L41-42: Translation and Protein Processing I-II Flashcards Preview

Biochemistry & Molecular Biology > L41-42: Translation and Protein Processing I-II > Flashcards

Flashcards in L41-42: Translation and Protein Processing I-II Deck (38):

Describe and contrast the composition of eukaryotic and prokaryotic ribosomes

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


Name ribosome sites

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


Describe the progression of ribosome assembly

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


Describe elongation and termination of translation

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


Names of 70S ribosome inhibitors

- Streptomycin - Neomycin - Gentamicin - Tetracycline - Chloramphenicol


Action of streptomycin

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


Action of neomycin

- 70 S ribosome inhibitor - Specifically causes mistranslation of codons


Action of gentamicin

- 70 S ribosome inhibitor - Specifically causes mistranslation of codons


Action of tetracycline

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


Action of chloramphenicol

- 70 S ribosome inhibitor - Specifically prevents peptidyl bond formation


Action of ricin

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


Action of diphtheria toxin

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


Explain the regulation of translation

- 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


Role of chaperones. Example

- 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


Charcot Marie Tooth Disease

- Congenital chaperone defects cause protein folding disorder


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

- 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


Describe the unfolded protein response

- 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


Function of protein glycosylation

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


How are glycosyltransferases specific?

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


Types of glycosylation. Describe

- 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


Describe N-linked glycosylation mechanism

- 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)


Disorders of glycosylation

- 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)


What is CDG-I?

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


What is CDG-II?

- Defective trimming of oligosaccharide chains – 6 variants


Where are O-glycosylated proteins seen?

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


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

- 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


Examples of post-translational modifications

- 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


Give examples for the post-translational modification of amino acids

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


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

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


How are proteins directed to lysosomes

- Proteins directed to lysosome require phosphomannose


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

- 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


Why are Hsp70 proteins necessary for mitochondrial proteins?

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


What is cystic fibrosis? What is the defect?

- 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


Name two protein sorting disorders

- Cystic fibrosis - I-cell disease


What is I-cell disease? Describe the defect

- 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


Compare and contrast protein degradation in lysosomes and proteasomes

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)


Explain the role of ubiquitin in protein degradation

- 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


Describe the factors that determine protein half-life

- 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