Functionas And Dysfunctions Of Protein Processing Flashcards
- Start codon, and what does it code for?
2. Stop codons, what do they code for?
- AUG—codes for methionine
2. UAA, UAG, UGA—code for nothing!
Silent mutation
Does not change the amino acid.
No consequence, does not make any “noise”
Missense mutation
Changes amino acid in the protein.
Depending on the amino acid, it could be severe or harmless.
Nonsense mutation
Codon changes into a stop codon (UAA, UAG, UGA), and truncated the protein.
“STOP this NONSENSE”
Frameshift mutation
One or more nucleotides are deleted/inserted into ORF.
Change in the codon sequence and consequently alteration in the amino acid sequence
Duchenne Muscular Dystrophy
Sickle cell anemia
- Type of mutation
- Change
- Consequence
- Missense mutation
- Hydrophilic Glu changes to hydrophobic Val
- Mutation alters the sharp of RBCs, causes them to aggregate and form rigid rod-like structures. Have poor oxygen capacity
Duchenne Muscular Dystrophy
- Type of mutation
- Consequence
- Characteristics
- Large in-frame and out-of-frame (OOF) deletions in dystrophin gene
- If OOF—little/no expression of dystrophin protein, give rise to severe Duchenne muscular dystrophy.
If in-frame—results in truncated dystrophin. Gives rise to milder Becker muscular dystrophy - Duchenne—present in 1:3500 males (common), leads to muscle wasting—confinement to wheel chair and respiratory failure.
What two modifications protect the mRNA from degradation during transport?
5’ 7-methylguanosine cap and a poly(A) tail
What is the secondary structure of tRNA?
Clover-leaf!
What is the function of the anticodon loop in the tRNA?
A set of 3 nucleotide that pair with the complementary codon in the mRNA
What is the function of the 3’ CCA terminal region in tRNA?
This region binds the amino acid that corresponds to the mRNA codon.
What is an aminoacyl tRNA, and how is it formed?
Aminoacyl tRNA: complex of tRNA with an amino acid.
Aminoacyl tRNA synthetase esterifies amino acid to CCA sequence using an ATP.
Each amino acid has its own aminoacyl tRNA synthetase—serves as a second genetic code.
What is the difference between prokaryotic and eukaryotic ribosomes?
Why is this important?
Prokaryotic ribosome: large subunit—50s, small unit—30s, total—70s.
Eukaryotic ribosome: large subunit—60s, small subunit—40s, total—80s
This is important because some antibiotics selectively target bacterial ribosomes.
Three important sites in the ribosomal complex
- Acceptor (A) site: mRNA codon exposed and able to receive aminoacyl tRNA. A for Amino Acid
- Peptidyl (P) site: where aminoacyl tRNA is attached. The polypeptide grows here.
- Empty (E) or exit site: location that is occupied by empty tRNA before exiting
Three steps of translation
- Initiation
- Elongation
- Termination
Shine-dalgarno sequence
In prokaryotes, the site at which protein synthesis can begin.
Steps of translation initiation
- Methionine binds to P site of small subunit, along with eIF2 and GTP
- A complex that includes the mRNA and two initiation factors (eIF4G and eIF4E), the small ribosomal unit scans the complex for AUG and “clicks” into place with the corresponding Met that is already in the P site.
- Initiation factors dissociate and the large ribosomal subunit binds.
- Next tRNA binds to A site, forms the first peptide bond (CO-NH)
Steps of translation elongation
- Aminoacyl tRNA is attached to GTPbound elongation factor, EF-Tu, and is brought to the A site
- A peptide bond is formed
- GTP-bound EF-G , assists in the translocation of the ribosome
Steps of translation termination
- A release factor (RF) binds to the stop codon
- Release factor cleaves the ester bond between the c terminus of the polypeptide and the tRNA
- Protein is released from the ribosome into the cytosol
- GTP hydrolysis dissociates ribosomal complex.
What is a polysome
Clusters of ribosomes simultaneously translating the same mRNA molecule.
More efficient
Energy requirement for initiation of translation
One hyrdolysis of one GTP
Energy requirement of elongation
Requires hydrolysis of two GTP per amino acid added
Energy requirement of termination
Requires hydolysis of one GTP
Prokaryotic elongation inhibitors (5)
- Tetracycline
- Chloramphenicol
- Clindamycin
- erythromycin
- Streptomycin
Mechanism of action of TETRACYCLINE
Binds to small 30s SU, clocks entry of aminoacyl-tRNA to ribosomal complex
Cannot make proteins
Mechanism of action for CHLORAMPHENICOL
Inhibits peptidyl tranferase
Mechanism of CLINDAMYCIN AND ERYTHROMYCIN
Binds to large 50s SU, blocks translocation of the ribosome
Erythromycin commonly used to treat pertussis**
Mechanism of action for STREPTOMYCIN
Binds to 30s SU, interferes with the binding of fmet-tRNA. Disrupts association with 50s subunit.
Eukaryotic elongation inhibitors (4)
- Cycloheximide
- Diphtheria toxin
- Shiga toxin
- Ricin
Mechanism of action of CYCLOHEXIMIDE
This is a toxin from streptomyces griseus that inhibits peptidyl transferase
Mechanism of action for DIPHTHERIA TOXIN
from corynebacterium diphtheriae that inactivated GTP-bound eEF-2, interfering with ribosomal translocation
Mechanism of action of SHIGA TOXIN and RICIN
Binds to large 60s SU, block energy of aminoacyl-tRNA to ribosomal complex.
Elongation inhibitor
What is its mechanism of action
Puromycin—from streptomyces alboniger
Causes premature chain termination in prokaryotes and eurkaryotes
It resembles the 3’ end of aminoacylayed-tRNA and enters the A cite. It adds to the peptide but forms a puromycylated chain which results in a premature release of the chain.
There are two major pathways for sorting proteins, what are they and where are their destinations?
Cytoplasmic pathway: proteins destined for the cytosol, mitochondria, nucleus, and peroxisomes. Ultimately, Andy protein that will live in the cell.
Secretory pathway: proteins destined from the ER, plasma membrane, or secretion. Anything that will be leaving the cell.
Proteins that do not have translocation signals will be sent where?
No where, they will stay in the cytosol
Mitochondrial translocation signal
N-terminal hydrophobic alpha-helix
Additionally, this sequence helps to interact with chaperone proteins.
Mitochondrial proteins must be brought across the membrane using which complexes?
How will they fit?
What else do they need?
Protein enters mitochondria via TOM, and the matrix of the mitochondria via TIM.
Proteins must unfold into their linear form to pass through these translocators.
The proteins will need the additional support of chaperones (specifically, heat shock protein HSP70) to refold after transport.
Nucleus translocation sequence
KKKRK signal sequence.
Rich in lysine and arginine
Peroxisome translocation signal
C-terminal SKL sequence
Serine, lysine, leucine
All proteins in the secretory pathway must first travel through the ER. What is the translocation signal?
N-terminal positively charged amino acid alpha helix
Translocation sequence for those proteins destined to stay in the ER
C-terminal KDEL retention signal
Lysine, aspartic acid, glutamic acid, leucine
Lysosome translocation signal
Mannose 6-phosphate
*this is the only one that is a sugar.
Linked to I-cell disease
Translocation sequence for secretory proteins
Tryptophan rich domain signal region+absence of retention motif
Membrane translocation sequence
N-terminal apolar region
Function of Signal Recognition Particle (SRP)
How does it work?
Bring ribosome-new protein complex to the ER
- SRP binds to the ER targeting signal and ribosome
- SRP wraps around the complex, tethers it to the ER membrane, temporarily halting translation
- Translation resumes when protein directed into the lumen
- Enzymes on luminal side cleave signal and release protein
I-cell disease
Inclusion cell disease
Severe from of lysosomal storage disease.
Tagging of lysosomal proteins with mannose 6P is defective, so they are not brought to the lysosome.
Leads to high plasma levels of lysosomal enzymes.
Small proteins can fold by themselves, but large ones cannot.
Why not, and what do they need to assist them?
Large proteins risk aggregation and proteolysis if fold unaided.
Helped by chaperones and chaperonins
Describe proteolytic cleavage
Convert inactive forms of enzymes to active by cleaving the unmasking site.
Convert precursor proteins to mature ones.
Types of covalent post translational modifications (4)
- Glycosylation
- Phosphorylation
- Disulfide bond formation
- Acetylation
Acetylation
- Modification
- Functional group
- Residue affected
- Covalent linkage to amine
- Amine (NH3+)
- Lys
Glycosylation
- Modification
- Functional group
- Residue affected
- O-glycosylation
- Hydroxyl (-OH)
- Serine/threonine
- N-glycosylation
- Acid-amine (-CONH2)
- Asn, Gln
Phosphorylation
- Modification
- Functional group
- Residue affected
- Phosphate linked via esterification
- Hydroxyl (-OH)
- Ser, Tyr, Ther; also Asp and His
Disulfide bond formation
- Modification
- Functional group
- Residue affected
- Oxidation to achieve covalent linkage of cysteine residues
- Sulfhydryl (-SH)
- Cys
Why do diabetics get cataracts, (potentially)?
The excess blood sugar in uncontrolled diabetics gets glycosylase death onto the proteins of the lens of the eye.
What enzyme phosphorylates?
Serine/threonine and tyrosine kinases
Phosphorylation is especially important during what events?
Cell signaling Cell growth Proliferation Differentiation Oncogenesis
Location of disulfide bond formation
ER lumen, facilitated by protein disulfide isomerases
Where are proteins typically acetylated?
On their lysine residues
Donor group for acetylation
Acetyl CoA
Collagen
- How is it post-translationally modified?
- Why?
- What do defects cause?
- Role of ascorbic acid
- Lysine in collagen is modified to make 5-hydrolysines and is then further glycosylated with glucose and galactose
- These mods are important for the assembly of collagen
- Skin, bone, and joint disorders: Ehlers-Danlos, Nevo Syndrome, Bruck Syndrome, Epidermolysis Bullosa Simplex
- Needed for activity of lysl and prolyl hydroxylases
Alzheimer’s disease (AD)
- Area of brain affected
- Cause
- Effects
- Hippocampus and frontal cortex
- Amyloid precursor protein (APP) breaks down into amyloid beta peptide (A-beta). The misfolding/aggregation of A-beta forms extracellular plaques in the brain
Additionally, the hyperphosphorylation of Tau (a microtubual stabiliazer) causes intracellular neurofibrillary tangles.
- Loss of memory, cognitive function, and language
Parkinson’s Disease (PD)
- Area of brain affected
- Cause
- Effects
- Substantia nigra (middle of the brain)
- Aggregation of alpha-synuclein (AS) forms fibrils that deposit as Lewy bodies in dopaminergic neurons of the substantia nigra. This results in selective death of these neurons and reduced availability of dopamine
- Impairment of fine motor control
Huntington’s disease (HD)
- Area of brain affected
- Cause
- Effects
- Basal ganglia
- Mutation in the Huntingtin gene results in excessive (36-121) repeats of CAG triplets. Results in polyglutamine repeats in abnormal HTT protein leading to aggregate
- Loss of movement and cognitive functions, psychiatric problems.
Creutzfeldt-Jakob disease
- Area of brain affected
- Cause
- Effects
- Entire brain—filled with holes, resmebles sponge.
- Misfolding of prion proteins. Are infective and convert normal proteins into prion form
- Failing memory, behavioral changes, lack of coordination, visual disturbances. Late stages—mental deterioration, blindness, weakness of extremities, coma.
