Pat O’FarrellPat O’FarrellDept. Biochem and Biophys.Dept. Biochem and Biophys.UCSFUCSFSan FranciscoSan Francisco

Characterizing expression differencesLooking for ONE difference (needle in the hay stack)Assaying ONE differenceDefining a complex (pleiotropic) response

S u p e r c e d e d

2D Gels/proteomics - what is it good forArrays Arrays

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Characterizing expression differencesLooking for ONE difference (needle in the hay stack)Assaying ONE differenceDefining a complex (pleiotropic) response

Characterizing expression differencesLooking for ONE difference (needle in the hay stack)Assaying ONE differenceDefining a complex (pleiotropic) response

2D Gels/proteomics - what is it good for

Characterizing expression differencesLooking for ONE difference (needle in the hay stack)Assaying ONE difference

Defining a complex (pleiotropic) response?

Assessing & comparing protein levels/tissues/secretions

Regulation of protein levels by turnover

Assessing modification of proteins

Finding surprisesFinding surprises

Defining a complex (pleiotropic) responseDefining a complex (pleiotropic) response

The Lac operon and catabolite repression

Glucose Genes for catabolism of other metabolites

Cyclic AMPGlucose Genes for catabolism of other metabolites

CrpCrpcAMP

cAMP cAM

PcAM

P

P Lac

-galactosidase

100

50

25

100

50

25

Control

+ cy AMPRepression by cy AMP

Control+ cy AMPControl+ cy AMPControl+ cy AMPControl+ cy AMPControl+ cy AMPControl+ cy AMPControl+ cy AMPControl

+ cy AMP

The catabolite repression (cyAMP) domain

Size: -about 10% of all genes respond to cyclic AMP

Heterogeneity: -responses vary - in direction (~1% repression 9% induction) - and magnitude

Mechanism: - all responses depend on the same receptor • i.e. crp mutants show no response to cyclic AMP - the receptor is inactive without cyclic AMP • i.e. adenyl cyclase mutants = crp mutants

Overlap - cyclic AMP responsive genes are downregulated by limitation for an amino acid

Warning!

• strains must be congenic

The analysis is EXTREMELY sensitive to conditions

• culture conditions must be precisely reproduced

•  Synthesis of ribosomes is a major part of bacterial growth• Ribosomes are half RNA and half protein

• aa starvation diminishes synthesis of ribosomal proteins• Stringent E. coli strains rRNA synthesis when short of aa• Relaxed strains lack the ability to down regulate rRNA

rel+coordinate rRNA & r-proteinsynthesisrel-lack coordination ofrRNA & r-proteinsynthesis

Coordinating growth

G tetraphos&

G pentaphosSignals for

aa starvationrel

ATP + GTP

AMPAMP + + ppppGpppGppp

ppppGppGpp

GDPspoT

A system that detects shortage of aa & signals starvation

rRNArRNA synthesissynthesis

Experiment to test effect of ppGpp on protein expression• ppGpp does not get into cells I could not just add it a test the consequences

Compare+ppGpp -ppGpp

rel+ strain

Restrict amino acids

Residual protein synthesis occurs in the presence of ppGpp

rel- strain

Restrict amino acids

Residual protein synthesis occurs in the absence of ppGpp

• My Plan

Contro

l

Residual protein synthesis in starved rel+ or rel -

35S-methionine incorporation

10 to 20% residual incorporation during starvation

-R induced proteins

Specific responses to particular -aa

-R -H -P -Lrel+

Contro

l

-R -H -P -L-R -H -P -Lrel+ rel-

Residual protein synthesis in starved rel+ or rel -

Specific responses to particular -aa

Global changes with in MW

The “Hungry Codon”

• a 20 residue protein with each of the aa • a step time, ST, is the time it normally takes to add one aa• its synthesis would take 20 ST

• simple thought experiment

C+1

D+1

E+1

F+1

G+1

H+1

I+1

K+1

L+1

M+1

N+1

P+1

Q+1

R+1

S+1

T+1

V+1

W+1

Y+1

A1

• starvation for histidine reduces protein synthesis to 10%

The “Hungry Codon”

• simple thought experiment

C+1

D+1

E+1

F+1

G+1

H+1

I+1

K+1

L+1

M+1

N+1

P+1

Q+1

R+1

S+1

T+1

V+1

W+1

Y+1

A1

• starvation for histidine reduces protein synthesis to 10%• rate of synthesis of average peptide is reduced 10x

H+181

I+1

K+1

L+1

M+1

N+1

P+1

Q+1

R+1

S+1

T+1

V+1

W+1

Y+1

C+1

D+1

E+1

F+1

G+1

A1

• it takes 10x as long to synthesis the average peptide• it takes 10x20=200 ST to make our 20 aa peptide• 19 of aa are normal and are added in 19 step times• translation of the hungry codon takes 200-19=181 ST

• a 20 residue protein with each of the aa • a step time, ST, is the time it normally takes to add one aa• its synthesis would take 20 ST

A

B

C

D

F

G

A steady state determines pool size

Sucking the pool dry (almost)

-histidine -proline

In rel- cells, aa starvation induces errors in translation

• during H starvation newly synthesized proteins are heterogeneous• interpretation: - an uncharged aa is occasionally incorporated in place of H - each substituted position removes a basic residue - the trail of spots is consistent with 3% misincorporation • starvation for different amino acids give different types of errors• interpretation: - termination occurs if an codon is not easily misread as another residue - charge errors occur if codon is easily misread as a differently charged residue

• •••• • •

Errors in translation are not seen in starved rel+ cells

Control -H rel+ -H rel-

How can ppGpp the fidelity of translation during starvation?

• ribosomal proteins L7 has no histidine

Starved rel+ cells behave as if they are not missing an aa

RelativeL7 expression

control +++ rel- - H +++++ rel- - I +

rel+ - H+++

rel+ - I+++

Difference consistent with the difference in H and I abundance in the protein

Expression insensitive to aa abundance

rel+ unstableppGpp

r R

N A

r R

N A

P r

o t e

i n

ppGpp Inhibits Protein Synthesis as well as rRNA Synthesis

rel- no ppGpp

rel+ spoT-

stableppGpp

Control - aa - + aa

Sharing the burden

• in a rel- strain all the slowing of translation occurs at the “hungry codon”

C+10

D+10

E+10

F+10

G+10

H+10

I+10

K+10

L+10

M+10

N+10

P+10

Q+10

R+10

S+10

T+10

V+10

W+10

Y+10

A10

H+181

I+1

K+1

L+1

M+1

N+1

P+1

Q+1

R+1

S+1

T+1

V+1

W+1

Y+1

C+1

D+1

E+1

F+1

G+1

A1

• ppGpp slows down translation at multiple steps of translation

• The aa-tRNA for the hungry codon is only reduced enough to generate a signal

ConclusionsConclusions

1. Protein synthesis has a precarious relationship with its substrates1. Protein synthesis has a precarious relationship with its substrates• imbalances in substrates are exaggerated as residues are incorporated according to the dictates of the code not availability

2. Substrate imbalance severely compromises fidelity2. Substrate imbalance severely compromises fidelity

• 3% missincorporation, tuncation & inactive enzymes

3. ppGpp makes translation more robust and accurrate3. ppGpp makes translation more robust and accurrate

• it adjusts protein synthesis rates to availability of the limiting aa (the weakest link)

• it acts as governor to coordinate translation with substrate supply

Generalization

• balanced substrate supply is universally important for translation

• a specific signaling system that senses substrate levels and modulates translation accordingly

Global requirement

The basis of this regulation outside of E coli is not known.It is not based on ppGpp, which is absent in eukaryotes.

rel+ spoT+

rel+ spoT-

rel-

- aa (I) +I (0-5 min) +I (12.5 min)ppGpp

t

t

t

[C]

[C]

[C]

ppGpp -aa

Specific responses to ppGpp

rel+ spoT+

rel+ spoT-

rel-

- aa (I) +I (0-5 min) +I (12.5 min)ppGpp

t

t

t

[C]

[C]

[C]

ppGpp -aa

Specific responses to ppGpp

- aa (I) +I (0-5 min) +I (12.5 min)

ppGpp -aa

Specific responses to ppGpp

rel-

t

[C]

rel+ spoT-

t

[C]

rel+ spoT+

ppGpp

t

[C]

Pat O’FarrellDept. Biochem and Biophys.UCSFSan Francisco