Tuning aminoacyl-tRNA Affinity to Elongation Factor Tu for Optimal Translation

Jared SchraderUhlenbeck Lab

Olke’s Lab tRNAs!ribozymes

DbpA

Genetic information is encoded in DNA

How does one interpret genetic information?

Source: National Human Genome Research Institute, by artist Darryl Leja

Molecular Biology of the Cell. 4th edition. Alberts B, Johnson A, Lewis J, et al. New York: Garland Science; 2002.

Decoding genetic information

DVDDigital signal Movie

Source: National Human Genome Research Institute, by artist Darryl Leja

Read the DVD

Decode the signal

0011000100

RNA Polymerase

Ribosome and tRNAs

decode signal

Biochemical Functionmessenger

RNA

DNA RNA Protein

Read the DNA

Decode the mRNA

Structures courtesy the PDB

tRNA is the key to mRNA decoding

GTPase Acti

vation

and GTP

Hydrolysis

Pi

Peptide Bond Formation

EF-Tu Release

EF-Tu•GTP

EF-Tu•GDP

EF-Tu•EF-Ts

EF-Ts

EF-Ts

aa-tRNA

Ternary Complex

Binding and DecodingNucleotide

Exch

ange

Accommodation

Ternary Complex Formation

EF-Ts Binding

43 tRNAs but only 1 EF-Tu and ribosome!

EF-Tu recognizes aminoacyl-tRNA along the acceptor and T stems

T. aquaticus EF-Tu•GDPNP•yeast Phe-tRNAPhe

Nissen et al. Science (1995)

EF-Tu

C CA

PheArg 274

His 273

His 67

Lys 52

Glu 226

Asp 227

Uniform affinity between all correctly acylated elongator aa-tRNAs

EF-Tu is specific for the esterified amino acid and the tRNA body

2

1Louie & Jurnak Biochemistry (1985) 2Dale et al Biochemistry (2004) 3Asahara & Uhlenbeck PNAS (2002)

3

DGo aa-tRNA = DGo amino acid + DGo tRNA body

D

-11.0

-10.8

-10.6

-10.4

-10.2

-10.0

-9.8

-9.6

-9.4

-9.2

-9.0

-8.8

C G

G U

G CAU

U AAU

U G

G U

G C

C G

G C

C G

AU

G UU GU A

tRNA Position

T-s tem

EF-Tu recognizes sequence elements in the T stem

Schrader et al. JMB (2009)

A Stem T Stem

tRNA specificity to EF-Tu is controlled by three base pairs in the T stem

Schrader et al. JMB (2009)

E. coli

Is this adaptation of the tRNA universal among bacteria?

Esterified amino acid binding pocket

tRNA specificity patch

Is tRNA affinity conserved across bacteria?

Model to calculate the affinity of any tRNA based upon its sequence

tRNAPhe tRNAVal

tRNAThr

E. coli EF-Tu

E. coli EF-TuT. Thermophilus EF-Tu

DGo Calculated = DGo

wild type tRNA + DDGo 49-65+ DDGo

50-64+ DDGo 51-63

• Data from different tRNAs and EF-Tu• Yeast tRNAPhe T. thermophilus EF-Tu• E. coli tRNAThr E. coli EF-Tu• E. coli tRNALeu T. thermophilus EF-Tu

Submitted

-11.0

-10.8

-10.6

-10.4

-10.2

-10.0

-9.8

-9.6

-9.4

-9.2

-9.0

-8.8

C G

G U

G CAU

U AAU

U G

G U

G C

C G

G C

C G

AU

G UU GU A

Calculating DGo of each bacterial tRNA

49-65 50-64 51-63Predicted DGo

(kcal/mol)

GC UA CG -10.0

GC CG GU -9.9

GU UA AU -9.9

GU GU GC -9.6

CG AU CG -9.6

GC UG CG -9.6

CG CG GU -9.5

CG CG CG -9.8

53 different T-stem sequences among species

Tight Medium Weak

-12

-11

-10

-9

-8

-12 -11 -10 -9 -8

E. coli tRNA Go (kcal/mol)

Mea

n

Go (

kca

l/mo

l)

tRNA affinity is conserved across all bacteria

Despite vast:Growth temperaturePressureFood sourcepHSalinity

and billions of years of evolution

All bacterial cells have this particular adaptation between EF-Tu and tRNA

How does having uniform aa-tRNA affinity to EF-Tu affect decoding on the ribosome?

GTPase Acti

vation

and GTP

Hydrolysis

Pi

Peptide Bond Formation

EF-Tu Release

Ternary Complex

Binding and Decoding

AccommodationEF-Tu•GDP

Too Weak Just RightToo Tight

■Wild Type▲T1 Mutants●Misacylated♦Val-tRNAVal

Mutants

0.01

0.1

1

10

100

0.1 1 10 100 1000

kp

ep (

s-1

)

koff

(s-1 x 10-4)

Tight and weak binding prohibit

decoding!

Schrader et al. PNAS (2011)

Tight

WeakNormal

EF-Tu affinity is like a pair of pants

Tight pants Loose pants Pants that fit

Very stable

Hard to remove

Not stable

Too easy to remove

Stable enough

Easy to remove

EF-Tu residue H66 controls esterified amino acid specificity

C CA

PheArg 274

His 273His 67

Lys 52

Glu 226

Asp 227

In Collaboration with Stephen Chapman

Tight tRNAs lower E. coli fitnesstufB promoter

WT tRNATyrrrnC

tufB promoterTight tRNATyr

rrnC

BsmFI site

Tight

WT

Grow 24 hrs

Dilute

Grow 24 hrs

Dilute

Grow 24 hrs

Dilute

In Collaboration with Margaret Saks, Laura Timmerman, and Mike Jewett

Watching EF-Tu release directly by FRET

GTPase Acti

vation

and GTP

Hydrolysis

Pi

Peptide Bond Formation

EF-Tu Release

Ternary Complex

Binding and Decoding

AccommodationEF-Tu•GDP

Labeled EF-Tu , Labeled aa-tRNA , or Labeled P-site tRNA

In Collaboration with

Barry Cooperman, Wei Liu U-Penn

In Conclusion:

EF-Tu and tRNA are highly evolved to optimally decode mRNAs.

You can think of it like a pair of pants.

Questions?

How the hell did I end up at Northwestern?

• My parent’s live 15 minutes away from campus– Rent free living

• Well known for biochemistry

Making a move to Evanston

• Theory Group wasn’t taking students• Tom Cech was at HHMI• Olke Uhlenbeck just left• At the Colorado interview weekend,

everyone said “Olke Uhlenbeck was great”

Larry Gold

“Olke is not an asshole.”

I really liked Olke’s lab

• went there for my 1st lab rotation and I never really left

• Doing experiments was way more fun than working on a computer

Ivy trying to ship me away

Uhlenbeck Group

• ?? Postdocs• 45 Students

>230 papers and countinga

>15000 citations and countinga

aFrom Google scholar

To be the last student

is lonely…but far more productive!

To be a student

is fun

Empty lab picture

Uhlenbeck Group

Productivity and Fun are anticorrolated

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Pro

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cti

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Ind

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Number of Students

Lee Graduated

Sarah Graduated

To all former students: How could you leave me here alone with this guy!

Sorry Peggy, Irina, and Emine, you will now have to fend for yourselves!

What to do next?• Bacterial habitat is not LB in liquid

culture!– Laboratory conditions are not the

natural growth conditions!

• Bacterial populations turn out to have multiple cell-types, spread roles around, and act cooperatively like a multicellular organism

• How do we understand this bacterial development, especially in the context of the natural habitat?

To Lucy Shapiro’s lab!

• 553 genes (19% of genome) are expressed at a specific phase of the cell cycle

• How does translation/sRNA mediated gene regulation affect development?

• Thank you Erin for letting me pursue this

James Watson?

Twitter updates

@Jmschrad

jmschrad@gmail.com

Keep in touch

AcknowledgementsLarry Gold – Great advice: 1) Olke is not an asshole 2) Lucy Shapiro is a

good advisor

Olke Uhlenbeck – Being a good advisor, giving me a great environment to grow intellectually, paying the bills!

Olke’s lab – Great group to work with• Sarah Ledoux – Getting me started on tRNAs• Lee Sanderson – Getting me started on EF-Tu• Peggy Saks – Getting me started on E. coli project• Stephen Chapman – Collaborating on a lot of experiments• Ivy Garcia – Telling me how to do a lot of stuff• Jenny Nelson• Lisa Sharpe-Elles• Irina Shepotinovskaya (Go Bulls)• Taraka Dale• Mikolaj Olejniczak• Eda Koculi• Merideth Burkhart• Ginger Leigh – Submitting all my papers and finding typos• Ehren Fournier• Kevin Keegan• Emine Yikilmaz

Thesis committee – Pushing Olke to read my manuscripts

• Jon Widom• Erik Sontheimer• Alfonso Mondragon

Lab Neighbors – Keeping the floor fun

• Andreas Matouschek• Matouschek Lab –

• Nordic Track• Nespresso

• John Marko• Marko Lab• Curt Horvath• Horvath Lab (Old

Neighbors)

IBIS Office – Reminding me to register for classes

• Sacha Patera• Cathy Prullage• Traci Galbaugh

Business Office – Paying me (usually)

ORS – Keeping me safe

Jewett Lab - Fun discussions at group meeting• (Too many to list!)• Mike & Laura – tRNA

toxicity experiments

Chicago RNA Club

Biophysics Club

Classmates• 2005 enrollees

My Family

My Friends

Beer Hour

ISOEveryone who is

reading this!