Lecture 12 - Translation

What occurs during translation?

mRNA is decoded by ribosomes to produce an amino acid chain that will fold into an active protein

What is an amino acid chain?

Polypeptide

What is Polypeptide?

Unfolded functional form of protein

What are the three components needed for translation?

1. Ribosome

2. tRNA

3. mRNA

What is the ribosome considered as?

Ribosome is the factory site for protein synthesis

What makes up the large subunit?

5S, 5.8S, and 28S rRNA with 47 proteins (60S total)

What is the function of the large subunit?

- Contains the enzymatic activity (FUNCTIONS AS A RIBOZYME)

- Orients mRNA and helps tRNA interact (anticodon-codon interaction)

What makes up the small subunit?

18S rRNA and 32 proteins (40S total)

What is the function of small subunit?

Responsible for reading the mRNA and monitoring complementarity between the codon and the anticodon

What are the sites of the ribosome?

1. Aminoacyl-site

2. Peptidyl site

3. Exit site

What resides in the amino-acyl site?

Charged tRNA (amino aid associated with tRNA)

What resides in the peptidyl-site?

Polypeptide chain

Forms peptide bonds between amino acids one in A site & other in P site.

What leaves the exit-site?

Uncharged tRNA (no amino acid associated with tRNA)

What is tRNA?

-Carriers of amino acids

-Small RNAs (73-93 bp) that are folded into 3d structure

What is the function of tRNAs?

Adapters in translating nucleic acids (mRNA) into proteins

What are the structural features of a tRNA?

1. Amino acid arm at the 3' end --attaches to its specific Amino Acid; CCA residue at the 3' end

2. Each tRNA carries a specific AA

3. 5' end is rich in poly guanylate (pG) residues

4. Anticodon arm: helps tRNA bind to a specific mRNA codon

5. D arm: has dihydrouridine residues

6. TΨC arm: has unusual bases ribothymidine (T) and pseudouridine (Ψ)

What does mRNA contain?

Genetic information

What are the structural features of mRNA?

1. Composed of numerous codons arranged in a linear, non-overlapping manner (codons don't share nucleotides)

2. Each codon codes for a specific AA

3. 64 (4^3) codons are present

4. 5' methylated cap and 3' poly a tail

What is the initiation codon?

AUG - methionine

What are the stop codons?

UAA, UAG, UGA

What is the genetic code considered?

-Degenerate - more than one codon codes for one specific amino acid

-universal

What are coding sequences considered?

linear and non-overlapping

What is the Wobble hypothesis?

-One tRNA (with the same anticodon) can bind to mRNA with ONE incorrect bp

- Non-Watson-Crick base pairing

In the wobble hypothesis, what base pairs can potentially act as non-Watson-Crick base-pairing?

1. I-U

2. I-A

3. I-C

4. U-G (and G-U)

5. A - does not base-pair with anything

Where is the wobble position in tNRA?

3rd base in an anti-codon --> closest to the 5' end

What is not possible for tRNA anticodon at 5' end?

No A's present

What are the 5 stages of translation?

Stage I: Activation of amino acids

Stage II: Initiation

Stage III: Elongation

Stage IV: Termination and ribosome recycling

Stage V: Folding and post-translational modifications

What direction does translation always occurs?

Reads from 5' to 3' direction

--OPPOSITE of polymerases (reads 3’ to 5’)

What are the steps of stage I: Activation of amino acids?

CHARGING tRNAs:

Step 1: Amino acid activation — use ATP to energize the amino acid.

Step 2: tRNA conjugation — use the energy associated with the ATP-AA to add to the tRNA.

Where does stage I occur?

At the aminoacyl-tRNA synthetase

What are the steps of stage II: Initiation?

1. Large and small subunits of the ribosome dissociate

2. Ternary complex is formed

3. 43S pre-initiation complex is formed

4. activation of mRNA

5. Hydrolysis of GTP on eIF2

6. Both subunits bind

7. A site opens for the next codon, coding the next AA

How is the ribosome dissociated in step 1 of stage II?

By binding of eukaryotic initiation factor 3 (eIF3) with the 40S subunit.

--once bound to the small subunit, eIF3 will prevent the large subunit from coming back together after being dissociated because only the small subunit is used for initiation.

What is the ternary complex made in step 2 of stage II?

eIF2 and GTP binds to methionyl-tRNA^Met

What is methionyl-tRNA^Met?

tRNA for methione that's charged

What is the 43 pre-initiation complex that is formed in step 3 stage II?

Combination of ternary complex +40S +eIF3 + eIF5 + eIF1 +eIF1a

What do eIF5 + eIF1 +eIF1a do?

Stabilize the 43 pre-initiation complex so it doesnt fall apart

How is mRNA activated in step 4 stage II?

Done by the binding of cap binding complex, eIF4F

What binds to the eIF4F?

5’ end of mRNA and then the 43S-pre-initiation complex

What does the cap binding complex do?

-Scans mRNA for the initiation codon

-Allows 5' cap to fall off the mRNA

-Acts as a sign for the 43S complex to know which frame to bind to

What is the cap binding complex, eIF4F, made up of?

-AGE backwards

eIF4E, eIF4G, eIF4A

How are all eIFs on the activated mRNA released from the 43S preinitiation complex?

By the hydrolysis of GTP on eIF2 with the help of eIF5

Once the eIFs are released, what does this allow for?

Binding of the small and large subunits and placing AUG bound to methionyl-tRNA^Met at the P site

What regulates the initiation step?

1. Insulin: stimulates protein synthesis by activating eIF4E of the cap-binding complex

2. Viral infections, starvation, heat shock, and phosphorylation of eIF2 inactivate it and inhibit protein synthesis.

What happens in the decoding stage of elongation?

1. Aminoacyl-tRNA (charged with the next AA) binds to elongation factor eEF1A and GTP

2. Complex above binds to the A site, and GTP is hydrolyzed

3. eEF1A-GDP (after hydrolysis) dissociates from the ribosome [occurs when the correct tRNA is in the A site)

4. eEF1A-GDP complex gets recharged with GTP in the cytosol

What makes up the elongation ternary complex?

eEF1A and GTP

Where is the peptide bond formed in the elongation stage?

covalent bond between carboxylic acid group of the AA in the P site and the amide group of the AA in the A site

What catalyzes peptide bond formation in the elongation stage?

Peptidyl transferase (rRNA w/ enzymatic activity) present in the 60S subunit

What occurs during the translocation step in elongation?

1. eEF2 that's bound to GTP binds to the mRNA-ribosome complex --> induces a conformational change

2. mRNA and attached tRNAs move with respect to the ribosome

3. Uncharged (empty) tRNA moves to E-site from P site because of the release of energy

4. tRNA with growing peptide chain moves to P site

5. Next aminoacyl-tRNA occupies the A site

What happens during termination stage IV?

1. Elongation continues until a stop codon enters the A site

2. Eukaryotic release factors eRF1 and eRF3 complex bind to the A site

3. Peptidyltransferase enzyme hydrolyzes the bond between the peptide chain and the tRNA at the P site

4. Ribosome dissociates into its subunits

What kind of activity does eRF3 have?

GTPase activity, so can release energy

What does peptidyltransferase do?

Hydrolyzes bond between peptide chain and tRNA at the P site

What does peptidyltransferase require?

1 GTP

How does translation proceed?

5' to 3'

What is the 5' end referred to as?

Amino terminus (N)

What is the 3' end referred to as?

Carboxyl terminus (C)

What are polyribosomes/polysomes?

-Multiple ribosomes reading a particular mRNA simultaneously

-As one ribosome moves along a mRNA translating it, a 2nd ribosome can bind to the 5' end of mRNA

What do polyribosomes/polysomes synthesize?

Multiple copies of the same protein

What is involved in the folding & post transcriptional modifs stage of translation?

--It is the folding & production of 3D structure of a protein

--Chaperons/heat shock proteins, disulfide isomerases, post-translational modifications

What do chaperons/heat shock proteins do?

Fold and refold the nascent (immature) polypeptide into its correct 3D conformation

What do disulfide isomerases introduce

disulfide bonds that stabilize the folded structure

what do post translational modifications add to the protein to make it functional

functional groups through methylation, glycosylation, hydroxylation, iodination, phosphorylation

what is phosphorylation (post transcriptional modifs)

**most common modif

-adds phosphate to serine, threonine, or tyrosine to control protein structure

what is lipidation (post transcriptional modifs)

attaches a lipid (fatty acid) to a protein chain and sticks protein into the membrane

what is glycosylation (post transcriptional modifs)

attaches a sugar usually to an "N" (asp) or "O" atom in an amino acid side chain

what is ubiquination (post transcriptional modifs)

adds ubiquitin to lysine residue of a target protein marking it for destruction

**don't want to make a protein anymore

what is acetylation (post transcriptional modifs)

adds an acetyl group to the N-terminus of a protein to increase stability

what is disulfide bond (post transcriptional modifs)

covalently links the "S" atoms of 2 diff cysteine residues

what is ribosomopathies

congenital human disorders resulting from defects in ribosomal proteins/rRNA/other genes involved in ribosome biogenesis

what are the most prominent targets of antibacterial drugs

ribosomes

if misfolded proteins aren't degraded, what do they form

amyloids - insoluble aggregates

where do amyloids accumulate and what are they associated with

-in cells of different organs or extracellular space

-pathology of amyloidosis

what is an example of localized amyloidosis

alzheimer's disease

what is alzheimer's caused by

deposits of beta amyloid protein deposits [amyloid plaques] in the brain

what is creutzfeldt-jakob disease (CJD)

-rare, degenerative, invariably fatal brain disorder

-form of spongiform encephalopathy

-300 cases/yr in US

-appears in 60+ years

-aggressive, death within a year

-can be sporadic, hereditary, or acquired

what are the characteristics of prion proteins, the causative agent of CJD

-glycoproteins

-harmless when structurally normal (PrPc)

-infectious when misfolded (PrPsc)

-cause of misfolding is sporadic, genetic, or transmitted

what are the characteristics of PrPsc

-insoluble, resistant to protease digestion

-amplification: one PrPsc can misfold several PrPc in chain rxn