Download I + rel + - UCSF Biochemistry & Biophysics

Pat O’Farrell
Dept. Biochem and Biophys.
UCSF
San Francisco
2D Gels/proteomics - what is it good for
Characterizing
Characterizingexpression
expressiondifferences
differences
for ONE difference (needle in the hay stack)
Looking
Looking for ONE difference (needle in the hay stack)
Assaying ONE difference


Assaying ONE difference
Defining
Defininga acomplex
complex(pleiotropic)
(pleiotropic)response
response
2D Gels/proteomics - what is it good for
Characterizing expression differences
 Looking 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 surprises
The Lac operon and catabolite repression
Genes for catabolism of
other metabolites
Glucose
Glucose
Cyclic AMP
Genes for catabolism of
other metabolites
-galactosidase
Crp
P Lac
100
Control
50
25
100
50
25
+ cy AMP
Repression
by cy AMP
+Control
cy AMP
+ 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!
The analysis is EXTREMELY sensitive to conditions
• strains must be congenic
• culture conditions must be precisely reproduced
Coordinating growth
• Synthesis of ribosomes is a major part of bacterial growth
• Ribosomes are half RNA and half protein
• Stringent E. coli strains
rRNA synthesis when short of aa
• Relaxed strains lack the ability to down regulate rRNA
rel+
coordinate
rRNA & r-protein
synthesis
rel-
lack coordination of
rRNA & r-protein
synthesis
A system that detects shortage of aa & signals starvation
AMP + ppGppp
rel
ATP + GTP
ppGpp
GDP
spoT
G tetraphos
&
G pentaphos
Signals for
aa starvation
rRNA
synthesis
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
• My Plan
rel+ strain
rel- strain
Restrict amino acids
Restrict amino acids
Residual protein synthesis
occurs in the presence of
ppGpp
Residual protein synthesis
occurs in the absence of
ppGpp
+ppGpp
Compare
-ppGpp
Residual protein synthesis in starved rel+ or rel rel +
-R -H -P -L
35S-methionine
incorporation
-R induced
proteins
10 to 20% residual
incorporation during
starvation
Specific responses
to particular -aa
Residual protein synthesis in starved rel+ or rel rel +
rel -R -H -P -L
Specific responses
to particular -aa
-R -H -P
-L
Global changes
with  in MW
The “Hungry Codon”
• simple thought experiment
• 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 C D E F G H I K L M N P Q R S T V W Y
1
+1
+1
+1
+1
+1
+1
+1
+1
+1
+1
+1
+1
+1
+1
+1
+1
+1
+1
+1
• starvation for histidine reduces protein synthesis to 10%
The “Hungry Codon”
• simple thought experiment
• 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 C D E F G H I K L M N P Q R S T V W Y
1
+1
+1
+1
+1
+1
+1
+1
+1
+1
+1
+1
+1
+1
+1
+1
+1
+1
+1
+1
• starvation for histidine reduces protein synthesis to 10%
• rate of synthesis of average peptide is reduced 10x
A C D E F G H I K L M N P Q R S T V W Y
1
+1
•
•
•
•
+1
+1
+1
+1
+181 +1
+1
+1
+1
+1
+1
+1
+1
+1
+1
+1
 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
+1
+1
A steady state determines
pool size
A
B
C
D
F
G
Sucking the pool dry (almost)
In rel cells, aa starvation induces errors in translation
-histidine
•••••
•
-proline
•
• 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?
Starved rel+ cells behave as if they are not missing an aa
• ribosomal proteins L7 has no histidine
Relative
L7 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
ppGpp Inhibits Protein Synthesis as well as rRNA Synthesis
Control
- aa
-  + aa
rel+
unstable
ppGpp
-
rel+ spoT
stable
ppGpp
Prot ein
relno ppGpp
Sharing the burden
• in a rel- strain all the slowing of translation occurs at
the “hungry codon”
A C D E F G H I K L M N P Q R S T V W Y
1
+1
+1
+1
+1
+1
+181 +1
+1
+1
+1
+1
+1
+1
+1
+1
+1
+1
+1
+1
• ppGpp slows down translation at multiple steps of translation
• The aa-tRNA for the hungry codon is only reduced enough to
generate a signal
A C D E F G H I K L M N P Q R S T V W Y
10
+10
+10
+10
+10
+10
+10
+10
+10
+10
+10
+10
+10
+10
+10
+10
+10
+10
+10
+10
Conclusions
1. 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 fidelity
• 3% missincorporation, tuncation & inactive enzymes
3. ppGpp makes translation more robust and accurrate
• it acts as governor to coordinate translation with
substrate supply
• it adjusts protein synthesis rates to availability of the
limiting aa (the weakest link)
Generalization
• balanced substrate supply is universally important for translation
Global requirement
• a specific signaling system that senses substrate levels and
modulates translation accordingly
The basis of this regulation outside of E coli is not known.
It is not based on ppGpp, which is absent in eukaryotes.
Specific
responses
to ppGpp
rel+ spoT+
- aa (I)
+I (0-5 min) +I (12.5 min)
ppGpp
[C]
ppGpp 
-aa
t

rel+ spoT-
[C]
t
rel-
[C]
t
Specific
responses
to ppGpp
rel+ spoT+
- aa (I)
+I (0-5 min) +I (12.5 min)
ppGpp
[C]
ppGpp 
-aa
t

rel+ spoT-
[C]
t
rel-
[C]
t
Specific
responses
to ppGpp
rel-
- aa (I)
+I (0-5 min) +I (12.5 min)
ppGpp
[C]
t
ppGpp 
-aa

rel+ spoT+
[C]
t
rel+ spoT-
[C]
t
Pat O’Farrell
Dept. Biochem and Biophys.
UCSF
San Francisco