Lect 2 Functions & Dysfunctions of Protein Processing Flashcards by Austin Collison

Start Codon

Methionine (Met, M)

AUG

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Stop Codon

UAA

UAG

UGA

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What is the genetic code?

How many combinations of codons are there?

What does degenerate mean?

Set of rules that convert nucleotide sequences of a gene into the AA sequence of a protein using mRNA as an intermediary
61 Triplet codons for 20 AAs, 3 Stop Codons
Some AAs can be coded by more than 1 codon

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Point Mutations

Silent Mutation

Missense Mutation

Nonsense Mutation

Frameshift Mutation

Silent: Does not change the AA
Missense: Changes AA in protein (no effect or vastly different function)
Nonsense: Codon –> Stop Codon (premature chain termination forms truncated version)
Frameshift: 1+ Nucleotides deleted or inserted into ORF
- Out of Frame: Change in codon sequence –> alteration in AA sequence

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How does Sickle Cell Anemia arise?

What AAs are changed?

What does this cause to happen?

What is the impact of having deformed RBCs?

Missense mutation
- GAG to GTG: substituting Val (hydrophobic) for Glu (negatively charged/hypophillic)
Causes HbA to aggregate and form rigid, rod-like structures and deforms the RBCs into sickle-like shape
Poor oxygen capacity and tend to clog capillaries

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What causes Duchenne Muscular Dystrophy?

Results of OOF Deletions

What is the physical result?

Results of IF Deletions

Large IF and OOF deletions to dystrophin gene

Little/no expression of dystrophin protein –> Severe form DMD

Muscle wasting

Truncated forms of dystrophin –> Milder form Becker muscular dystrophy

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After transcription, pre-mRNA edited to form mRNA and exported to cytoplasm for translation

What are the components of Eukaryotic mRNA?

Coding region

5’ and 3’ untranslated regions

7-methylguanosine 5’ Cap

Poly(A) tail at 3’ end

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What is the general structure of tRNA?

What are the two regions of unpaired nucleotides and their functions?

Cloverleaf secondary structure

Anticodon Loop: pairs with complementary codon in mRNA

3’ CCA Terminal Region: Binds the AA matching the corresponding codon

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Aminoacyl tRNAs are complexes of tRNA with AA

Where is the AA attached?

What catalyzes this process?

What are the steps?

AA esterified to CCA sequence at 3’ end of tRNA catalyzed by aminoacyl tRNA synthetases (unique to each AA)
- Aminoacyl tRNA synthetase catalyzes addition of AMP to COOH end of AA
- AA transferred to cognate tRNA

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What composes Ribosomes?

How is the structure of prokaryotic and eukaryotic ribosomes medically relevant?

What size subunits in each?

Proteins and rRNA

Antibiotics target prokaryotic translational machinery (ribosomes)

Eukaryotic (80S) = 60S + 40S

Prokaryotic (70S) = 50S + 30S

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What are the three important sites on the Ribosomal complex?

Acceptor (A) Site
- mRNA codon exposed to receive aminoacyl tRNA, except Met tRNA
Peptidyl (P) Site
- aminoacyl tRNA is attached
Empty (E) Site
- occupied by empty tRNA before exiting ribosome

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Translation occurs in this direction

It occurs in 3 steps

5’ –> 3’ direction

Initiation –> Elongation –> Termination

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Initiation

What first binds to the P site?

What is the initiator tRNA-methionine complex?

What else is added?

Translation begins with what?

Initiator tRNA (bound GTP) attaches to P site of small su

Methioninyl tRNA in eukaryotes is loaded on the small subunit of ribosomes on the P-site

Initiation factors (IF in prokaryotes) and eukaryotic initiation factors (eIFs) are added

Initiation codon AUG (Met)

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Elongation:

What is this process?

What catalyzes peptide bond formation?

Activated AA attaches to initiating methionine via peptide bond
- aminoacyl tRNA anticodon base pairs with codon on A site
- Prior to loading, aminoacyl tRNA attached to GTP bound elongation factor
- Loading accompanied by GTP hydrolysis and release of factor from aminoacyl tRNA
Peptide bond formation between AA in A and P site catalyzed by Peptidyl Transferase

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Termination:

What triggers termination?

Stop codons are recognized by _ which bind to the _ site and causes what?

What dissociates the ribosomal complex?

Triggered by Stop Codons (UAA, UAG, and UGA)

Release Factors (RFs) bind to A site and cleaves ester bond (via addition of water) between C-terminus of polypeptide and tRNA

GTP Hydrolysis

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What are polysomes?

Clusters of ribosomes simultaneously translating a single mRNA molecule

GTP Hydrolysis during Translation Steps

Initiation: Hydrolysis of 1 GTP

Elongation: Hydrolysis of 2 GTP per AA added

Termination: Hydrolysis of 1 GTP

Prokaryotic Elongation Inhibitors

Streptomycin

Clindamycin/Erythromycin

Tetracyclines

Chloramphenicol

Streptomycin: 30S su to interfere with binding fmet-tRNA (interferes with 30S association with 50S)

Clindamycin and Erythromycin (Pertussis): 50S su to disrupt translocation of ribosome

Tetracyclines: 30S su to disrupt elongation (block entry of aminoacyl-tRNA to ribosomal complex)

Chloramphenicol: inhibits peptidyl trasferase

Eukaryotic Elongation Inhibitors

Shiga Toxin/Ricin

Diptheria Toxin

Cycloheximide

Shiga Toxin & Ricin: 60S su to disrupt elongation (block entry of aminoacyl-tRNA to ribosomal complex)

Diptheria Toxin: inactivates GTP-bound eEF-2 interfering with ribosomal translocation

Cycloheximide: Inhibits peptidyl transferase (activity in large subunits)

Eukaryotic/Prokaryotic Elongation Inhibitor

Puromycin

Puromycin: Causes premature chain termination (prok/euk)

Cytoplasmic Sorting Pathway destinations and process

Secretory Sorting Pathway destinations and process

Cytoplasmic:
- Destined for cytosol, mitochondria, nucleus, peroxisomes (No translocation signals - stay in cytoplasm)
Secretory:
- Proteins destined for ER, lysosomes, plasma membranes, or secretion

Proteins with specific signals trafficked to various organelles:

Mitochondrial signals include what?

The sequence helps them interact with what?

N terminal hydrophobic a-helix

Chaperone proteins

Mitochondrial Protein Import includes the usage of these transporters

What type of proteins are the chaperones?

Transporter in Inner Membrane (TIM) and Transporter in Outer Membrane (TOM)

Heat Shock Proteins 70 (HSP70)

Nuclear Import is performed with what?

Small proteins can pass through specific pores but large proteins require what?

Proteins destined for nucleus have this feature

Nuclear Pores

Nuclear Localization Signals

Four continuous basic residues (Lys,K and Arg,R)

_Signal Sequences:_ Peroxisomes ER Lumen Lysosomes Secretion Membranes

Peroxisome: **SKL signal sequence** ER Lumne: **KDEL (Lys, Aspartate, Glutamate, Leucine)** Lysosome: **Mannose-6-Phosphate** Secretion: **Tryptophan domain** Membranes: **N terminal apolar region (stop transfer sequence)**

**Secretory Pathway:** Each protein in the pathway has what? Two properties of each protein Describe the pathway

* **ER-targeting signal peptide** * **1 or 2 basic AA (Lys or Arg)** near N terminus & an extremely **hydrophobic sequence** (10-15) on C terminus of basic residues 1. **SRP** binds to ER-targeting signal and ribosome 2. **SRP wraps** around **ribosome-mRNA-peptide complex**, tethering to ER membrane and **halting translation** temporarily 3. Translation resumes when protein directed into ER lumen 4. **Enzymes** on luminal side **cleave the signal** to release protein 5. Protein undergoes PTMs in ER and/or Golgi 6. Additional signal sequences serves to guide each protein to final destination

**Lysosomal Proteins: I-Cell Disease** Tagging of lysosomal proteins with _ is \_, leading to \_

Tagging mechanism with Mannose 6 Phosphate is defective leading to high plasma levels of lysosomal enzymes

**Protein Folding** Which proteins can fold into native conformations spontaneously, and which are at risk for aggregation and proteolysis? What is the function of chaperones (HSP70)? What is the function of chaperonins (HSP60)?

Small proteins can fold spontaneously Large proteins are at risk Protect the protein and help fold into proper tertiary structure Barrel shaped compartments that catalyze folding of proteins in an ATP-dependent manner

Proteolytic Cleavage function:

Conversion from inactive to active form by unmasking active site

**Glycosylation** Differences between O-glycosidic and N-glycosidic

**O-Glycosidic: O-links** with **-OH groups** of **Ser/Thr** residues **N-Glycosidic: N-links** with **Asparagine**. Precursor sugar transferred from **phospho Dolichol**

Phosphorylation is the formation of an _ bond between _ and _ of an AA via the activity of this enzyme How are phosphates removed? Phosphorylation function?

**Ester bond** between **phosphate and OH** of an AA via the activity of **serine/threonine** and **tyrosine kinase** **Phosphatases** Regulates enzyme **activity** and protein **function** particularly in signaling

Disulfide Bond Formation function and locations Facilitated by which enzymes?

**Inter** and **intra-molecular** disulfide bonds **stabilize** proteins and form between **thiol (SH) group** of **2 cysteine** residues (**formation** occurs in **ER lumen)** ## Footnote **Protein Disulfide Isomerase**

**Acetylation** Proteins are typically acetylated on what AA? What is the acetyl group donor? Reactions are catalyzed by what enzymes?

**Lysine Residues** **Acetyl CoA** **Histone acetyltransferase (HAT)** or **Histone Deacetylase (HDAC)**

**Post Translational Modifications are important for assembly of Collagen** What is required for activity of lysyl and prolyl hydroxylases? What results in skin, bone, and joint disorders (Ehlers-Danlos Syndrome)?

**Ascorbic Acid** **Defects in lysyl hydroxylases**

**Alzheimer's Disease** Amyloid precursor protein (APP) breaks down to form \_ Misfolding/Aggregation of AB results in \_ Neurofibrillary tangles results from what? Mutations in APP and Tau cause _ of AD Brain aging is the common denominator for \_

**Amyloid Beta Peptide (AB)** **Plaques** in brain (extracellular) **Hyperphosphorylation of Tau** (intracellular) **Familial Forms** **Sporadic Form**

**Parkinson's Disease (PD)** Aggregation of _ protein forms insoluble fibrils which deposit as _ and results in selective death in neurons Symptoms are due to \_ Mutations in AS cause _ of PD Brain aging is the common denominator for the \_

**a-synuclein (AS)** **Lewy bodies in dopaminergic neurons in substantia nigra** **Reduced availability of dopamine** **Familial forms** **Sporadic form**

**Huntington's Disease (HD)** Cause of disease and consequences What causes the symptoms?

* Mutation in **Huntingtin gene** --\> expansion of **CAG triplet repeats** * Causes **polyglutamine repeats** in abnormal HTT protein * **Selective death of cells in BG cause the symptoms**

**Creutzfeldt-Jakob Disease** Caused by what? What does Transmissible mean?

* Caused by **misfolding of prion proteins** * Infection by misfolded proteins **convert normal proteins to misfolded form** * **Transmissible spongiform encephalopathies (TSEs)**