10 chapter 3
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McGill McGill BIO200 - Fall 201 BIO200 - Fall 201 © R. Roy, 2010 © R. Roy, 2010 10 10 chapter 3
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10 chapter 3. 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 - PowerPoint PPT Presentation
Transcript of 10 chapter 3
McGillMcGill BIO200 - Fall 2010BIO200 - Fall 2010
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10 10
chapter 3
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50kDa
600nt
400nt
Western Blot
Northern Blot
mRNA levels and protein levels that do notmRNA levels and protein levels that do notcorrelate: Translational Regulationcorrelate: 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
Time0h 2h 4h 8h 12h
McGillMcGill BIO200 - Fall 2010BIO200 - Fall 2010
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Translation of some mRNAs can be inhibitedTranslation of some mRNAs can be inhibited
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.
FERRITIN is an intracellular protein that binds iron ions, thereby preventing the accumulation of toxic levels of free iron ions.
ferritin binds excess amounts of free iron ions
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mRNA translation inhibition has a pivotal mRNA translation inhibition has a pivotal role in role in DrosophilaDrosophila embryo development embryo development
wild type:
nanos mutant:
Despite its uniform mRNA distribution, hunchback protein is present in an anterior to posterior gradient!
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.
This is due to repression of translation of hunchback mRNA by nanos protein, as observed in a nanos mutant!
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Post-translational RegulationPost-translational Regulation
Protein modification -Phosphorylation
-Glycosylation-Lipid moieties-Small peptides
Protein Folding -Chaperones
Protein Stability -Ubiquitin-mediated proteolysis
-Proteasome
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Kinases accept two substrates to transfer a Kinases accept two substrates to transfer a phosphate from one to the otherphosphate from one to the other
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Phosphorylation of proteins can sometimes Phosphorylation of proteins can sometimes cause mobility shiftscause 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.
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Phosphorylation mediates many cellular Phosphorylation mediates many cellular processesprocesses
• 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
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Ubiquitin is the founding member of a Ubiquitin is the founding member of a family of polypeptide modifiersfamily of polypeptide modifiers
Ubiquitin is a76 amino acid polypeptide
that can be covalentlylinked to lysine (K)
residues
Polyubiquitination targetsproteins for degradatioin
via the proteasome
Mono-ubiquitination does not
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Ubiquitination requires 3 classes of enzymeUbiquitination requires 3 classes of enzyme
• E1-Ubiquitin activating enzymeUses ATP to link itself to
ubiquitin through a thiolester bond with a Cysteine residue
• E2-Ubiquitin conjugating enzymeUbiquitin is transferred to this
enzyme to associate with the appropriate E3.
• E3-Ubiquitin ligaseSubstrate specificationCell may use many E3 enzymesAPC, HECT, SCF
McGillMcGill BIO200 - Fall 2010BIO200 - Fall 2010
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Ubiquitination requires 3 classes of enzymeUbiquitination requires 3 classes of enzyme
• E1-Ubiquitin activating enzymeUses ATP to link itself to
ubiquitin through a thiolester bond with a Cysteine residue
• E2-Ubiquitin conjugating enzymeUbiquitin is transferred to this
enzyme to associate with the appropriate E3.
• E3-Ubiquitin ligaseSubstrate specificationCell may use many E3 enzymesAPC, HECT, SCF
McGillMcGill BIO200 - Fall 2010BIO200 - Fall 2010
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Ubiquitination requires 3 classes of enzymeUbiquitination requires 3 classes of enzyme
• E1-Ubiquitin activating enzymeUses ATP to link itself to
ubiquitin through a thiolester bond with a Cysteine residue
• E2-Ubiquitin conjugating enzymeUbiquitin is transferred to this
enzyme to associate with the appropriate E3.
• E3-Ubiquitin ligaseSubstrate specificationCell may use many E3 enzymesAPC, HECT, SCF
McGillMcGill BIO200 - Fall 2010BIO200 - Fall 2010
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The Proteasome looks like a garbage can!The Proteasome looks like a garbage can!
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……and works like one too! and works like one too! Protein target is
polyubiquitinated on Lysine residues
Polyubiquitin chain is recognised by subunits in the
proteasome cap
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……and works like one too! 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 -
subunits --> amino acids
