the rotated state pause
1 2 3 4
Uncoupled translocation leaves the ribosome in a
rotated state with exposed A site
Chen, Jin, et al. Nature 512.7514 (2014)
Binding of tRNA to the A site actively drives the shift to
the -1 frame
Binding of tRNA to the A site actively drives the shift to
the -1 frame
Reading frame changes during long
“equilibration” period
Normal translation
No stall
Stall
Uncoupled translocation
No frameshift
Frameshift to -1 frame
No frameshift
Normal translation
Hairpin open/close
rRNA-SD engagement
tRNA slippage into -1 frame
Or redefining into 0 frame
EF-G
Changes in frame/decoding may occur generally during long pauses observed
here
100-150 s
Bypassing induced by mRNA rearrangements and
nascent peptide interactions
Bypassing induced by mRNA rearrangements and
nascent peptide interactions
Factors affecting local translational dynamics
1. mRNA sequence
2. mRNA structure
3. nascent peptide chain sequence
The choreography is both simple and complex
Using the RS for dynamics
1. Fuchs, G., Petrov, A. N., Marceau, C. D., Popov, L. M., Chen, J., O'Leary, S. E., Wang, R., Carette, J. E., Sarnow, P., Puglisi, JD. (2015). Kinetic pathway of 40S ribosomal subunit recruitment to hepatitis C virus internal ribosome entry site. Proceedings of the National Academy of Sciences of the United States of America 112 (2): 319-325. PMC4299178.
2. Chen, J, Coakley, A, O’Connor, M, Petrov, A, O’Leary, SE, Atkins, JA, Puglisi, JD (2015). Coupling of mRNA Structure Rearrangement to Ribosome Movement during Bypassing of Non-coding Regions. Cell, 163, 1267-80
3. Nilsson, OB, Hedman, R, Marino, J, Wickles, S, Bischoff, L, Johansson, M, Müller-Lucks, A, Trovato, F, Puglisi, JD, O’Brien, EP, Beckmann, R, von Heijne, G (2015). Co-translational protein folding inside the ribosome exit tunnel. Cell Rep. 12, 1533-40.
4. Choi, J., Ieong, KW, Demerci, H, Chen, J, Petrov, A, Prabhakar, A, O’Leary, SE, Dominissini, D, Rechavi, G, Soltis, SM,Ehrenberg, M, Puglisi, JD (2015). N6-methyladenosine in mRNA disrupts tRNA selection and translation elongation dynamics.
Nature Struct. Mol. Biol. (in press).
5. Noriega, T. R., Chen, J., Walter, P., Puglisi, JD. (2014). Real-time observation of signal recognition particle binding to actively translating ribosomes ELIFE, Oct 30;3. PMC4213662.
6. Chen J, Petrov A, Johansson M, Tsai A, O'Leary SE, Puglisi JD. (2014). Dynamic pathways of -1 translational frameshifting. Nature. 2014 Jun 11. doi: 10.1038/nature13428. PMC4472451.
7. Tsai A, Kornberg G, Johansson M, Chen J, Puglisi JD. (2014). The Dynamics of SecM-Induced Translational Stalling. Cell Rep. 7(5):1521-33. PMC4059775.
8. Johansson M, Chen J, Tsai A, Kornberg G, Puglisi JD. (2014). Sequence-dependent elongation dynamics on macrolide-bound ribosomes. Cell Rep. 7(5):1534-46. PMC4387896.
9. Tsai A, Uemura S, Johansson M, Puglisi EV, Marshall RA, Aitken CE, Korlach J, Ehrenberg M, Puglisi JD. (2013). The Impact of Aminoglycosides on the Dynamics of Translation Elongation. Cell Rep. 3(2):497-508. PMC3766726.
10. Chen J, Dalal RV, Petrov AN, Tsai A, O'Leary SE, Chapin K, Cheng J, Ewan M, Hsiung PL, Lundquist P, Turner SW, Hsu DR, Puglisi JD. (2014). High-throughput platform for real-time monitoring of biological processes by multicolor single-molecule fluorescence. Proc Natl Acad Sci U S A. 111(2):664-9. PMCID: PMC3896158.
I believe all complicated phenomena can be
explained by simpler scientific principles.
-Linus Pauling
Puglisi Group
Colin E. Aitken
R. Andrew Marshall
Sotaro Uemura
Albert Tsai
Alexey Petrov
Jin Chen
Junhong Choi
Guy Kornberg
Arjun Prabhakar
Alex Johnson
Magnus Johansson
Sean O’Leary
Hasan Demirci (SLAC)
NIH GM099687, GM51266
Stanford
Peter Sarnow
Gaby Fuchs
PacBio
David Hsu
Ravi Dalal
Pei-Lin Hsiung
Karen Chapin
Janice Cheng
Mark Ewan
Paul Lundquist
Steve Turner
Jonas Korlach
Sweden
Mans Ehrenberg
Kaweng Leong
Gunnar von Heinje
Magnus Johansson
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