Lecture 7 - Protein Precursor Processing Flashcards
Purpose and location of 3’ UTR?
Untranslated region to the 5’ of the poly A tail on mRNA
Contains regulatory elements with particular structures (like hairpins) for proteins to bind and regulate gene expression
Purpose and location of 5’ UTR?
Untranslated region to the 3’ of the 5’ cap
Contains regulatory elements with particular structures (like hairpins) for proteins to bind and regulate gene expression
What are the 5 fates of the soluble proteins produced by free ribosomes? What kind of transport brings them to all 5 places?
- Nucleus
- Mito
- Plastids
- Peroxisome
- ER
Gated transport
What is the 1 fate of the proteins once they are processed by the ER?
Golgi
What are the 4 fates of the proteins that have been processed by the Golgi? What do these depend on?
- Lysosome
- Endosome
- Secretory vesicles
- Cell surface
Different glycosylations
How are proteins transported from the ER to the Golgi?
Vesicles
What are 5 examples of the regulatory elements (repressors or enhancers) on the mature mRNA in the untranslated regions? What do these have in common?
- Hairpin
- Hairpin-like secondary structures
- uORF = upstream open reading frame
- IRES = internal ribosome entry site
- CPE = cytoplasmic polyadenylation element
Many of these are palindromic
What part of the newly synthesized protein directs it to the ER?
Signal peptide at the protein N-terminal
When does the protein translocation into ER happen?
Either co-translationally or post-translationally
What does co-translational protein translocation in the ER involve?
Binding of the ribosome to the ER membrane
Describe the signal peptide.
13-36 AA (usually 20) hydrophobic peptide with N-terminal portion containing at least 1 positive AA (R or K) and cleavage site preceded by 4-5 polar residues except positions 1 and 3 have neutral and small R groups
What is another name for cotranslational protein translocation into the ER?
Vectorial transport
What is the signal hypothesis? 6 steps
- SRP binds the signal peptide which causes a pause in translation
- SRP-bound ribosome attaches to an SRP receptor in the ER membrane close to a translocator that forms a pore in the membrane
- Translation continues and translocation begins through the pore
- SRP and its receptor are displaced and recycled
- A signal peptidase is closely associated with the translocator and clips off the signal sequence during translation
- The mature protein is released into the lumen of the ER immediately after synthesis.
In what conformation are the translocators on the ER membrane? Why?
The translocators are closed until the ribosome has bound, so that the permeability barrier of the ER membrane is maintained at all times
What happens to the signal peptide after it is cleaved off?
It remains in the ER membrane for a period of time (N-terminal on cytosolic side) and then either goes into ER lumen (if soluble) or remains in membrane
What is SRP?
Signal recognition particle: riboprotein (cytoplasmic RNA + proteins)
What is the single pool of ribosomal subunits for?
Common pool of cytosolic ribosomal subunits form ribosomes that synthesize the proteins that stay in the cytosol and those that are transported into the ER
Describe how an ER membrane bound polyribosome works.
The mRNA molecule remains permanently bound to the ER as part of a polyribosome, while the ribosomes that move along it are recycled; at the end of each round of protein synthesis the ribosomal subunits are released and rejoin the common pool in the cytosol
Describe the structure of the ER membrane translocator.
Alpha helices with two parts attached at a seam surrounding an aqueous pore with a plug that seals it
Describe how the ER membrane translocator opens.
To open, the complex rearranges itself to move the plug helix out of the way and the pore can also open sideways at a seam
What are the 2 fates of the protein as it goes through the ER membrane translocator? What does the fate depend on?
- ER lumen
- ER membrane
Based on the characteristics of the R groups of the AAs, free energy would decide
Describe the formation of an ER transmembrane protein.
There are hydrophobic stop and start transfer peptide binding sites that are read by the translocator and direct the protein stretch either in the membrane or outside (cytosol or ER lumen) (either stopping or starting translocation) depending on whether the AAs are hydrophilic or hydrophobic
Where is the N-terminal of an ER transmembrane protein?
Lumen
Where is the C-terminal of an ER transmembrane protein?
Cytosol
What side of the ER membrane is the N-terminal of the signal peptide?
Cytosol
What do the sequences on the upper part of the hydropathy plot represent? How long are these?
Hydrophobic, transmembrane domains of the protein (~20 AAs long)
What is another name for the signal sequence?
The leader sequence
What is the purpose of the 1 (+) charged residue near the N terminal of the signal peptide?
Binding to the SRP
Where is the signal peptidase?
Transmembrane protein with activity on the ER lumen side
Where does glycosylation of proteins happen?
On the ER lumen side
What are the 2 ways in which the Golgi sends proteins to the cell surface? Describe each.
- Regulated exocytosis: involves signal transduction and ligand-receptor signaling to secretory granules or vesicles that are mobilized to the cell surface for release
- Constitutive exocytosis: dependent on receptor-mediated transcription events (aka gene expression regulation) in the nucleus, leading to trafficking via Golgi through recycling endosomes to the cell surface
What does constitutive exocytosis include?
Neuronal and endocrine secretions (paracrine and autocrine)
How is the C-terminal of biologically active peptides protected? Protect against what?
AMIDATION: amide group left over from peptide bond cleavage to glycine
to protect the protein from carboxy-peptidases
How is the N-terminal of biologically active peptides protected (3 ways)? Protect against what?
- DISULFIDE BOND: forms a cyclic structure
- Acetylated (once or twice)
- Pyroglutamyl moiety (ring structure)
Protect from amino-peptidases
What kind of AAs usually surround the biologically active protein sequence in the pre-pro-peptide form?
Pairs of basic AAs
What are the 3 enzymes that process a prepropeptide into a biologically active protein? List them in order of activity.
- Proprotein convertases (PC)
- Carboxypeptidase H
- Peptidyl-glycine-α-mono-oxygenase (PAM)
Explain how and where proprotein convertases (PC) work.
They cut on the C-terminal side of a basic AA within a pair of basic AA at both the C-terminal and N-terminal of the proprotein
What kind of peptidases are proprotein convertases?
Endopeptidases
Explain how and where carboxypeptidases H work.
They cut off C-terminal basic AAs (C-terminal of proprotein)
Explain how and where peptidyl-glycine-α-mono-oxygenases (PAMs) work. Write out the reaction.
Cuts off part of a C-terminal glycine residue to amidate the C-terminal.
-X-Gly + O2 + ascorbate (VC) = -X-NH2 + glyoxylate + dehydroascorbate + H2O
What does the “pre” nomenclature mean?
Signal peptide + protein
What does the “pro” nomenclature mean?
Protein without signal peptide but not fully processed
What is an example of a protein whose N-terminal is revealed by a signal peptidase? Instead of what?
Vasopressin
Instead of it being revealed by a proprotein convertase
Are peptide hormones usually glycosylated?
Usually not
When does preproprotein processing usually happen?
During vesicular transport from Golgi
What is an example of a protein that can be processed in many different ways to produce many different biologically active proteins? What does the different processing depend on?
POMC
Depends on location
What is an example of a protein processing cascade in the blood? Provide details.
Pre-pro-renin => pro-renin => renin => converts angiotensinogen (prohormone) to angiotensin I (not a hormone) => ACE converts it to angiontensin II (hormone)
What does it mean when Py is read in a recognition DNA sequence?
Pyrimidine
