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1

What are the principles of the genetic code?

1. Degenerate: Since there are 64 possible codons (43) but only 20 amino acids, one amino acid is usually coded for by multiple codons (synonyms). Exceptions are Met and Trp residues.

2. Non-overlapping: The codons are read in a particular order consecutively as opposed to the previous codon making up part of the current.

3. Non-universal: The genetic code is not the same for all organisms.

2

What are the properties of the genetic code?

1. XY(U/C) always code for the same codon and XY(A/G) usually code for the same codon.

2. Changes in the first letter of the codon tend to code for the same or amino acids with similar chemical properties.

3. Codons with 2nd position pyrimidines usually code for hydrophobic amino acids.

4. Codons with 2nd position purines usually code for hydrophilic amino acids.

5. There are 3 stop codons.

3

What are the 3 stop codons?

UGA, UAG, UAA

4

What is the name given to 2 or more codons coding for the same amino acid?

Synonyms

5

What are the functions of tRNA?

1. Reads and interprets genetic code

2. Shuttles amino acids to the ribosomes

6

What are the common structural features of amino acids?

1. They are 75-90 nucleotides long.

2. Contain high number of modified nucleotides (e.g. pseudouridine, inosine…).

3. Acceptor stem containing unpaired CCA sequence on 3’ end that is covalently bonded to specific amino acids.

4. The tRNA contains 5 arms.

7

What are the 5 arms of the tRNA molecule?

1. Acceptor arm: Contains acceptor stem

2. D arm

3. TψC (T) arm

4. Anticodon arm: Contains anticodon

5. Variable arm

8

What shape does the tRNA fold into in 3D?

L-shape

9

What is the process of charging a tRNA?

Attaching a tRNA to its associated amino acid.

10

What are the sequence of events that occur during tRNA charging?

1. Amino acid is reacted with ATP to form aminoacyl-adenylate, releasing PPi group. PPi is hydrolysed to provide energy for this reaction.

2. The aminoacyl-adenylate is then reacted with specific tRNA to form aminoacyl-tRNA.

11

What is the overall reaction for tRNA charging?

Amino acid + tRNA + ATP → aminoacyl-tRNA + AMP + PPi

12

What enzymes mediate tRNA charging?

- Aminoacyl-tRNA synthetase (aaRS).

- There are 20 unique aaRSs, one for each amino acid.

13

What is the specificity of aaRSs determined by?

At least for class I aaRSs, specificity is determined by the ability for the aaRS to interact with both the acceptor stem and the anti-codon.

14

What is the proofreading mechanism for aaRSs?

- aaRSs have intrinsic proof-reading mechanisms to ensure that the right amino acid is attached to the right tRNA.

- Step 1 of charging is less specific but step 2 is highly specific.

15

What is a example of the proofreading capability of aaRSs?

- IleRS can ract Val residue with ATP in first step, but fails in the second step and hydrolyses it to Val and AMP in the presence of tRNAIle.

- This is due to that fact that IleRS has 2 active sites, one for step 1 and one for step 2. Step 2 active site specific to Ile-adenylate.

16

What is wobble?

1. The 1st and 2nd bases are recognised by complementary base pairing between mRNA and anticodon.

2. Degree of flexibility in sugar-phosphate backbone (wobble) allows 3rd base to pair with non-complementary bases.

17

What are the laws governing wobble?

1. Anticodon with A/T at 3rd position pairs with only complementary pairs.

2. Anticodon with U at 3rd position recognises G/A.

3. Anticodon with G at 3rd position recognises C/U.

4. Anticodon with I at 3rd position recognises U/C/A.

18

What are the different sizes of prokaryotic and eukaryotic ribosomes?

- Prokaryotic (70S): 30S + 50S

- Eukaryotic (80S): 40S + 60S

19

What are the binding sites on RNA molecules?

1. A (aminoacyl) site

2. P (peptidyl) site

3. E (exit) site

20

What are the 3 stages of translation?

1. Initiation

2. Elongation

3. Termination

21

What is the sequence of events that occur during prokaryotic translation initiation?

1. IF-3 binds to the 70S ribosome complex and causes the 30S subunit to dissociate from the 50S subunit.

2. tRNAfMet is bound to 30S subunit through the action of IF-2, which is a G-protein. IF-1 binds to A-site to block premature tRNA binding.

3. In prokaryotes, there is a consensus sequence just upstream of the initiating AUG codon called the Shine-Dalgarno sequence (GGAGG), which is recognised by the 30S subunit (via complementary base pairing between it and UUTUU on 30S) and defines the starting site of translation.

4. IF-1 and IF-3 are released. 50S subunit joins onto 30S subunit, causing IF-2 to hydrolyse GTP to GDP, which is also subsequently released.

22

What is the sequence of events that occur during eukaryotic initiation of tranlation?

1. eIF-2 mediates the binding of tRNAiMet onto 40S subunit

2. eIF-4F recognises the 5’ cap and binds to it via cap binding proteins (eIF-4E).

3. tRNAiMet-40S complex is recruited to the 5’ cap by eIF-4F complex.

4. The complex scans towards the 3’ end until it detects the first AUG codon.

5. Once AUG found, eIF-2 hydrolyses bound GTP to GDP, causing release of all bound initiation factors.

6. 60S subunit recruited to 40S subunit in process mediated by eIF-5B.

23

What are the sequence of events that occur during elongation in translation?

1. Decoding: The aa-tRNA complementary to the codon in the A-site is recruited. It binds onto the codon in the A-site via the action of an elongation factor (e.g. eEF-1 in eukaryotes), coupled with hydrolysis of GTP. This process is highly monitored by the ribosome to ensure correct decoding.

2. Traspeptidation: Peptidyl transferase activity in the ribosome catalyses the transfer of the peptide in peptidyl-tRNA in P-site to amino acid in aminoacyl-tRNA in A-site to form n+1 peptidyl-tRNA. This peptidyl transferase activity is provided by ribozyme.

3. Translocation: The process whereby the ribosome complex moves along the mRNA molecule by exactly 3 nucleotides so that the new peptidyl-tRNA moves into the P-site, the empty tRNA moves into the E-site (later released in step 1) and the A-site is empty to allow for repeat of cycle. This process requires elongation factor eEF-2 and GTP.

24

When does translation terminate?

When a stop codon is encountered by the A-site.

25

What is the sequence of events that take place during termination of translation?

1. A release factor (eRF1) to be recruited into the A-site.

2. This causes the peptidyl-tRNA to be ‘transferred’ to a water molecule by the peptidyl transferase, effectively hydrolysing the ester bond between the peptide and tRNA.

3. Free tRNA and polypeptide released from the ribosome.

26

What are the 2 types of translational control?

1. Global: Affects translation of all mRNA molecules.

2. Specific: Affects translation of certain mRNA molecules.

27

What is the sequence of events that occur in the interferon response (general)?

1. Viral dsRNA is detected by the cell.

2. elF2 is phosphorylated.

3. elF2 is responsible for delivering Met-tRNAi to the 40s chromosome in order to initiate all translational events. Phosphorylation inhibits its activity by preventing the exchange of bound GDP for another GTP, which prevents it from taking part in another initiation reaction.

4. Initiation of translation is inhibited and thus all protein synthesis in the cell stops.

5. This prevents the viruses from hijacking the cellular protein synthesis machinery for its own reproduction.

28

What is the sequence of events that occur during the regulation of ferritin synthesis (specific)?

1. Ferritin mRNA contains unique hairpin structure in its 5’ & 3’ UTR called the iron response element (IRE).

2. When cellular [Fe] concentrations are low, iron regulatory protein (IRP) binds to IRE, which prevents the 40S subunit and Met-tRNAi complex from binding to the initiation codon, preventing the initiation of translation.

3. This reduces the production of ferritin, as it is not needed.

4. In addition, binding of IRPs to 3’ IREs stabilise mRNA and prevents it from being degraded. This ensures that mRNA is available for when [Fe] is high and response is quick.

5. When [Fe] high, IRP affinity for IRE decreases, thus allowing ferrin translation to occur.

29

What is the sequence of events that occur during siRNA interference?

1. dsRNA is chopped into short double stranded fragments called siRNA by RNase called dicer.

2. siRNA binds to RISC (RNA-induced silencing complex).

3. RISC unwinds siRNA using intrinsic helicase activity. This creates 2 strands of ssRNA. One strand is the passenger strand and is broken down. The other strand, called the guide strand, remains bound.

4. RISC complex binds to mRNA sequence complementary to guide strand.

5. AGO (Argonaute) component of RISC cleaves the mRNA, which allows it to be degraded by nuclease enzymes.

30

What are the main differences between siRNA and miRNA interference?

1. siRNA is synthetic while miRNA is natural.

2. There is perfect complementary base pairing between siRNA and mRNA while base pairing is not perfect between miRNA and mRNA.

3. miRNA does not mediate the AGO-cleavage of mRNA (due to imperfect base pairing). Instead, it is beleived to promote decapping an deadenylation of poly-A tail.

31

What are additional regulators of RNA translation?

1. Antitermination

2. Riboswitches

32

What is an example of antitermination?

HutP regulates Hut expression by preventing the formation of terminating hairpin loops on mRNA.

33

What is an example of riboswitches?

TTP (thiamine pyrophsophate) binds to TTP-sensing riboswitch (thi box) in the 5'UTR. This causes conformational change masking thw Shine-Dalgarno sequence.

34

What are the ways in which proteins are degraded?

1. Lysosomes

2. Proteasomes

35

What types of proteins are degraded through the lysosomal pathway?

Proteins with the KFERQ sequence.

36

What types of proteases are found in lysosomes?

Cathepsin proteases (only active in low pH environments)

37

What are the steps in ubiquitination?

1. C-terminus Gly residue in Ub is linked to SH-group on E1 (ubiquitin activating enzyme). This process requires ATP.

2. The Ub is then transferred onto another SH-group in E2 (ubiquitin-conjugating protein). There are numerous enzymes in this family for specificity.

3. E3 (ubiquitin-protein ligase) transfers the Ub from E2 to Lys residues of a condemned protein. Each specific E3 ubiquinates a specific set of proteins.

4. Polyubiquination occurs whereby a chain of ubiquitins are linked together in a linear fashion (by Gly-Lys bonds). This increases the efficiency by which the proteins are degraded by the proteasome.

38

What is the structure of a proteasome?

1. 20S barrel: Main site of protein degradation.

2. 19S cap: Recognition of ubiquitinated protein.

39

What is the sequence of events that take place during proteasome-mediated proteolysis?

1. Recognition: Ubiquinated protein binds to the 19S cap.

2. Dissociation: ATPase activity I the cap results in the unwinding of the protein and the elimination of the bound ubiquitin molecules.

3. Translocation: The unwound protein is fed into the barrel, which contains proteins that catalyse hydrolysis of peptide bonds.

4. Degradation: Protein is degraded into 8 amino acid fragments that are released into the cytosol.

40

What types of proteins are degraded by the proteasome pathway?

1. N-end rule: Proteins with Asp, Arg, Leu, Lys, Phe sequence at the N-terminus once Met residue removed are degraded very quickly. Proteins with Ala, Gly, Met, Ser, Thr, Val sequence at the N-terminus are degraded very slowly. This is due to their interactions with RING finger domains on E3.

2. PEST sequences: Proteins rich in Pro, Glu, Ser, Thr can be phosphorylated on Ser or Thr, which allows for ubiquination and targeted destruction.

3. D-box: Specific N-terminus sequences that promote ubiquitination and are responsible for cyclin ubiquination and degradation.