L16 + 17: Translation Flashcards
1
Q
Formation of a peptide bond
A
- AAs joined by peptide bonds; covalent bond formed between carboxyl group of one AA and amino grp of an adjacent aa
2
Q
Universal genetic code
A
- 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
3
Q
tRNA
A
- 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
4
Q
Charging
A
- 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
5
Q
Ways of achieving site specificity in tRNA synthetase enzymes
A
- size exclusion
- editing pre-transfer
- editing post-transfer
6
Q
Wobble pairing
A
- 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
7
Q
Which amino acid is incorporated into the protein first?
A
- W/ rare exception, methionine is the first; may be removed later
- tRNAfmet(prok) or tRNAimet is always used at the start and another for elongation involving methionine (tRNAmmet)
8
Q
Bacterial vs Eukaryotic ribosome
A
-
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)
9
Q
Shine-Dalgarno (bacteria)
A
- 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
10
Q
Attachment of ribosome to mRNA (Initiation) (bacteria)
A
- 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
11
Q
Role of IF1 an IF3 (bacteria)
A
- 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
12
Q
Sites in the ribosome
A
- Aminoacyl site
- Peptidyl site
- Exit site
12
Q
Elongation (bacteria)
A
- 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)
13
Q
Recycling of EFTu (bacteria)
A
- ‘EFTs’ exchanges GDP for GTP on EFTu
- Can be reused
14
Q
Peptide bond formation in ribosome (bacteria)
A
- 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
15
Q
Ribozymes (bacteria)
A
- RNA section of the 50S subunit
- Involved in number of cellular processes including splicing of rRNA molecules and removal of introns form mRNA
16
Q
Translocation in the ribosome (bacteria)
A
- 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) - EFG is then released (requires GTP hyrolysis). Can be reused
17
Q
Structure of EFG (bacteria)
A
- 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
18
Q
Termination (release factors) (bacteria)
A
- 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
19
Q
Recycling step in translation (bacteria)
A
- Ribosome recycling factor (RRF) and EFG-GTP promote complex disassembly
- IF3 binds to small subunit to stabilise it in its dissociated state
20
Q
Coupling of transcription and translation (prok vs euk)
A
- 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
21
Q
Translation (eukaryotes - compare to bacteria)
A
- 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)
22
Q
Initiation factors (eukaryotes)
A
- ‘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
23
Q
Close loop complex, Pre-I complex formation (eukaryotes), binding of large subunit
A
- 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 - 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 - 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
24
Q
Elongation (eukaryotes) - analogues to bacteria
A
- 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
25
Q
Termination (eukaryotes)
A
- 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
26
Q
Protein synthesis inhibitors as antibiotics (7 examples w/ action)
A
- 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)
27
Q
Fusidic acid
A
- 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
28
Q
Puromycin
A
- 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
29
Q
Diptheria toxin
A
- 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
30
Q
Ricin
A
- 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
