L41-42: Translation and Protein Processing I-II

Question 1

Describe and contrast the composition of eukaryotic and prokaryotic ribosomes

Answer 1

1.) Eukaryotic - small subunit = 40 S - large subunit = 60 S - assembled size = 80 S * Mitochondrial: small = 30-35S, large = 40-45S, total = 55S 2.) Prokaryotic - small subunit = 30 S - large subunit = 50 S - assembled size = 70 S

Question 2

Name ribosome sites

Answer 2

• E: exit site - P: peptidyl site - A: acceptor site

Question 3

Describe the progression of ribosome assembly

Answer 3

1.) GTP binds eIF2a 2.) GTP:eIF2a becomes bound to Met-tRNA to form ternary complex 3.) 40S:eIF3 binds ternary complex (with eIF1 and eIF1alpha) 4.) mRNA now binds small subunit and pre-initiation complex is formed (with aid of eIF-4a, eIF-4b, eIF-4f, eIF-5 and PAB) 5.) eIF-5b:GTP are added to this complex displacing hydrolyzed GDP:eIF2a and 60 S subunit is recruited and positioned with met-tRNA in P site. Elongation can now ensue

Question 4

Describe elongation and termination of translation

Answer 4

1.) EF-1-GTP charges tRNA molecule (EF1-GDP = product). AA-tRNA moves into A site 2.) Peptide bond formation occurs 3.) EF-2-GTP hydrolysis allows ribosomal complex to move one codon down with mRNA-peptidyl-complex now occupying the P site and the A site is empty. Uncharged tRNA leaves through E site 4.) Ribosome is now ready to repeat the cycle 5.) Once the stop codon is moved into the A site, eRF bound to GTP is hydrolyzed and the ribosomal complex dissociates

Question 5

Names of 70S ribosome inhibitors

Answer 5

• Streptomycin - Neomycin - Gentamicin - Tetracycline - Chloramphenicol

Question 6

Action of streptomycin

Answer 6

• 70 S ribosome inhibitor - Specifically binds to small subunit (30 S) and inhibits initiation and causes mistranslation of codons

Question 7

Action of neomycin

Answer 7

• 70 S ribosome inhibitor - Specifically causes mistranslation of codons

Question 8

Action of gentamicin

Answer 8

• 70 S ribosome inhibitor - Specifically causes mistranslation of codons

Question 9

Action of tetracycline

Answer 9

• 70 S ribosome inhibitor - Specifically blocks A site and prevents tRNA binding

Question 10

Action of chloramphenicol

Answer 10

• 70 S ribosome inhibitor - Specifically prevents peptidyl bond formation

Question 11

Action of ricin

Answer 11

• potent ribosome inactivating protein (RIP) found in castor beans - it removes adenine bases from rRNA

Question 12

Action of diphtheria toxin

Answer 12

• protein produced by C. diphtheriae that inactivates EF-2 by ADP ribosylation, preventing elongation

Question 13

Explain the regulation of translation

Answer 13

• Points of regulation are at 1.) recognition of start codon and 2.) activity of initiation factors 1.) Recognition of start codon: bind of regulatory protein in 5’ UTR can mask start codon 2.) eIF-2a can be inactivated by phosphorylation

Question 14

Role of chaperones. Example

Answer 14

• Proteins emerging from ribosome need to fold correctly - Folding is aided by chaperones – example = Hsp 90. Hsp90 binds ATP and misfolded proteins, loosens up protein and gives it another chance to fold correctly - These are important for survival of stress – heat shock

Question 15

Charcot Marie Tooth Disease

Answer 15

• Congenital chaperone defects cause protein folding disorder

Question 16

Explain the synthesis of exported proteins. Where does this occur?

Answer 16

• Synthesis of exported proteins begins at ER - Protein emerging from ribosome has signal peptide sequence - Signal recognition particle binds to signal peptide (stalls translation), binding ribosome to docking protein and positioning exiting protein sequence within translocon to ER lumen - Translation resumes, protein sequence is fed into ER lumen and signal peptidase inside lumen cleaves signal peptide - Protein is modified in ER and Golgi apparatus

Question 17

Describe the unfolded protein response

Answer 17

• Accumulation of unfolded proteins in ER due to physiological stressors triggers unfolded protein response, which is general inhibition of translation, specific induction of HSP and / or apoptosis

Question 18

Function of protein glycosylation

Answer 18

• Protein glycosylation confers: - 1.) physical changes: solubility, structure and bulk - 2.) generation of individual surface signatures

Question 19

How are glycosyltransferases specific?

Answer 19

1.) specific for activated sugar 2.) specific for acceptor 3.) specific for linkage formed

Question 20

Types of glycosylation. Describe

Answer 20

• N-linked: starts in ER before protein folding is complete, adds sugars to Asn residues in protein in predictable fashion, modification of this can occur in Golgi - O-linked: starts in Golgi after protein folding is complete, adds sugars to serine or threonine residues, but not in predictable fashion

Question 21

Describe N-linked glycosylation mechanism

Answer 21

• Dolichol phosphate in ER membrane acts as site for oligosaccharide in ER - Glycosyltransferase adds two GlcNAcs to dolichol - Glycosyltransferase adds five mannose - Dolichol phosphate linked to above CHOs reorientates from cytoplasm into ER lumen - 4 more mannoses are added onto oligosaccharide using dolicholphosphomannose - 3 glucoses are added to mannose forming universal oligosaccharide containing 14 sugars - Highly specific modification of universal oligosaccharide occurs in Golgi apparatus by addition or removal of CHOs, yielding high mannose type or complex type (sialic acid, fucose, N-acetyl glucosamine, N-acetyl-galactosamine, galactose)

Question 22

Disorders of glycosylation

Answer 22

• CDG = congenital disorders of glycosylation - CDG-I: defective synthesis of lipid-linked oligosaccharide precursor (12 variants) - CDG-II: defective trimming of oligosaccharide chain (6 variants)

Question 23

What is CDG-I?

Answer 23

• Defective synthesis of lipid-linked oligosaccharide precursors – 12 variants

Question 24

What is CDG-II?

Answer 24

• Defective trimming of oligosaccharide chains – 6 variants

Question 25

Where are O-glycosylated proteins seen?

Answer 25

• Proteoglycans of the ECM - H-antigen on surface of RBCs (predictable)

Question 26

RBC surface antigens. Core? Sugar residues for O, A and B antigen?

Answer 26

• Core: Serine – Gal – GlcNAc – Gal X – Fucose - O: Serine – Gal – GlcNAc – Gal X – Fucose - A: Core – Gal X attached to GalNAc as well as Fucose - B: Core – Gal X attached to Gal as well as Fucose

Question 27

Examples of post-translational modifications

Answer 27

• Glycosylations - Hydroxylation of proline - Acetylation of lysine - Cysteine to formylglycine - N-terminal trimming (removal of methionine) - Addition of hydrophobic moieties - Addition of GPI anchor to C-terminus

Question 28

Give examples for the post-translational modification of amino acids

Answer 28

• Hydroxylation of proline - Acetylation of lysine - Cysteine to formylglycine

Question 29

Explain the four ways by which hydrophobic molecules are added to proteins. What is the function of this?

Answer 29

1.) N-terminal myristoylation 2.) Palmitoylation at cysteine 3.) Prenylation at cysteine close to C-terminus 4.) Addition of GPI anchor to c-terminus - Function: anchor proteins to PM for hanging structure into cytoplasm or into ECF

Question 30

How are proteins directed to lysosomes

Answer 30

• Proteins directed to lysosome require phosphomannose

Question 31

Describe protein import into mitochondria and insertion of proteins into mitochondrial membranes

Answer 31

• Mitochondrial pre-sequence is present on polypeptide sequence - Hsp 70 chaperones prevent premature folding in cytoplasm - TOM/TIM complex spanning outer to inner MM are present and feed polypeptide into mitochondrial matrix - Presequence is cleaved by matrix proteases. Hsp 60 proteins associate with sequence inside the mitochondrial matrix - Other signal sequences mediate their insertion into the mitochondrial membranes and what direction they hang

Question 32

Why are Hsp70 proteins necessary for mitochondrial proteins?

Answer 32

• Hsp70 proteins associate with mitochondrial polypeptide sequences and prevent folding within the cytoplasm. - Folding will prevent movement of these proteins into the mitochondrial matrix

Question 33

What is cystic fibrosis? What is the defect?

Answer 33

• CF is most often caused by deletion prevents correct glycosylation of CFTR1 protein. As a result, it isn’t moved to the cell surface and is degraded in the cytosol

Question 34

Name two protein sorting disorders

Answer 34

• Cystic fibrosis - I-cell disease

Question 35

What is I-cell disease? Describe the defect

Answer 35

• Surface mannose residues are not phosphorylated and therefore lysosomal proteins don’t reach the correct compartment and appear in serum - Result: lysosomal degradation of proteins and CHOs is impaired, inclusion bodies are seen in lysosomes making them appear dense

Question 36

Compare and contrast protein degradation in lysosomes and proteasomes

Answer 36

1.) Lysosomes - Lysosomes contain acid hydrolases for all types of molecules - Functions in autophagy and in the endocytic pathways where clathrin associates with endocytosed vesicles directing them to lysosome 2.) Proteasomes - Proteasome is multiprotein complex in cytoplasm and nucleus - It selectively degrades poly-ubiquinated proteins (ubiquitination regulates protein activity ie. cyclins and misfolded proteins)

Question 37

Explain the role of ubiquitin in protein degradation

Answer 37

• Ubiquitin is a small protein that is transferred to target proteins, specifically an amide bond is formed bw c-terminal glycine and a lysine in target molecule - Ubiquitination is regulatory (mono-ubiquitin protein are not necessary degraded) - Poly-ubiquitination leads to proteasomal destruction of proteins, using activation and transfer of ubiquitin by E1, E2 and E3 enzymes

Question 38

Describe the factors that determine protein half-life

Answer 38

• Conformation: eg. Misfolding results in hydrophobic domains being placed on surface and leads to degradation - N-terminus: Ser/Met proteins are more stable than Arg/Lys proteins - Other sequences: eg. PEST sequences shorten lifespan