Functions/Dysfunctions Of Protein Processing Flashcards by Sarah Gillen

A protein is synthesized from the __________ to _______________

N-terminus

C-terminus

[sequentially adding aa’s to the C-terminus of the growing peptide chain

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True or false: the mRNA sequence will be complementary to the coding strand

False, the mRNA will be identical, but replace the Ts with Us

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How many codons code for amino acids?

61, 3 are stop codons that terminate translation

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What is the start codon?

AUG - codes for methionine

[fMet in prokaryotes]

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Why is the genetic code considered unambiguous and degenerate?

Unambiguous - each codon codes for only 1 aa

Degenerate - each amino acid can be coded for by more than one codon (exceptions = Met, Trp)

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What type of mutation is this and what is the effect on the resulting protein?

New codon –> same amino acid

Silent; no effect on protein

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What type of mutation is this and what is the effect on the resulting protein?

New codon –> new aa

Missense; variable effect on protein

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What type of mutation is this and what is the effect on the resulting protein?

New codon –> stop codon

Nonsense; nonfunctional protein

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What type of mutation is this and what is the effect on the resulting protein?

1+ nucleotides deleted or inserted

Frameshift; nonfunctional protein

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Sickle cell anemia results from what type of mutation?

Missense mutation of 6th codon in allele of gene for human beta-globin, from GAG to GTG (substitutes Val for Glu)

The mutation alters the conformation of HbA, causing it to aggregate and form rigid rod-like structures which deform RBCs into sickle like shape

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Duchenne Muscular Dystrophy results from what type of mutation?

Large in-frame and out of frame deletions to the dystrophin gene, leading to partially or non-functioning dystrophin protein

Leads to muscle wasting and wheelchair confinement by age 12, death by respiratory failure within 10 years

Muscle is replaced with fat and fibroid, elevated CK

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What post translational modifications occur to pre-mRNA and what is the purpose of these?

5’ cap and polyA tail

Makes mRNA more soluble for transport to cytoplasm for translation

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Each type of tRNA carries only one __________ amino acid, which is determined by its ___________ sequence

Activated; anticodon

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Amino acid activation is catalyzed by _____________ ___________, which requires ______ high energy bonds from ATP

Aminoacyl-tRNA synthetase; 2

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tRNA has an anticodon sequence that allows pairing with the mRNA codon, and transfers the activated amino acid on the ______ end of the correct tRNA

3’

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How many aminoacyl tRNA synthetases are there?

20, one for each amino acid

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What are the 2 uses of aminoacyl-tRNA synthetase?

Transfers AMP to the amino acid to form enzyme-bound aminoacyl-AMP

Transfers the aminoacyl group to the tRNA to activate the aa

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There are 2 subunits to a ribosome that assemble around the mRNA: the _______ subunit positions the mRNA and the _______ removes each amino aicd and adds it to the growing peptide chain

Small; large

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Compare/contrast the different ribosome structures in prokaryotes vs. eukaryotes

Prokaryotes = 70S (50S and 30S)

Eukaryotes = 80S (60S and 40S)

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The ribosomal complex consists of 3 sites. At which site is the aminoacyl-tRNA attached to the growing peptide chain?

Peptidyl (P) site

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The ribosomal complex consists of 3 sites. At which site does the mRNA codon accept the aminoacyl-tRNA?

Acceptor (A) site

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The ribosomal complex consists of 3 sites. At which site does the tRNA sit prior to exiting the ribosome?

Empty (E) site

[aka Exit site]

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Translation occurs in the 5’ to 3’ direction and consists of what 3 general steps?

Initiation
Elongation
Termination

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What are the initiation sequences in prokaryotes vs. eukaryotes?

P = Shine-Dalgarno sequence (AGGAGG)

E = 5’cap, 3’polyA tail, Kozak sequence, and ATP dependent mRNA scan

[the initiation site determines the reading frame for the whole length of the mRNA]

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The pre-initiator complex consists of: an initiator tRNA to which _____ is bound, which attaches to the P site of a small subunit (_______________ tRNA in prokaryotes and ____________ tRNA in eukaryotes) mRNA strand with ______ start codon Other initiation factors (____ in prokaryotes, _____ in eukaryotes) The large subunit is then added to complete the complex, which is driven by hydrolysis of ______.

GTP; N-formyl-methionyl tRNA; methionyl AUG IF; eIF GTP

How can prokaryotic vs. eukaryotic translation be determined in a figure?

If the mRNA does not have a 5' cap or polyA tail, it is prokaryotic

During elongation, the polypeptide chain is extended by the catalytic action of the ribosome: Loading of an aminoacyl tRNA onto the ribosome, its anticodon base pairs with codon positioned on A site Prior to loading, the aminoacyl tRNA is attached to GTP-bound ___________ _____________ Loading of tRNA is accompanied by _______ ___________ and release of GDP-bound EF from aminoacyl tRNA Peptide bond formation is catalyzed by __________ __________

Elongation factor (EF) GTP hydrolysis Peptidyl transferase

What are 4 the prokaryotic elongation inhibitors?

Tetracycline Chloramphenicol Clindamycin Erythromycin

______________ is a prokaryotic elongation inhibitor the inhibits peptidyl transferase in prokaryotes and mitochondria

Chloramphenicol

_____________ is a prokaryotic elongation inhibitor that binds to the small 30S subunit, blocking entry of aminoacyl tRNA to the ribosomal complex

Tetracycline

______________ and ___________ are prokaryotic elongation inhibitors that bind to large 50S subunit and block translocation of the ribosome

Clindamycin; erythromycin [note that erythromycin is commonly used to treat pertussis]

What are the 4 eukaryotic elongation inhibitors?

Cyclohexamide Diphtheria toxin Shiga toxin Ricin

Which eukaryotic elongation inhibitor inactivates GTP-bound eEF-2, interfering with ribosomal translocation?

Diphtheria toxin

Which eukaryotic elongation inhibitor inhibits peptidyl transferase?

Cyclohexamide

Which eukaryotic elongation inhibitors bind to large 60S subunit and block entry of aminoacyl tRNA to ribosomal complex?

Shiga toxin and Ricin

Which elongation inhibitor causes premature chain termination in both prokaryotes and eukaryotes?

Puromycin [resembles the 3' end of the aminoacyl tRNA, enters A site and adds to growing chain, leads to premature release, mroe resistant to hydrolysis, stops the ribosome]

What are the 3 stop codons?

UAA UAG UGA

Termination is triggered when a stop codon resides in the ____ site, and ______ ________ are loaded

``` A Release factors (RFs) ``` This triggers peptidyl transferase to cleave the ester bond between the C terminus of the chain and the tRNA

What is the last event prior to dissociation of the ribosomal complex?

GTP hydrolysis

The __________ pathway of protein sorting typically consists of proteins destined for the cytoplasm, mitochondria, nucleus, and peroxisomes. Synthesis usually begins and ends on ribosomes in the cytoplasm.

Cytoplasmic

The __________ pathway consists of proteins destined for the ER, lysosomes, secretion, or membranes. The first 20 AA of the polypeptide contains an ER-targeting signal

Secretory

If any protein lacks a translocation signal, it will remain in the _____________

Cytoplasm

If there is an N-terminal hydrophobic alpha helix signal peptide, where is that protein destined to go?

Cytoplasmic pathway - mitochondria

If there is a KKKRK translocation signal sequence, where is the protein destined to go?

Cytoplasmic pathway - nucleus

If there is a C-terminal SKL translocation signal sequence, where is the protein destined to go?

Cytoplasmic pathway - Peroxisomes

If there is a C-terminal KDEL retention signal, where is the protein destined to go?

Secretory pathway - ER lumen [each protein in secretory pathway contains the N-terminal hydrophobic alpha-helix ER signal peptide]

If there is a mannose 6-phosphate translocation signal, where is this protein destined to go? What pathological condition is associated with this?

Secretory pathway - Lysosomes I-cell disease [each protein in secretory pathway contains the N-terminal hydrophobic alpha-helix ER signal peptide]

If there is a tryptophan-rich domain signal region and absence of retention motifs, where is the protein destined to go?

Secretory pathway - secretion [each protein in secretory pathway contains the N-terminal hydrophobic alpha-helix ER signal peptide]

If there is an N-terminal apolar region (stop-transfer sequence), where is the protein destined to go?

Secretory pathway - membranes [each protein in secretory pathway contains the N-terminal hydrophobic alpha-helix ER signal peptide]

What is the purpose of the N terminal hydrophobic alpha-helix on mitochondrial proteins?

Allows interaction with chaperone proteins (HSP70 family) Translocation sequences for mitochondria are recognized by TIM and TOM

Proteins in the secretory pathway contain an ER-targeting signal peptide with 1 or 2 basic amino acids like _______ or _______, and a 10-15 residue ____________ sequence

Lys; Arg | Hydrophobic

With the secretory pathway, post-translational modifications occur in the ER and/or golgi. This occurs when the _________ wraps itself around the ribosome-mRNA-peptide complex, tethering to the membrane and temporarily halting translation. Once the protein is directed into the ER lumen, translation resumes and is directed to the ____________ space. Enzymes then cleave the _________ to release the protein

SRP (signal recognition particle) Luminal Signal

I-cell disease, or Mucolipidosis II, is a severe form of _____________ storage disease where the tagging of lysosomal proteins with _________ is defective, resulting in high plasma levels of those enzymes.

Lysosomal | Mannose 6P

What are the clinical manifestations of I cell disease?

By 6 months: failure to thrive, developmental delays, physical manifestations Abnormal skeletal development, coarse facial features, restricted joint movement, stiff claw-shaped hands, dwarfism, clouding on corneas, hepatomegaly, splenomegaly, recurrent respiratory tract infections Death usually by age 7 due to CHF or respiratory tract infection.

________ proteins can spontaneously fold into native conformations, ________ proteins cannot due to risk of aggregation and proteolysis.

Small; large

______________ are auxiliary proteins that protect the protein, and help it to fold into its proper tertiary structure A subset of these proteins are called ____________, which are barrel-shaped compartments that admit unfolded proteins, and catalyze their folding in an ATP-dependent manner

Chaperones Chaperonins

What disease results from a mutation in the gene for the human B-globin, which alters the conformation of HbA, causing it to aggregate and form rigid, rod-like structures?

Sickle cell anemia

Alzheimer's disease results from a mutation in the gene for __________ __________ __________, which predisposes it to misfold and aggregate to form neuritic plaques in the brain. Supplementation with ____________ has recent clinical significance.

Amyloid precursor protein (APP) Lysine

What are prions?

Transmissible misfolded proteins that convert their properly folded host counterparts to the same misfolded structure that they posses Cause diseases like CJD, Kuru, and Mad Cow

What causes Huntington disease?

Mutation in the Huntington gene results in the expansion of CAG triplet repeats which results in a polyglutamine tract, resulting in an abnormal HTT protein. mHTT forms intramolecular H-bonds, which eventually misfold and aggregate

_______________ disease results from mutated a-synuclein that aggregates to form insoluble fibrils called Lewy bodies This is caused by an N-terminal apolar sequence (the functional form has ___________, and the __________ form has insoluble beta sheets. Results in neuronal death in the midbrain, reducing the availability of _____________

Parkinson Alpha-helix; fibril Dopamine

Proteolytic cleavage converts inactive forms (zymogens) to active enzymes by "unmasking" the active site. How does this apply to digestive enzymes?

Trypsinogen --> trypsin Chymotrypsinogen --> chymotrypsin

______________ is a post-translational modification where the N-terminus is bonded to an acetyl or long-chain acyl residue, or thioesterification with a long-chain acyl group Affects N-terminus and Cys residues

Acylation

______________ is a post-translational modification involving covalent linkage to an amine. What residue does this affect?

Acetylation Lysine

______________ is a post-translational modification with a covalent bond to sugar residues and occurs in the ER as they are translated What residues does this effect?

Glycosylation O-glycosylation affects Ser, Thr N-glycosylation affects Asn, Gln

______________ is a post-translational modification where a phosphate is linked via formation of an ester bond between phosphate and an -OH of an amino acid What residues are affected?

Phosphorylation Ser, Tyr, Thr, Asp, His

______________ is a post-translational modification with the addition of a -COOH group? What residue is affected?

g-carboxylation Glu

______________ is a post-translational modification with the covalent modification with ubiquitin? What residue is affected?

Ubiquitination Lysine

______________ is a post-translational modification with the addition of an -OH group? What residues are affected?

Hydroxylation Pro and Lys

______________ is a post-translational modification where oxidation achieves covalent linkages of Cys residues?

Disulfide bonds

______________ is a post-translational modification where an isoprenoid group such as farnesyl or geranyl-geranyl groups are added to cysteins in C-terminal CAAX or CC motifs?

Prenylation

What post-translational modification is thought to play a role in diabetic cataract formation?

Glycosylation of lens crystallins

What is the most common glycosylation?

N-linked: -NH of Asn

Glycosylation occurs when a precursor oligosaccharide (14-residue) is synthesized on membrane-embedded dolichol, catalyzed by glycotransferases. These glycotransferases can be inhibited by antibiotic _____________

Tunicamycin

What is the general result of disulfide bond formation on protein stability?

Makes them more stable, but can be reduced in certain conditions

Where does the formation and reorganization of disulfide bonds occur?

In the ER lumen