10 chapter 3

1. 10 chapter 3

2. mRNA levels and protein levels that do not correlate: Translational Regulation The synthesis of some polypeptides is under strict regulatory control. Protein synthesis often reflects mRNA abundance such that: more mRNA = more Protein When this relationship appears skewed it may indicate that protein synthesis off the mRNA template is controlled by cellular mechanims 50kDa Western Blot 600nt Northern Blot 400nt 0h 2h 4h 8h 12h Time

3. ferritin binds excess amounts of free iron ions In the low iron induced confirmation, IRE-BP can bind to the IREs in the 5’ UTR of ferritin mRNA, inhibiting translation of the mRNA. This results in a drop of ferritin protein production and therefore prevents depletion of iron from important cellular processes. Translation of some mRNAs can be inhibited • FERRITIN is an intracellular protein that binds iron ions, thereby preventing the accumulation of toxic levels of free iron ions.

4. Unlike nanos mRNA, hunchback mRNA is present throughout the egg. • nanos mRNA is located at the posterior pole of a Drosophila egg. Translation of nanos mRNA results in a gradient of nanos protein throughout the egg, since the protein diffuses anteriorly from its site of production. • Despite its uniform mRNA distribution, hunchback protein is present in an anterior to posterior gradient! • This is due to repression of translation of hunchback mRNA by nanos protein, as observed in a nanos mutant! mRNA translation inhibition has a pivotal role in Drosophila embryo development wild type: nanos mutant:

5. Post-translational Regulation Protein modification -Phosphorylation • -Glycosylation • -Lipid moieties • -Small peptides • Protein Folding -Chaperones • Protein Stability-Ubiquitin-mediated proteolysis -Proteasome

6. Kinases accept two substrates to transfer a phosphate from one to the other

7. Phosphorylation of proteins can sometimes cause mobility shifts During SDS-PAGE electrophoresis proteins migrate according to molecular mass because they are coated with highly negatively charged SDS. It is presumed that when proteins are phosphorylated sometimes the amount of SDS that binds can be reduced and thus they do not carry the same negative charge as their non-phosphorylated counterparts. This results in a mobility shift in the gel that is detectable following colouration or in a western blot.

8. Phosphorylation mediates many cellular processes • Phosphorylation can induce changes in protein conformation due to increased local charge. • Activation of an enzymatic activity by bringing catalytic sites into proximity of substrates …CDKs • Exposure of new recognition domains …nuclear localisation • Targeting for degradation via proteolysis …-catenin, cyclins

9. Ubiquitin is a 76 amino acid polypeptide that can be covalently linked to lysine (K) residues Ubiquitin is the founding member of a family of polypeptide modifiers Polyubiquitination targets proteins for degradatioin via the proteasome Mono-ubiquitination does not

10. Ubiquitination requires 3 classes of enzyme • E1-Ubiquitin activating enzyme • Uses ATP to link itself to ubiquitin through a thiolester bond with a Cysteine residue • E2-Ubiquitin conjugating enzyme • Ubiquitin is transferred to this enzyme to associate with the appropriate E3. • E3-Ubiquitin ligase • Substrate specification • Cell may use many E3 enzymes • APC, HECT, SCF

11. Ubiquitination requires 3 classes of enzyme • E1-Ubiquitin activating enzyme • Uses ATP to link itself to ubiquitin through a thiolester bond with a Cysteine residue • E2-Ubiquitin conjugating enzyme • Ubiquitin is transferred to this enzyme to associate with the appropriate E3. • E3-Ubiquitin ligase • Substrate specification • Cell may use many E3 enzymes • APC, HECT, SCF

12. Ubiquitination requires 3 classes of enzyme • E1-Ubiquitin activating enzyme • Uses ATP to link itself to ubiquitin through a thiolester bond with a Cysteine residue • E2-Ubiquitin conjugating enzyme • Ubiquitin is transferred to this enzyme to associate with the appropriate E3. • E3-Ubiquitin ligase • Substrate specification • Cell may use many E3 enzymes • APC, HECT, SCF

13. The Proteasome looks like a garbage can!

14. …and works like one too! Protein target is polyubiquitinated on Lysine residues Polyubiquitin chain is recognised by subunits in the proteasome cap

15. …and works like one too! The ubiquitin moieties are removed and the target protein is unfolded and threaded through the tunnel (ATP dependent) The protein is degraded into peptides in the central chamber consisting of the b-subunits --> amino acids