(3) Functions and Dysfunctions of Protein Processing Flashcards
What does AUG code for?
Met (M), START codon
What are the three STOP codons?
UAA, UAG, UGA
What is a codon?
A group of 3 consecutive nucleotides
What are the four different categories of mutations?
- Silent mutation
- Missense mutation
- Nonsense mutation
- Frameshift mutation
What happens with a silent mutation?
Does not change the amino acid
What happens with a missense mutation?
Changes amino acid in the protein
What happens with nonsense mutation?
Codon changes into a STOP codon;
protein either degraded or formed as a truncated version
What happens with a frameshift mutation?
Change in the codon sequence and consequently alteration in the amino acid sequence
*MAJOR EXAMPLE: Duchenne Muscular Dystrophy
CLINICAL CORRELATION:
Sickle Cell anemia:
MISSENSE mutation
- Substitutes Val (hydrophobic) for Glu (neg., hydrophilic)
- Deforms RBCs
- Deformed erythrocytes have poor oxygen capacity and clog capillaries
CLINICAL CORRELATION:
Duchenne Muscular Dystrophy
FRAMESHIFT MUTATION
- Deletions in dystrophin gene
- Muscle wasting
- In-frame deletions result in expression of truncated forms of dystrophin: milder form BECKER muscular dystrophy
What is the mRNA 5’ cap made of?
7 Methyl Guanosine at 5’ end
What is the mRNA poly(A)tail?
Repeating A’s at the 3’ end
What are tRNA?
“Transfer” RNAs
- Have binding site for both codons (in mRNA) and amino acid)
- Match amino acids to codons in mRNA
What is the structure of tRNA?
Cloverleaf Shape:
ONE END=Anticodon loop
OTHER END= 3’ CCA Terminal region, binds amino acid that matches corresponding codon
Aminoacyl tRNAs?
- complex of tRNA w/ AA
- catalyzed by AMINOACYL tRNA synthetases
- each tRNA charged with the correct AA to maintain fidelity of protein synthesis
Ribosomes have ____ subunits
2; a large and small subunit
What is the difference b/w prokaryotic and eukaryotic ribosomes?
Prokaryotic: 70S, large=50S, small=30S
Eukaryotic: 80S, large 60S, small=40S
What are the three important sites of the ribosomal complex?
- Acceptor site (A) site
- Peptidyl (P) site
- Empty (E) exit site
What are the three steps of translation?
- Initiation
- Elongation
- Termination
Describe INITIATION of translation, elongation and termination via WHITEBOARD GO!
Did you do it? Hope so.
What are polysomes?
- Clusters of ribosomes simultaneously translating a single mRNA molecule
- Makes protein synthesis more efficient
What are the two major pathways for protein sorting?
- Cytoplasmic pathway
2. Secretory pathwy
What is the function of the cytoplasmic pathway?
*For proteins destined for CYTOSOL, MITOC., NUCLEUS, and PEROXISOMES
What is the function of the secretory pathway?
*For proteins destined for ER, LYSOSOMES, PMs, or for FAT SECRETION
Proteins that are targeted for the mitochondria have…
N-terminal hydrophobic alpha helix
Proteins that are signaled to the nucleus have…
Lots of Lys(K) and Arg(R)
Proteins that are signaled to the peroxisome have…
-SKL
Secretory pathway:
What is the ER targeting signal?
(+) charged aa sequence at N terminus
Secretory pathway:
What signals proteins to stay in RER?
K(Lysine)
D(Aspartic acid)
E(Glutamic Acid)
L(Leucine)
Secretory pathway
What signals protein to lysosome?
Mann 6-P
Secretory pathway:
What signals protein to secrete protein out into cytosol?
Trp-rich domain
Nuclear Import:
How do materials get into the nucleus?
- Imported via nuclear pores
- Small proteins able to pass through specific pores
- Large proteins >40kDa require a NUCLEAR LOCALIZATION SIGNALS
- FOUR continuous basic residues (Lys and Arg)
What is Alzheimer’s disease?
Loss of memory, cognitive function, language
What is Parkinson’s disease?
Impairment of fine motor control
What is Huntington’s disease?
Loss of movement and cognitive functions and psychiatric problems
What is Crutzfeldt-Jacob disease?
Failing memory, behavioral changes, lack of coordination and visual disturbances. Late stages involve mental deterioration blindness, weakness of extremities and coma
What is the manifestation of Alzheimer’s disease?
- Amyloid beta peptide (ABeta)
- Amyloid beta plaques in brain
- Hyperphosphorylation of Tau (neurofibrillary tangles
What is the manifestation of Parkinson’s disease (PD)?
- Aggregation of alpha-synuclein (AS) –> deposit as LEWY bodies in dopaminergic neurons in substantia nigra
- Sx. due to reduced availability of dopamine
What is the manifestation of Huntington’s disease (HD)?
- Mutation in Hungingtin gene; results in expansion of CAG triplet repeats
- Results in Polyglutamine repeats
- Selective death of cells in basal ganglia cause the sxs
What is the manifestation of Creutzfeldt-Jakob disease (HD)?
- Caused by misfolding of PRION proteins
- **Transmissible
- Belongs to Transmissible spongiform encephalopathies (TSEs)
- SPONGIFORM
What is the central dogma of molecular biology?
Replication - Transcription - Translation
How are tRNAs activated?
AMINOACYL tRNA SYNTHETASE catalyzes the addition of AMP to COOH at the end of AA
Ribosome structures differ between prokaryotes and eukaryotes. How is this medically relevant?
Use antibiotics to target prokaryotic translational machinery
Translation occurs in the ___’ to ____’ direction
5’ –> 3’
Translation initiation:
Where does it occur in prokaryotes?
Shine-Delgarno (SDG) sequence (AGGAGG)
Translation initiation:
Where does it occur in eukaryotes?
5’ cap, 3’ poly-A tail
Translation initiation:
All known mRNA molecules contain …
signals that define the beginning of each encoded polypeptide chain
Translation initiation:
Where does the MET add?
Small SU of ribosomes on the P site
Translation initiation:
What initiation factors are included in prokaryotes vs eukaryotes?
Proks: IF
Euks: eLFs
Translation begins with the initiation codon _____
AUG (Methionine)
WHITEBOARD: Explain ELONGATION
GOOOOoooOOOoo
Elongation:
Activated aa attached to initiating methionine via a ______ bond
peptide
Elongation:
How is the polypeptide chain extended?
Loading of aminoacyl tRNA onto the ribosome so that the anticodon base pairs with codon positioned on A site
Elongation:
Prior to loading, the aminoacyl tRNA is attached to a ____ bound elongation factor
GTP
Elongation:
Loading accompanied by ____ and _____ from aminoacyl tRNA
GTP hydrolysis; release of factor
Elongation:
What is the peptide formation between aa in the A and P site catalyzed by?
Peptidyl transferase
Termination:
What is termination triggered by?
STOP CODONS
UAA, UAG, UGA
*Release factor binds to the A site
Termination:
Stop codons are recognized by:
Release factors (RFs)
Termination:
RFs bind to the _____ of the ribosome containing the stop codon and cleaves the ______
A site; ester bond b/w the C terminus of the polypeptide and the tRNA
Termination:
______ dissociates ribosomal complex
GTP Hydrolysis
What are the ribosome subunits for prokaryotes?
30S, 50S [70S]
What are the ribosome subunits for eukaryotes?
40S, 60S [80S]
Ribosome key components to remember:
What binds to the 30S subunit to disrupt initiation of translation?
Streptomycin
Ribosome key components to remember:
What binds to the 60S subunit to disrupt elongation?
Shiga Toxin
Ribosome key components to remember:
What does Streptomycin do?
Binds to the 30S subunit to disrupt initiation of translation
Ribosome key components to remember:
What does Shiga toxin do?
Binds to the 60S subunit to disrupt elongation
Ribosome key components to remember:
What two medications bind to the 50S subunit to disrupt translocation of the ribosome?
Clindamycin and erythomycin
Ribosome key components to remember:
What to Clindamycin and erythomycin do?
Bind to the 50S subunit to disrupt translocation of the ribosome
Ribosome key components to remember:
________ bind to the 30S subunit to disrupt elongation
Tetracyclines
Ribosome key components to remember:
What does Tetracycline do?
Binds to the 30S subunit to disrupt elongation
Ribosome key components to remember:
_________ is housed in the large subunits
Peptidyl transferase activity
Initiation requires hydrolysis of:
ONE GTP
Elongation requires hydrolysis of:
TWO GTP per amino acid added
Termination requires hydrolysis of:
ONE GTP
What does Diphtheria do?
Inactivates EF2-GTP and inhibits elongation
What are the (4) prokaryotic elongation inhibitors?
- Tetracycline
- Chloramphenicol
- Clindamycin/Erythromycin
- Streptomycin
What does Chloraphenicol do?
Inhibits peptidyl transferase
*Prok
What does Clindamycin/Erythromycin do?
Binds to large 50S SU blocking translocation of the ribosome
*Prok
What does Streptomycin do?
Binds to 30S subunit, interferes with the binding fmet-tRNA. Interferes with 30S subunit association with 50S subunit
*Prok
What are the four eukaryotic elongation inhibitors?
- Cycloheximide
- Diptheria toxin
- Shiga Toxin
What does Cycloheximide do?
Inhibits peptidyl transferase
*Euk
What does Diphtheria toxin do?
Inactivates GTP bound- eEF-2 interfering with ribosomal translocation
*Euk
What does Shiga toxin/Ricin do?
Binds to large 60S SU, blocking entry of aminoacyl tRNA to ribosomal complex
What does Puromycin do?
Causes premature chain termination
*elongation inhibitor
Cytoplasmic Pathway:
Proteins synthesized in this pathway have _________. Stay in cytoplasm
NO TRANSLOCATION SIGNALS
What are TIM and TOM?
Transporters in the mitochondrial membrane
TOM: Transporter in Outer membrane
TIM: Transporter in Inner membrane
Unfolded proteins are protected by binding to chaperones, in particular _______
Heat Shock Proteins 70 (HSP70)
What is the fx of signal recognition particle (SRP)?
Binds to the ER-targeting signal and the ribosome during translation;
SRP wraps itself around the ribosome-mRNA-peptide complex, halts translation temporarily
What are HSP60 and HSP70 examples of?
Chaperone proteins
Post-translational processing:
What is proteolytic cleavage?
Converts inactive forms to active enzymes by unmasking active site
Converts nascent precursor proteins to mature ones
What are the 4 post translational covalent modifications?
- Glycosylation
- Phosphorylation
- Disulfide bond formation
- Acetylation
Post Translational Modifications:
Acetylation:
What? Functional Group? Residue Affected?
Covalent linkage to amine
Amine (-NH3)
Lys
Post Translational Modifications:
Glycosylation
What? Functional Group? Residue Affected?
O-glycosylation(hydroxyl -OH) ; Ser, Thr
N-glycosylation (Acid Amide (-CONH2) ; Asn (Asparagine)
Post Translational Modifications:
Phosphorylation
What? Functional Group? Residue Affected?
Phosphate linked via esterification
Hydroxyl (OH)
Ser, Thr & Tyrosine Kinase
*regulated enzyme activity and protein function
Post Translational Modifications:
Disulfide bonds
What? Functional Group? Residue Affected?
Oxidation to achieve covalent linkage of cysteine residues
Sulfhydryl (-SH)
Cys
Phosphate removed by:
Phosphatases
Post Translational Modifications:
How are acetylation reactions catalyzed?
Histone acetyltransferases (HAT)
of
Histone deacetylase (HDAC)
CORRELATION BOX:
Sickle cell anemia
MISSENSE MUTATION THAT….
changes HYDROPHOBIC (VAL) to HYDROPHILIC (GLU)
- Aggregate and form rigid, rod-like structures
- Deforms RBCs
- Poor oxygen capacity, clog capillaries
CORRELATION BOX:
Duchenne muscular dystrophy (DMD)
- Large in-frame and out of frame deletions to the DYSTROPHIN GENE
- Muscle wasting
Initiation requires hydrolysis of _______
One GTP
Elongation requires hydrolysis of _______
2 GTP per amino acid added
Termination requires hydrolysis of _____
1 GTP
