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

---GACTAGAAAG----

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