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Pat O’Farrell Dept. Biochem and Biophys. UCSF San Francisco

Pat O’Farrell Dept. Biochem and Biophys. UCSF San Francisco. Characterizing expression differences. Characterizing expression differences. S u p e r c e d e d. . Looking for ONE difference (needle in the hay stack). Looking for ONE difference (needle in the hay stack). .

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Pat O’Farrell Dept. Biochem and Biophys. UCSF San Francisco

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  1. Pat O’Farrell Dept. Biochem and Biophys. UCSF San Francisco

  2. Characterizing expression differences Characterizing expression differences S u p e r c e d e d  Looking for ONE difference (needle in the hay stack) Looking for ONE difference (needle in the hay stack)  Assaying ONE difference Assaying ONE difference  Defining a complex (pleiotropic) response Defining a complex (pleiotropic) response Characterizing expression differences Looking for ONE difference (needle in the hay stack) Assaying ONE difference Defining a complex (pleiotropic) response 2D Gels/proteomics - what is it good for Arrays Arrays  Arrays Arrays  Arrays Arrays  Arrays

  3.  Defining a complex (pleiotropic) response Defining a complex (pleiotropic) response 2D Gels/proteomics - what is it good for ? Characterizing expression differences  Looking for ONE difference (needle in the hay stack)  Assaying ONE difference Assessing & comparing protein levels/tissues/secretions Regulation of protein levels by turnover Assessing modification of proteins Finding surprises

  4. Genes for catabolism of other metabolites Glucose Cyclic AMP cAMP cAMP -galactosidase Crp P Lac The Lac operon and catabolite repression Genes for catabolism of other metabolites Glucose

  5. 100 50 25 100 50 25 Control Repression by cy AMP + cy AMP

  6. Control Control Control Control Control Control Control + cy AMP + cy AMP + cy AMP + cy AMP + cy AMP + cy AMP + cy AMP Control + cy AMP

  7. 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

  8. Warning! The analysis is EXTREMELY sensitive to conditions • strains must be congenic • culture conditions must be precisely reproduced

  9. Coordinating growth

  10. G tetraphos & G pentaphos Signals for aa starvation rel rRNA synthesis A system that detects shortage of aa & signals starvation AMP + ppGppp ppGpp spoT GDP ATP + GTP

  11. 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 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 +ppGpp -ppGpp Compare

  12. + rel -R -H -P -L Residual protein synthesis in starved rel+ or rel- Control 35S-methionine incorporation -R induced proteins 10 to 20% residual incorporation during starvation Specific responses to particular -aa

  13. + rel -R -H -P -L Residual protein synthesis in starved rel+ or rel- - rel Control -R -H -P -L Specific responses to particular -aa Global changes with  in MW

  14. 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 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 • starvation for histidine reduces protein synthesis to 10%

  15. A A C C D D E E F F G G H H I I K K L L M M N N P P Q Q R R S S T T V V W W Y Y 1 1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +181 +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 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 • starvation for histidine reduces protein synthesis to 10% • rate of synthesis of average peptide is reduced 10x •  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

  16. A steady state determines pool size A B C D F G

  17. Sucking the pool dry (almost)

  18. 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

  19. Errors in translation are not seen in starved rel+ cells -H rel+ -H rel- Control How can ppGpp  the fidelity of translation during starvation?

  20. Relative L7 expression +++ control +++++ Difference consistent with the difference in H and I abundance in the protein rel- - H + rel- - I +++ rel+ - H Expression insensitive to aa abundance +++ rel+ - I Starved rel+ cells behave as if they are not missing an aa • ribosomal proteins L7 has no histidine

  21. P r o t e i n r R N A ppGpp Inhibits Protein Synthesis as well as rRNA Synthesis - aa -  +aa Control rel- no ppGpp rel+ unstable ppGpp rel+ spoT- stable ppGpp

  22. A A C C D D E E F F G G H H I I K K L L M M N N P P Q Q R R S S T T V V W W Y Y 10 1 +10 +1 +10 +1 +1 +10 +10 +1 +10 +1 +181 +10 +1 +10 +1 +10 +10 +1 +10 +1 +10 +1 +1 +10 +1 +10 +1 +10 +10 +1 +10 +1 +1 +10 +1 +10 +1 +10 Sharing the burden • in a rel- strain all the slowing of translation occurs at the “hungry codon” • ppGpp slows down translation at multiple steps of translation • The aa-tRNA for the hungry codon is only reduced enough to generate a signal

  23. 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)

  24. 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.

  25. t t t Specific responses to ppGpp -aa (I) +I (0-5 min) +I (12.5 min) ppGpp rel+ spoT+ [C] ppGpp -aa  rel+ spoT- [C] rel- [C]

  26. t t t Specific responses to ppGpp -aa (I) +I (0-5 min) +I (12.5 min) ppGpp rel+ spoT+ [C] ppGpp -aa  rel+ spoT- [C] rel- [C]

  27. t t t Specific responses to ppGpp -aa (I) +I (0-5 min) +I (12.5 min) ppGpp rel- [C] ppGpp -aa  rel+ spoT+ [C] rel+ spoT- [C]

  28. Pat O’Farrell Dept. Biochem and Biophys. UCSF San Francisco

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