Sponsored by:

Participating Experts:Joshua Plotkin, Ph.D.University of PennsylvaniaPhiladelphia, PA

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Brought to you by the Science/AAAS Business Office

Christine Vogel, Ph.D.University of Texas at AustinAustin, TX

Mark Welch, Ph.D.DNA2.0Menlo Park, CA

28 October, 2009

From Genes to ProteinsFrom Genes to ProteinsThe Impact of Gene Sequence onTranslation and Expression

coding-sequence determinants of gene expression

joshua b. plotkinuniversity of pennsylvania

grzegorz kudla

andrew murray

david tollervey

in collaboration with

the genetic code

the expression code

High CAI =>High expression

Low CAI =>Low expression

codon adaptation (codon bias)

what features of coding sequences influence expression levels?

• synthesize library of synonymous GFP genes

• systematically interrogate effects of codon usage on transcription, mRNA stability, translation (starting in e. coli)

ATG TAA

ATG TAA

ATG TAA

experimental plan

5’...GGGNGTNCTNCARG

CANGANGTYCTYCA...5’

N = {25%A, 25%C, 25%G, 25%T}R = {50%A, 50%G}Y = {50%C, 50%T}

GFP library - synthesis

GFP library - alignment

Completely random mutations (cf Welch et al)

synthetic GFPs

0

0.2

0.4

0.6

0.8

1

0 0.2 0.4 0.6 0.8 1

GC3

CA

I

all E.coli genes

0

0.2

0.4

0.6

0.8

1

0 0.2 0.4 0.6 0.8 1

GC3

CA

I

GFP library – sequence diversity

in vitro recombination

reaction

(Gateway entry vector -> Gateway expression vector)

grow overnight to saturation

dilute

1/15

grow 1hgrow 3hinduce expression(T7 polymerase)

1 mM IPTGmeasure GFP fluorescence

protocol (E. coli)

E. coli

Inoculate mediumwith 4 replicates of the

same GFP

GFP library – protein levels

0

2000

4000

6000

8000

10000

12000

GFP ID

fluor

esce

nce

GFP library – protein levels

codon adaptation and protein levels

cf Bulmer (1991)

fluor

esce

nce

codon adaptation (CAI)

mRNA folding and protein levels

cf Andersson & Kurland (1990)Eyre-Walker & Bulmer (1993)

fluor

esce

nce

mRNA folding energy (nt -4 to +37)

mRNA folding and protein levels

mRNA folding energy (nt -4 to +37)

fluor

esce

nce

mRNA folding and protein levelsS

igni

fican

ce (-

log

p)

window center (nt)

mRNA folding and protein levels

endogenous e. coli genes may have already undergone selection for reduced 5’ mRNA structure:

energy (-4 to +37) vs (+38 to +79): Wilcoxon p

R2 = 0.231

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

0.2 0.3 0.4 0.5 0.6

codon adaptation and cellular fitness

codon adaptation (CAI)

optic

al d

ensi

ty

codon adaptation and cellular fitness

Toxic mistranslation-induced misfolding (Drummond et al 2005)?

r(CAI, fluor/mRNA) = 0.09 (ns)

r(CAI, fluor/coomassie)= -0.07 (ns)

…but even an undetectably small amount of mistranslation-induced toxicity could impose a large fitness cost

• codon adaptation does not correlate with expression in E. coli

• 5’ mRNA structure had a predominant effect on gene expression in our data (based on random mutations)

• significant residual variation yet unexplained

• poor codon adaptation reduces cellular fitness, likely by imposing a load on ribosome pool

summary

grzegorz kudla

andrew murray

david tollervey

thanks!

The relationship between protein and mRNA expression

levels

Christine Vogel

University of Texas at Austin

The Central Dogma of Biology

ProteinProtein

RNARNA

DNADNA

structures: http://www.molecularstation.com

Transcription

Translation

mRNA degradation

Protein degradation

The Central Dogma of Biology

ProteinProtein

RNARNA

DNADNATranscription

Translation

mRNA degradation

Protein degradation

structures: http://www.molecularstation.com

Multiple mechanisms regulate protein expression

RNARNA

ProteinProtein

AAA(A)nCap

Abreu, Molecular BioSystems, 2009, DOI: 10.1039/b908315d

Translation

poly(A) tail

Multiple mechanisms regulate protein expression

RNARNA

ProteinProtein

AAA(A)nCap

Ribosome

Translation

poly(A) tailInternal entry sitesuORFs

Abreu, Molecular BioSystems, 2009, DOI: 10.1039/b908315d

Multiple mechanisms regulate protein expression

RNARNA

ProteinProtein

AAA(A)nCapNucleotide composition

Codon usage

miRNARNA-binding protein

Ribosome

Translation

poly(A) tailInternal entry sitesuORFs

Abreu, Molecular BioSystems, 2009, DOI: 10.1039/b908315d

Multiple mechanisms regulate protein expression

RNARNA

ProteinProteinUb

UbUbPEST

NH2

K

AAA(A)nCap

Protein degradation

poly(A) tail

Abreu, Molecular BioSystems, 2009, DOI: 10.1039/b908315d

Multiple mechanisms regulate protein expression

RNARNA

ProteinProtein

AAA(A)nCap

Protein degradation

poly(A) tail

Ubiquitinylation

N-degrons

Degradation signals

Ub

UbUbPEST

NH2

K

Abreu, Molecular BioSystems, 2009, DOI: 10.1039/b908315d

Multiple mechanisms regulate protein expression

RNARNA

ProteinProtein

Amino acid compositionStructure

AAA(A)nCap

Protein degradation

poly(A) tail

Ubiquitinylation

N-degrons

Modifications

Degradation signals

Ub

UbUbPEST

NH2

K

Abreu, Molecular BioSystems, 2009, DOI: 10.1039/b908315d

Methods to study protein expression regulation

ProteinProtein

RNARNA

DNADNA

Time course,poly(A)

Polysomal profiling, Ribosome footprinting

Cycloheximide, Pulse chase

Microarrays, SAGE, RNA-seq

Methods to study protein expression regulation

ProteinProtein

RNARNA

DNADNA

Microarrays, SAGE, RNA-seq

Time course, poly(A)

Polysomal profiling, Ribosome footprinting

Tagged proteinsShotgun proteomics

Lu, Nat Biotech, 2007 25(1)Braisted, BMC Bioinf, 2008 (8)

Cycloheximide, Pulse chase

Protein ~ mRNA across organisms

N=2468R2=0.58

Yeast

Abreu, Molecular BioSystems, 2009

Protein ~ mRNA across organisms

N=423R2=0.47

N=2468R2=0.58

E. coliYeast

Abreu, Molecular BioSystems, 2009

Protein ~ mRNA across organisms

N=511R2=0.22

N=423R2=0.47

N=2468R2=0.58

E. coli

Human

Yeast

Abreu, Molecular BioSystems, 2009

Protein expression regulation in humans

HumanDaoy medulloblastoma cell lysate

efficient translationstable protein

inefficient translationunstable protein

HumanDaoy medulloblastoma cell lysate

mRNA

Pro

tein

27%

What explains the rest?

Protein variance explained

Protein expression regulation in humans

Translation and protein degradation regulation are encoded in sequence features

ProteinProtein

RNARNAAAA(A)nCap

translation and protein degradation regulation

Ub

UbUbPEST

NH2

K

Translation and protein degradation regulation are encoded in sequence features

ProteinProtein

RNARNAAAA(A)nCap

translation and protein degradation regulation

Sequence lengthuORFs

NucleotidesALU

KozakCodon usage

miRNA bindingPoly-adenylation sites

Amino acids and PropertiesUnstructuredness

Degradation SignalsSecondary Structure

etc. etc. etc.sequence signatures

PEST

NH2

K

Ub

UbUb

Sequence length correlates strongly (and inversely) with protein expression

3’5’-0.53*** -0.19***-0.10

Spearman rank (Protein vs. feature, fixing mRNA)

mRNA

Pro

tein

Leng

th

Sequence length correlates strongly (and inversely) with protein expression

• ribosome fidelity [Ingolia, Science 2009 324(5924)]• protein folding [Drummond, PNAS 2003 102(40)]

• miRNA [Sandberg, Science 2008 320(5883)]• alternative cleavage [Mayr, Cell 2009 138(4)]

• secondary structures [e.g. Ringner, Plos CompBio 2005 1(7)]

3’5’-0.53*** -0.19***-0.10

Spearman rank (Protein vs. feature, fixing mRNA)

mRNA

Pro

tein

Leng

th

Protein stability is a significant factor to regulate expression levels

Spearman rank

PEST regions -0.37***Unstructuredness -0.18*

Protein Stability Index# 0.09

Ser, Glu, Leu (Polar amino acids) -0.24***Glycine 0.17***

Phosphorylation$ 0.06

# Yen, Science 2009, 322(5903)$ http://www.phosphopep.org/

Ub

UbUb

+

mRNAPro

tein

Length

Stability

Amino acids

PEST

NH2

K

Translation initiation efficiency influences protein production (per mRNA)

Spearman rank

AUG and uORFs (5’UTR) -0.21***Secondary structures (5’UTR) -0.20***

3’5’

mRNAPro

tein

Tran

slatio

n in

itiatio

n

-

Translation initiation efficiency influences protein production (per mRNA)

Spearman rank

AUG and uORFs (5’UTR) -0.21***Secondary structures (5’UTR) -0.20***

3’5’

mRNAPro

tein

Tran

slatio

n in

itiatio

n

-

>Putative Transcription Factor ZNF462 5’UTRGGAGAGGGAGGGAGGGAGAGAGAGAGAGAGGGAGAGAGACGGATATCTCAGGTCATCTGC AGCTGCAGCGAGTCTGAGGAGCCGAGGAAGGCAGGGAAGATGGCGATCCTCCATTGCTG AGACCCGGCAGAAGCACATGAGACTCCCAAACAACTTCCACAACAATAACCCGAGCAGGAA GAGGAGAAAGAGAAAGAGGATAAGGAGGCGGTGGGGCTGGAGAACCCGAAGCACCTCCCG GCGCCGGGACGCTTCTTCTGTTCCTAATGTGAGAGGCTAGACCCAGATC

mRNA expression and sequence characteristics explain two-thirds of protein expression variation

mRNA

Pro

tein

27%Protein variance explained:

mRNA expression and sequence characteristics explain two-thirds of protein expression variation

mRNA

Pro

tein

27%Protein variance explained:

mRNAP

rote

in

Leng

th

46%

mRNA expression and sequence characteristics explain two-thirds of protein expression variation

mRNA

Pro

tein

27%Protein variance explained:

mRNAP

rote

in

Leng

th

46%

271911

5

Combined contributions: mRNALengthAmino acids (and properties)Nucleotides (and structure)

…mRNA

Pro

tein

…

67%…

Summary and conclusions

The protein vs. mRNA correlation varies widely across organism.

We can explain and predict ~2/3 of the variation in protein expression in a human cell system.

ProteinProtein

RNARNA

DNADNA

We can use such models to:

- Identify ‘hotspots’ of extreme translation and degradation regulation

- Characterize human cell types

- Understand the relationship between transcription, translation and degradation

AcknowledgmentsCollaborators and Co-authors:

Edward Marcotte, Dan Boutz(UT Austin, TX)

Luiz Penalva, Raquel de Sousa Abreu, Daijin Ko, Devraj Sandhu(UT San Antonio, TX)

Dan Miranker, Smriti Ramakrishnan(Computer Science, UT Austin, TX)

John Braisted, Srilatha Kuntumalla, Rembert Pieper(JC Venter Institute, DC)

Bruce A. Shapiro, Shu-Yun Le(National Cancer Institute)

Funding:

Synthetic Gene Design for Heterologous Expression

Mark WelchDNA2.0, Inc

October 28, 2009

Navigating Gene Design Space

Max CAI

MatchHost Bias

Codon PairBias

mRNAStructure

TargetGC%

Min RareCodons

Min RNaseE

Min SD-like

RemoveSplice

Min polyA Sites

HarmonizeCodons

?

~10100 codings for a 30kDa protein!

Interrogating E. coli Preferences NSF SBIR Funded Study

• Only synonymous codon usage varied

• Two different genes studied: a DNA polymerase and an scFv

In silico design (DoE) Gene synthesis Express in E. coli(pET, BL21)

scFV Gene Variant Set

Increased biasDecreased bias

Expression(% cell mass)

0%

15%

30%

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

scFv Variants

Variables 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24GCA 0.025 0.011 0.014 0.007 0.018 0 0.025 0.018 0.011 0.011 0.021 0.014 0.014 0.021 0 0.014 0.007 0.025 0.021 0.028 0.007 0.018 0.007 0.014GCC 0.021 0.021 0 0.021 0.011 0 0.007 0.011 0 0.007 0.007 0.007 0.011 0.014 0 0.014 0.007 0.011 0.007 0 0.014 0.018 0.007 0.004GCG 0.018 0.025 0.028 0.018 0.014 0.028 0.032 0.032 0.021 0.021 0.036 0.028 0.028 0.028 0.021 0.025 0.032 0.011 0.014 0.025 0.032 0.018 0.028 0.032GCT 0 0.007 0.021 0.018 0.021 0.036 0 0.004 0.032 0.025 0 0.014 0.011 0 0.043 0.011 0.018 0.018 0.021 0.011 0.011 0.011 0.021 0.014AGA 0 0 0 0 0 0 0 0 0 0 0 0 0.018 0 0 0 0 0 0 0 0 0 0 0CGA 0 0.007 0 0 0 0 0 0.007 0 0 0 0.014 0 0 0 0 0 0 0 0 0 0 0 0CGC 0.014 0.007 0.021 0.014 0.007 0.014 0.018 0.007 0.018 0.018 0.021 0.011 0.018 0.021 0.021 0.011 0.007 0.011 0.018 0.025 0.028 0.014 0.018 0.018CGG 0 0.004 0 0 0 0 0 0.014 0 0 0 0 0.004 0 0 0 0 0 0 0 0 0.011 0 0CGT 0.032 0.028 0.025 0.032 0.039 0.032 0.028 0.018 0.028 0.028 0.025 0.021 0.007 0.025 0.025 0.036 0.039 0.036 0.028 0.021 0.018 0.021 0.028 0.028AAC 0.011 0.007 0.021 0.018 0.018 0.021 0.007 0.011 0.021 0.011 0.011 0.011 0.014 0.014 0.021 0.014 0.014 0.011 0.018 0.021 0.011 0.014 0.021 0.011AAT 0.011 0.014 0 0.004 0.004 0 0.014 0.011 0 0.011 0.011 0.011 0.007 0.007 0 0.007 0.007 0.011 0.004 0 0.011 0.007 0 0.011GAC 0.028 0.028 0.028 0.028 0.018 0.011 0.018 0.028 0.046 0.036 0.021 0.018 0.025 0.021 0.032 0.032 0.036 0.036 0.036 0.032 0.043 0.032 0.028 0.028GAT 0.032 0.032 0.032 0.032 0.043 0.05 0.043 0.032 0.014 0.025 0.039 0.043 0.036 0.039 0.028 0.028 0.025 0.025 0.025 0.028 0.018 0.028 0.032 0.032T GC 0.007 0 0.011 0.007 0.004 0.007 0.004 0.007 0.007 0.004 0.007 0.014 0.007 0.014 0.011 0.011 0 0.007 0.007 0.007 0.004 0.011 0.007 0.007T GT 0.007 0.014 0.004 0.007 0.011 0.007 0.011 0.007 0.007 0.011 0.007 0 0.007 0 0.004 0.004 0.014 0.007 0.007 0.007 0.011 0.004 0.007 0.007CAA 0.021 0.018 0.004 0.018 0.021 0.004 0.028 0.032 0.004 0.021 0.032 0.039 0.021 0.021 0.004 0.014 0.004 0.018 0.007 0.004 0.028 0.028 0.014 0.011CAG 0.028 0.032 0.046 0.032 0.028 0.046 0.021 0.018 0.046 0.028 0.018 0.011 0.028 0.028 0.046 0.036 0.046 0.032 0.043 0.046 0.021 0.021 0.036 0.039GAA 0.011 0.011 0.011 0.014 0.014 0.014 0.014 0.021 0.014 0.014 0.011 0.014 0.021 0.014 0.021 0.018 0.014 0.011 0.018 0.014 0.007 0.007 0.014 0.011GAG 0.011 0.011 0.011 0.007 0.007 0.007 0.007 0 0.007 0.007 0.011 0.007 0 0.007 0 0.004 0.007 0.011 0.004 0.007 0.014 0.014 0.007 0.011GGA 0 0.018 0 0.007 0 0 0 0.011 0 0.032 0 0.011 0.018 0 0 0.004 0 0 0 0 0 0.021 0 0GGC 0.039 0.039 0.046 0.039 0.046 0.064 0.064 0.043 0.046 0.032 0.071 0.032 0.053 0.05 0.046 0.046 0.043 0.053 0.068 0.036 0.021 0.036 0.06 0.064GGG 0 0.014 0 0.021 0.004 0 0 0.014 0 0.021 0 0.021 0.011 0 0 0 0 0 0 0 0 0.014 0 0GGT 0.078 0.046 0.071 0.05 0.068 0.053 0.053 0.05 0.071 0.032 0.046 0.053 0.036 0.068 0.071 0.068 0.075 0.064 0.05 0.082 0.096 0.046 0.057 0.053CAC 0.025 0.011 0.014 0.011 0.018 0.018 0.018 0.014 0.011 0.021 0.014 0.014 0.014 0.018 0.021 0.018 0.021 0.025 0.028 0.011 0.014 0.011 0.025 0.011CAT 0.007 0.021 0.018 0.021 0.014 0.014 0.014 0.018 0.021 0.011 0.018 0.018 0.018 0.014 0.011 0.014 0.011 0.007 0.004 0.021 0.018 0.021 0.007 0.021AT A 0 0.007 0 0 0 0 0 0.004 0 0 0 0.007 0 0 0 0 0 0 0 0 0 0.004 0 0AT C 0.021 0.018 0.028 0.021 0.028 0.036 0.018 0.021 0.028 0.036 0.032 0.018 0.025 0.025 0.025 0.039 0.021 0.036 0.028 0.021 0.014 0.011 0.032 0.032AT T 0.021 0.018 0.014 0.021 0.014 0.007 0.025 0.018 0.014 0.007 0.011 0.018 0.018 0.018 0.018 0.004 0.021 0.007 0.014 0.021 0.028 0.028 0.011 0.011CT A 0 0 0 0 0 0 0 0.004 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0CT C 0 0.007 0 0.011 0.004 0 0 0.007 0 0.014 0 0.011 0.007 0 0 0.004 0 0 0 0 0 0.018 0 0CT G 0.071 0.028 0.071 0.036 0.039 0.071 0.071 0.043 0.071 0.043 0.071 0.007 0.021 0.071 0.071 0.053 0.071 0.071 0.071 0.071 0.071 0.014 0.071 0.071CT T 0 0.007 0 0.004 0.011 0 0 0.004 0 0.004 0 0.025 0.014 0 0 0.011 0 0 0 0 0 0.011 0 0T T A 0 0.011 0 0.007 0.007 0 0 0.007 0 0.007 0 0.018 0.007 0 0 0 0 0 0 0 0 0.011 0 0T T G 0.004 0.021 0.004 0.018 0.014 0.004 0.004 0.011 0.004 0.007 0.004 0.014 0.025 0.004 0.004 0.007 0.004 0.004 0.004 0.004 0.004 0.021 0.004 0.004AAA 0.021 0.021 0.025 0.025 0.032 0.039 0.028 0.032 0.025 0.028 0.025 0.028 0.036 0.032 0.039 0.025 0.036 0.032 0.028 0.021 0.018 0.018 0.025 0.036AAG 0.018 0.018 0.014 0.014 0.007 0 0.011 0.007 0.014 0.011 0.014 0.011 0.004 0.007 0 0.014 0.004 0.007 0.011 0.018 0.021 0.021 0.014 0.004T T C 0.021 0.021 0.018 0.018 0.028 0.025 0.004 0.018 0.025 0.025 0.018 0.011 0.011 0.021 0.025 0.021 0.014 0.018 0.025 0.011 0.021 0.018 0.032 0.028T T T 0.011 0.011 0.014 0.014 0.004 0.007 0.028 0.014 0.007 0.007 0.014 0.021 0.021 0.011 0.007 0.011 0.018 0.014 0.007 0.021 0.011 0.014 0 0.004CCA 0 0.011 0 0.007 0.007 0 0 0.014 0 0.007 0 0.007 0.011 0 0 0 0.007 0.014 0.004 0 0.011 0.011 0.018 0.007CCC 0 0.004 0 0 0 0 0 0 0 0 0 0.014 0.007 0 0 0 0 0 0 0 0 0.011 0 0CCG 0.036 0.011 0.036 0.021 0.025 0.036 0.036 0.018 0.036 0.021 0.036 0.007 0.011 0.036 0.036 0.036 0.025 0.018 0.028 0.036 0.007 0.007 0.018 0.025CCT 0 0.011 0 0.007 0.004 0 0 0.004 0 0.007 0 0.007 0.007 0 0 0 0.004 0.004 0.004 0 0.018 0.007 0 0.004AGC 0.121 0.025 0.039 0.018 0.039 0.004 0.121 0.028 0.028 0.039 0.121 0.032 0.032 0.121 0.004 0.021 0.036 0.028 0.032 0.043 0.036 0.032 0.053 0.032AGT 0 0.014 0 0.025 0.004 0 0 0.018 0 0.007 0 0.018 0.021 0 0 0.004 0 0 0 0 0 0.018 0 0T CA 0.004 0.025 0.004 0.011 0.007 0.004 0.004 0.028 0.004 0.025 0.004 0.021 0.021 0.004 0.004 0.007 0.004 0.004 0.004 0.004 0.004 0.014 0.004 0.004T CC 0 0.011 0.05 0.032 0.021 0.05 0 0.025 0.046 0.028 0 0.021 0.021 0 0.06 0.039 0.036 0.039 0.05 0.039 0.05 0.021 0.025 0.05T CG 0 0.025 0 0.014 0.004 0 0 0.004 0 0.011 0 0.021 0.004 0 0 0.014 0 0 0 0 0 0.021 0 0T CT 0 0.025 0.032 0.025 0.05 0.068 0 0.021 0.046 0.014 0 0.011 0.025 0 0.057 0.039 0.05 0.053 0.039 0.039 0.036 0.018 0.043 0.039ACA 0 0.025 0 0.011 0 0 0 0.018 0 0.004 0 0.014 0.014 0 0 0.007 0 0 0 0 0 0.028 0 0ACC 0.039 0.032 0.043 0.028 0.05 0.057 0.05 0.018 0.057 0.036 0.043 0.021 0.028 0.046 0.043 0.032 0.05 0.043 0.05 0.043 0.018 0.007 0.043 0.046ACG 0.043 0.018 0.004 0.025 0.007 0.004 0.032 0.039 0.004 0.021 0.039 0.036 0.028 0.036 0.004 0.011 0.007 0.021 0.014 0.004 0.036 0.021 0.011 0.004ACT 0 0.007 0.036 0.018 0.025 0.021 0 0.007 0.021 0.021 0 0.011 0.011 0 0.036 0.032 0.025 0.018 0.018 0.036 0.028 0.025 0.028 0.032T AC 0.036 0.036 0.025 0.046 0.046 0.043 0.043 0.014 0.028 0.036 0.039 0.025 0.025 0.021 0.039 0.043 0.043 0.039 0.028 0.025 0.039 0.025 0.053 0.06T AT 0.028 0.028 0.039 0.018 0.018 0.021 0.021 0.05 0.036 0.028 0.025 0.039 0.039 0.043 0.025 0.021 0.021 0.025 0.036 0.039 0.025 0.039 0.011 0.004GT A 0 0.007 0.014 0.011 0.014 0 0 0 0.011 0.004 0 0.014 0.007 0 0 0.018 0 0.007 0.007 0.014 0.014 0.007 0.004 0GT C 0.021 0.018 0 0.007 0.004 0 0.014 0.021 0 0 0.011 0.004 0 0.014 0 0 0 0.007 0.007 0 0.014 0.014 0.004 0.011GT G 0.011 0.011 0.011 0.014 0.007 0.025 0.018 0.011 0.018 0.025 0.018 0.014 0.025 0.014 0.021 0.018 0.011 0.011 0.011 0.011 0.007 0.018 0.025 0.021GT T 0.011 0.007 0.018 0.011 0.018 0.018 0.011 0.011 0.014 0.014 0.014 0.011 0.011 0.014 0.021 0.007 0.032 0.018 0.018 0.018 0.007 0.004 0.011 0.011

GC% 5 7 .4 5 1 .7 5 4 .9 5 3 .3 5 2 .0 5 4 .9 5 6 .5 5 1 .6 5 5 .4 5 4 .2 5 8 .0 4 9 .7 5 0 .7 5 7 .1 5 4 .3 5 4 .6 5 3 .7 5 4 .2 5 6 .0 5 3 .7 5 4 .0 5 1 .5 5 6 .7 5 6 .3

5' AT 0 .4 0 0 0 .0 6 7 0 .2 0 0 0 .6 6 7 0 .6 6 7 0 .0 6 7 0 .4 0 0 0 .1 3 3 0 .4 0 0 0 .2 0 0 0 .0 6 7 0 .8 0 0 0 .7 3 3 0 .0 6 7 0 .4 0 0 0 .1 3 3 0 .4 0 0 0 .3 3 3 0 .2 6 7 0 .2 0 0 0 .3 3 3 0 .4 0 0 0 .3 3 3 0 .2 6 7

CAI 0 .7 2 5 0 .4 6 0 0 .8 0 8 0 .5 6 7 0 .7 1 2 0 .8 6 8 0 .7 2 3 0 .4 8 7 0 .8 2 8 0 .5 6 3 0 .7 2 5 0 .4 1 7 0 .4 7 8 0 .7 4 7 0 .8 6 3 0 .7 2 2 0 .8 2 2 0 .7 6 8 0 .8 0 5 0 .7 9 3 0 .6 7 7 0 .4 2 1 0 .8 0 0 0 .8 0 3

Rare Codons 2 5 3 2 3 6 1 5 2 2 4 2 2 3 7 2 5 9 4 8 2 2 1 6 2 2 2 2 2 5 7 2 2

GC Clusters 2 3 1 2 1 4 2 5 1 3 1 0 0 1 1 4 1 4 1 3 1 6 1 0 8 7 9 2 0 2 8 2 0

AT Clusters 5 1 1 1 1 8 1 0 4 5 2 1 1 0 9 8 1 3 8 1 3 6 7 5 4 1 3 1 2 6 1 0 3 2

mRNA Strucutre -1 1 .8 -9 .6 -1 0 .8 -1 0 .2 -9 .1 -1 0 .0 -1 1 .7 -9 .2 -1 0 .4 -1 0 .7 -1 2 .1 -9 .4 -9 .6 -1 2 .2 -9 .5 -1 0 .4 -1 0 .5 -1 0 .2 -1 0 .7 -1 0 .6 -1 0 .4 -9 .1 -1 0 .9 -1 0 .3

5' RNA Structure -1 3 .0 -1 5 .0 -1 2 .7 -1 1 .6 -1 2 .2 -1 2 .9 -1 3 .3 -1 0 .1 -1 2 .3 -1 1 .5 -1 4 .5 -1 0 .0 -1 0 .0 -1 6 .2 -1 2 .1 -1 7 .8 -1 0 .9 -1 3 .6 -1 4 .6 -1 2 .8 -1 1 .8 -1 0 .0 -1 3 .3 -1 1 .9

RNaseE Sites 7 .0 6 .0 3 .0 7 .0 7 .0 6 .0 1 0 .0 5 .0 3 .0 5 .0 6 .0 7 .0 7 .0 6 .0 5 .0 4 .0 5 .0 2 .0 3 .0 5 .0 6 .0 7 .0 1 .0 3 .0

F

P

S

T

Y

V

E

G

HI

L

K

A

R

NDCQ

Codon Frequencies

OtherFeatures

Protein Expression not Correlated to CAI or 5’ mRNA Structure

R²(scFv) = 0.1003

R²(Pol) = 0.0109

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

-11.0 -9.0 -7.0 -5.0 -3.0 -1.0

Rel

ativ

e E

xpre

ssio

n

Free Energy (kcal/mol)

R²(scFv) = 0.0013

R²(Pol) = 0.0029

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0

Rel

ativ

e E

xpre

ssio

n

CAI

CAI 5’ mRNA Structure(Window: -4 to +37)

Polymerase

scFv

Variant Relative Expression

15 7 ± 1

Hyb 15-15-19 17 ± 6

Hyb 15-19-15 7 ± 1

Hyb 19-15-15 10 ± 1

Hyb 15-19-19 34 ± 2

Hyb 19-15-19 35 ± 4

Hyb 19-19-15 25 ± 3

19 100 ± 22

Variant Hybrids Distributed Coding Effects – Polymerase Hybrid Set #1

75 325 575

• Search for any local effects (deleterious motifs, etc.)• Further diversification along useful trajectories

Multivariate Regression Analysis of Codon Usage

• Partial least squares regression (PLS) used to identify relationships between codon use and expression

• Input: individual codon frequencies in genes

• Output: optimal set of weights for each codon that best predict expression

Other directions of codon bias?

CAI

Exp

ress

ion

X

Multivariate Analysis of Expression Combined Model

• Validated with random subset cross validation (20% left out)

New Designs

Polymerase

scFv

Preference for ‘Reserve’ tRNAs?

AA%AA, scFv

%AA, coli Codon

Preference Ratio

tRNA sensitivity1

Ser 12.5 4.7 AGC 2.1 3.4AGU 0.0 3.4

UCA 0.4 7.5

UCC 0.6 35.5

UCG 0.6 4.4

UCU 0.4 7.9

Thr 8.2 5.4 ACA 0.0 5.7ACC 0.9 20.9

ACG 2.4 2.4ACU 0.5 6.6

1Elf, et al (2003) Science,300:1718

tRNA sensitivity dependent on Fci[tRNAi ]

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33NEHKY 0 1 0 ‐1 0 ‐1 0 1 0 ‐1 0 1 0 1 0 ‐1 0 1 0 ‐1 0 ‐1 0 1 0 ‐1 0 1 0 1 0 ‐1 1

A 0 0 ‐1 ‐1 0 0 ‐1 ‐1 0 0 ‐1 ‐1 0 0 ‐1 ‐1 0 0 1 1 0 0 1 1 0 0 1 1 0 0 1 1 1R 0 1 ‐1 0 0 1 ‐1 0 0 1 ‐1 0 0 1 ‐1 0 0 ‐1 1 0 0 ‐1 1 0 0 ‐1 1 0 0 ‐1 1 0 1D 0 0 0 0 ‐1 ‐1 1 1 0 0 0 0 ‐1 ‐1 1 1 0 0 0 0 1 1 ‐1 ‐1 0 0 0 0 1 1 ‐1 ‐1 1C 0 1 0 ‐1 ‐1 0 1 0 0 1 0 ‐1 ‐1 0 1 0 0 ‐1 0 1 1 0 ‐1 0 0 ‐1 0 1 1 0 ‐1 0 1Q 0 0 ‐1 ‐1 1 1 0 0 0 0 ‐1 ‐1 1 1 0 0 0 0 1 1 ‐1 ‐1 0 0 0 0 1 1 ‐1 ‐1 0 0 1G 0 1 ‐1 0 1 0 0 ‐1 0 1 ‐1 0 1 0 0 ‐1 0 ‐1 1 0 ‐1 0 0 1 0 ‐1 1 0 ‐1 0 0 1 1I 0 0 0 0 0 0 0 0 ‐1 ‐1 1 1 1 1 ‐1 ‐1 0 0 0 0 0 0 0 0 1 1 ‐1 ‐1 ‐1 ‐1 1 1 1L 0 1 0 ‐1 0 ‐1 0 1 ‐1 0 1 0 1 0 ‐1 0 0 ‐1 0 1 0 1 0 ‐1 1 0 ‐1 0 ‐1 0 1 0 1F 0 0 ‐1 ‐1 0 0 ‐1 ‐1 1 1 0 0 1 1 0 0 0 0 1 1 0 0 1 1 ‐1 ‐1 0 0 ‐1 ‐1 0 0 1P 0 1 ‐1 0 0 1 ‐1 0 1 0 0 ‐1 1 0 0 ‐1 0 ‐1 1 0 0 ‐1 1 0 ‐1 0 0 1 ‐1 0 0 1 1S 0 0 0 0 ‐1 ‐1 1 1 1 1 ‐1 ‐1 0 0 0 0 0 0 0 0 1 1 ‐1 ‐1 ‐1 ‐1 1 1 0 0 0 0 1T 0 1 0 ‐1 ‐1 0 1 0 1 0 ‐1 0 0 ‐1 0 1 0 ‐1 0 1 1 0 ‐1 0 ‐1 0 1 0 0 1 0 ‐1 1V 0 0 ‐1 ‐1 1 1 0 0 1 1 0 0 0 0 ‐1 ‐1 0 0 1 1 ‐1 ‐1 0 0 ‐1 ‐1 0 0 0 0 1 1 1

5'AT 1 ‐1 1 ‐1 ‐1 1 ‐1 1 ‐1 1 ‐1 1 1 ‐1 1 ‐1 ‐1 1 ‐1 1 1 ‐1 1 ‐1 1 ‐1 1 ‐1 ‐1 1 ‐1 1 1RCO 1 1 ‐1 ‐1 ‐1 ‐1 1 1 ‐1 ‐1 1 1 1 1 ‐1 ‐1 ‐1 ‐1 1 1 1 1 ‐1 ‐1 1 1 ‐1 ‐1 ‐1 ‐1 1 1 1

Gene Variant Set for Yeast with Dr. Robert Stroud, UCSF

Gene Variants

Des

ign

Varia

bles

Protein

1 Increased bias (use more frequent codons)0 Host bias‐1 Decreased bias (use more infrequent codons)

• Human membrane protein expressed in S. cerevisiae• Total protein in membrane fraction analyzed• WT gene shows no detectable expression• Top expression level ~1mg/L

Yeast Bias, high frequency cut-off

Yeast Bias, low frequency cut-off

High frequency codon biased

PLS Model of Yeast Expression Data

R2=0.891R2 (CV)=0.809

Conclusions, Ongoing Work

• Systematic gene diversification useful to identify coding parameters relevant to expression

• Heterologous gene expression is correlated to codon usage• Codon preferences may reflect tRNA sensitivity to over-

consumption• Several ongoing studies with academic and industry

collaborators: E. coli, plants, yeasts, fungi, mammalian cells, trypanosomes, cell-free systems and more

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Webinar SeriesWebinar SeriesScienceScience

28 October, 2009

From Genes to ProteinsFrom Genes to ProteinsThe Impact of Gene Sequence onTranslation and Expression

Slide Number 1coding-sequence determinants of gene expressionSlide Number 3Slide Number 4Slide Number 5Slide Number 6Slide Number 7Slide Number 8Slide Number 9Slide Number 10Slide Number 11Slide Number 12Slide Number 13Slide Number 14Slide Number 15Slide Number 16Slide Number 17Slide Number 18Slide Number 19Slide Number 20Slide Number 21Slide Number 22Slide Number 23The relationship between protein and mRNA expression levels��Christine Vogel�The Central Dogma of BiologyThe Central Dogma of BiologyMultiple mechanisms regulate protein expressionMultiple mechanisms regulate protein expressionMultiple mechanisms regulate protein expressionMultiple mechanisms regulate protein expressionMultiple mechanisms regulate protein expressionMultiple mechanisms regulate protein expressionMethods to study protein expression regulationMethods to study protein expression regulationProtein ~ mRNA across organismsProtein ~ mRNA across organismsProtein ~ mRNA across organismsProtein expression regulation in humansSlide Number 39Translation and protein degradation regulation are encoded in sequence featuresTranslation and protein degradation regulation are encoded in sequence featuresSequence length correlates strongly (and inversely) with protein expressionSequence length correlates strongly (and inversely) with protein expressionProtein stability is a significant factor to regulate expression levelsTranslation initiation efficiency influences protein production (per mRNA) Translation initiation efficiency influences protein production (per mRNA) mRNA expression and sequence characteristics explain two-thirds of protein expression variationmRNA expression and sequence characteristics explain two-thirds of protein expression variationmRNA expression and sequence characteristics explain two-thirds of protein expression variationSummary and conclusionsAcknowledgmentsSynthetic Gene Design for Heterologous ExpressionNavigating Gene Design SpaceInterrogating E. coli Preferences �NSF SBIR Funded StudyscFV Gene Variant SetProtein Expression not Correlated to CAI or 5’ mRNA StructureVariant Hybrids�Distributed Coding Effects – Polymerase Hybrid Set #1Multivariate Regression Analysis of Codon UsageSlide Number 59Preference for ‘Reserve’ tRNAs?Gene Variant Set for Yeast�with Dr. Robert Stroud, UCSFPLS Model of Yeast Expression DataConclusions, Ongoing WorkSlide Number 64