L16 + 17: Translation

Question 1

Formation of a peptide bond

Answer 1

• AAs joined by peptide bonds; covalent bond formed between carboxyl group of one AA and amino grp of an adjacent aa

Question 2

Universal genetic code

Answer 2

• 20 aas but 61 codons; 3 stop codons
  -> some degeneracy (all except Met and Trp are encoded by multiple codons)
• AUG used in the initiation of protein synthesis

Question 3

tRNA

Answer 3

• Couples the region of ribosomes which binds mRNA codon and aa
• Regions of self-complentarity form hairpin loops (D-loop, T, loop, anticodon loop) -> clover structure
• Also has 3’ CCA tail, added post-transcriptionally
• Unusual modified bases:
  e.g. Dihydrouridine (D-loop)
  e.g. Pseudouridine (T-loop)
  -> added post-transcriptionally by enzymes

Question 4

Charging

Answer 4

• Adding tRNA to its cognate amino acid
• Carried out by aminoacyl-tRNA synthetase (using ATP as a cofactor)
  -> produces aminoacyl-tRNA (very specific)
• Reaction proceeds by IM using ATP as a cofactor…
  a. Activation of amino acid
  b. Transfer of amino acid to tRNA
  -> AMP
• Amino acid added to 3’ or 2’ OH group of the 3’ terminal adenine nt of the tRNA

Question 5

Ways of achieving site specificity in tRNA synthetase enzymes

Answer 5

• size exclusion
• editing pre-transfer
• editing post-transfer

Question 6

Wobble pairing

Answer 6

• For the binding between 1st base of anticodon and 3rd base of codon, non-watson crick base pairing allowed
  -> wobbles
• Provides part of the basis for degeneracy of the genetic code

Question 7

Which amino acid is incorporated into the protein first?

Answer 7

• W/ rare exception, methionine is the first; may be removed later
• tRNA_f^met(prok) or tRNA_i^met is always used at the start and another for elongation involving methionine (tRNA_m^met)

Question 8

Bacterial vs Eukaryotic ribosome

Answer 8

• Bacterial: 70S (50S large subunit w/ 2 rRNA molecules, 30S small subunit w/ 1 rRNA)
  *S is related to volume:SA so doesn’t directly add up
• Eukaroytic: 80S (60S large subunit w/ 3 rRNA molecules, 40S small subunit w/ 1 rRNA molecule)

Question 9

Shine-Dalgarno (bacteria)

Answer 9

• Critical to identification of polycistronic mRNA
• Polypurine sequence, AGGAGGU (E.coli)
• Most efficiently allows binding of the ribosome
• Binds to anti-shine-dalgrano sequence found on 16S rRNA of small subunit

Question 10

Attachment of ribosome to mRNA (Initiation) (bacteria)

Answer 10

• Shine-Dalgarno binds to anti-Shine-Dalgarno by base-pairing (at small subunit), allowing small subunit to bind and f-met to recognise translation start site (AUG)
• Small subunit will already have IF3 and IF1 bound (initiation factors)
• IF2 (GTPase accessory factor) which binds and provides energy for substrate to bind
• Large subunit binds, GTP hydrolysis, dissociation of IFs

Question 11

Role of IF1 an IF3 (bacteria)

Answer 11

• Help guide the initiator tRNA into the right place (peptidyl site)
• Protects the site either side of the initiator binding site
• Also prevent large subunit from binding

Question 12

Sites in the ribosome

Answer 12

• Aminoacyl site
• Peptidyl site
• Exit site

Question 12

Elongation (bacteria)

Answer 12

• The next aminoacyl-tRNA molecule (in complex w/ EFTu and GTP) binds to the exposed codon in the A site
  -> initial selection (ensures fidelity)
• The EFTu undergoes GTP hydrolysis and leave the site
• Additional proofreading step (further fidelity)

Question 13

Recycling of EFTu (bacteria)

Answer 13

• ‘EFTs’ exchanges GDP for GTP on EFTu
• Can be reused

Question 14

Peptide bond formation in ribosome (bacteria)

Answer 14

• Catalysed on ribosome by peptidyl transferase centre
• Nucleophilic attach followed by hyrolysis
• Parts of ribosomal large subunit facilitate this by helping to coordinate; bring aas into proximity

Question 15

Ribozymes (bacteria)

Answer 15

• RNA section of the 50S subunit
• Involved in number of cellular processes including splicing of rRNA molecules and removal of introns form mRNA

Question 16

Translocation in the ribosome (bacteria)

Answer 16

1. Ribosome moves one codon along in the 3’ direction
   - Requires EFG and GTP
   - Peptidyl RNA moves form A site to P site (uncharged tRNA moves from P site to E site)
2. EFG is then released (requires GTP hyrolysis). Can be reused

Question 17

Structure of EFG (bacteria)

Answer 17

• Structural mimic of EFT:tRNA
• Binds to ribosome competitively w/ EFTu (binds to A site)
• GTP hydrolysis is coupled to conformational change in EFG and ribosome
  -> forces movement of peptidyl-tRNA from A to P site, pushing deacetylated tRNA into exit site

Question 18

Termination (release factors) (bacteria)

Answer 18

• When one of the 3 stop codons is reached, there is no tRNA available to enter the A site, instead a release factor binds to the stop codon
• RF1 (UAA, UAG)
• RF2 (UAA, UGA)
• Enables hydrolysis of bond linking peptide chain to P site

Question 19

Recycling step in translation (bacteria)

Answer 19

• Ribosome recycling factor (RRF) and EFG-GTP promote complex disassembly
• IF3 binds to small subunit to stabilise it in its dissociated state

Question 20

Coupling of transcription and translation (prok vs euk)

Answer 20

• In prokaryotes, coupling of transcription and translation can occur
  -> mutiple ribosomes loaded onto one mRNA
• In euk, however, this can’t occur since they occur in different parts of cell

Question 21

Translation (eukaryotes - compare to bacteria)

Answer 21

• Met is still first aa (but not formylated), special form of initiator tRNA only binds first AUG codon
• No S-D sequence; the 7-methylG cap assists in binding of ribosome and ribosome moves along to first AUG encountered
• Kozak sequence: preferred sequence context for start codons in mammals (RXX_AUG_G)

Question 22

Initiation factors (eukaryotes)

Answer 22

• ‘eIFs’
• Assist the start of translation; 12 identified
• eIFs are numbered 1-6, each no. w/ different types further names w/ a letter
• Numbers are associated with a different step

Question 23

Close loop complex, Pre-I complex formation (eukaryotes), binding of large subunit

Answer 23

1. The mRNA is bound by the eIF4 family of initiation factors. eIF4E recognises the cap, eIF4G acts as scaffold between cap and polyA tail
   -> closed loop complex
   -> 40S, already bound to initiator tRNA (contrasts w/ bacteria) and several IFs is recruited via eIF4G/eIF3 interaction
2. The Pre-I complex scans for start codon: the eIF4A/4B complex has ATP-dependent helicase activity; draws mRNA through until ‘AUG’ located
   -> since there are UTR regions at both ends to bypass
3. Once a suitable start codon has been identified, the GTPase activity of eIF2 is activated, causing a conformational shift
   -> large subunit (60S) binds, majority of remaining IFs released
   -> closed loop complex aids further rounds of translation; enhances translatability of mRNA

Question 24

Elongation (*eukaryotes*) - analogues to *bacteria*

Answer 24

- eEF1A ~ EFTu (binds aminoacyl tRNA and GTP) - eEF1B ~ EFTs (exchanges GTP for GDP; recycling of eEF1A) - eEF2 ~ EFG (involved in ribosome translocation) ...process almost exactly identical

Question 25

Termination (*eukaryotes*)

Answer 25

- eRF1 recognises all termination codons - mimics the structure of tRNA - Results in ribosome w/ one uncharged tRNA and RF w/ ABCE1 attached (w/ ATP) - Ribosome then recycled for future re-use; ABCE1 binds eRF1, starts hydrolysis, protein released - eIF1,1A and 3 recruited to protect A and E sites

Question 26

Protein synthesis inhibitors as antibiotics (7 examples w/ action)

Answer 26

- A range of compounds found to inhibit translation on *prokaryotic* 70S rib.s but not *euk* 80S rib.s - They prevent growth of *bacteria*, shouldn't affect human and animal protein synthesis - However, 70S rib.s present in mitochondria; can have detrimental side effects e.g.... - kasugamycin (X initiation) - tetracyclin (X aminoacyl tRNA binding) - kirromycin (X release of EFTu) - aminoglycosides (cause miscoding in ribosome; incorrect tRNAs bind A site, nonfunctional proteins produced) - chloramphenicol (X peptidyl transfer) - thiostrepton (X translocation) - erthyromycin (sterically block exit tunnel)

Question 27

Fusidic acid

Answer 27

- Narrow spectrum steroid antibiotic - Principally used to target staphylococci (e.g MRSA) - Also used in structural studies of rib. function - Inhibits elongation at the EG mediated translocation step - Binds to EFG-GDP and prevents its release from the ribosome - Blocks A site for next round of aa synthesis

Question 28

Puromycin

Answer 28

- Wide spectrum; can even affect *euk* - Mimics an aminoacyl-tRNA; resembles an aromatic aa linked to a sugar-base - Treated by rib. as if it were an incoming aminoacyl-tRNA -> binds to A site but ONLY on large subunit; peptidyl transferase reaction occurs, product not anchored , so peptidyl-puromycin adduct is released in the form of a polypeptidyl-puromycin - Can' be reused, essentially a suicide inhibitor

Question 29

Diptheria toxin

Answer 29

- 63 KDa pp produced by *Corynebacterium diptheriae* - Catalyses the ADP-ribosylation of eEF-2 (NAD⁺ as a cofactor) - Blocks *euk* protein synthesis by inactivating elongation factor; specialist histidine on protein is (post-T) modified into dipthamide - Catalytic inactivation; one molecule can have widespread effects - could theoretically wipe out synthesis in whole cell ->**very toxic**

Question 30

Ricin

Answer 30

- Very toxic by inhalation, injection or ingestion (LD₅₀ is 5-10 microg/Kg via inhalation/injection, 30-40 orally - Type 2 ribosome inactivating protein (RIP) - Initially synthesised as pre-pro-polypeptide w/ A and B chains, split by proteolysis linked by disulphide bridge - Chain B is a lectin glycoprotein, binds galactose, enters cell through membrane and ER - Chain A is an RNA N-glycosidase, binds and depurinates specific adenine of 28S rRNA -> becomes sandwiched between 2 tyrosine rings in catalytic cleft -> hydrolysed -> completely inactivates ribosome - Enzymatic; one molecule has widespread effects