Translation & Regulation of Translation

Question 1

Prokaryotic ribosome subunits

Answer 1

50S subunit: 23S +5S rrNA 30S subunit: 16SrRNA Total: 70S ribosome

Question 2

Eukaryotic ribosome subunits

Answer 2

60S subunit: 28S + 5.8 S + 5S rRNA 40S subunit: 18SrRNA Total: 80S ribosome

Question 3

degenerate (genetic code)

Answer 3

more than 1 codon for some aa’s

Question 4

non-ambiguous (genetic code)

Answer 4

1 codon can only specify 1 aa

Question 5

silent mutation

Answer 5

a change that specifies the same amino acid

Question 6

missense mutation

Answer 6

a change that specifies the same amino acid

Question 7

nonsense mutation

Answer 7

a change that produces a stop codon

Question 8

insertion/deletion

Answer 8

addition or deletion of one or more bases; if 1 or 2 bases only–> will cause a frameshift

Question 9

wobble

Answer 9

• base pairing between codon/anticodon in 5’ and middle codon bases must be perfect
• some flexibility in 3’ base of codon *allows mRNA to be translated with fewer than 64 tRNAs

Question 10

tRNAs

Answer 10

adaptor molecules with bind both amino acids and mRNA codons

Question 11

aminoacyl tRNA synthetases

Answer 11

protein synthesis requires energy amino acids are activated with ATP prior to tRNA attachment (this enzyme carries out both steps) synthetase is specific for a given amino acid

Question 12

prokaryotic initiation

Answer 12

• Shine-Dalgarno sequence positions 30S subunit so initiator tRNA is in P site
• Shine-Dalgarno base-pairs with 3’ end of 16S rRNA -formyl-met is first aa incorporated

Question 13

prokaryotic elongation

Answer 13

• aa-tRNA -EF-Tu-GTP enters the A site, start of the cycle (also the rate-limiting step)
• EF-Tu-GDP is recycled (with EF-Ts) (correct positioning involves the TYC loop, which is complementary to the sequence in 5S RNA)
• Formation of peptide bond with transfer of growing peptide chain to the tRNA in the A site, leaving free tRNA in P site (catalyzed by peptidyl transferase; ribozyme component of 23SrRNA)
• Translocation from P–>E mediated by elongation factor EF-G-GTP (requires energy, hydrolyzed)

Question 14

prokaryotic termination

Answer 14

1. Stop codon (UAA, UAG, UGA appears in A site)
2. RFe-GTP binds ribosome
3. Hydrolysis of GTP
4. Cleavage of ester bond (peptidyl transferase)
5. Release of protein, tRNA, mRNA, ribosomal subunits

Question 15

tetracycline

Answer 15

inhibitor of protein synthesis; blocks binding of aminoacyl-tRNA to A-site of ribosome (prokaryotes only)

Question 16

chloramphenicol

Answer 16

resembles peptide bond; inhibits peptidyl transferase activity (prokaryotes only)

Question 17

puromycin

Answer 17

• looks like a tRNA
• enters A site and accepts polypeptide chain; translocation is blocked (prokaryotes and eukaryotes)

Question 18

differences between prokaryotic and eukaryotic initiation

Answer 18

• capped mRNA -initiation factors (eIF)
• no fmet tRNA (met-tRNA initiator or i)
• locate Kozak sequence (which includes AUG start codon in it, almost always first AUG after cap)
• small subunit binds at cap

Question 19

eukaryotic initiation steps

Answer 19

how much to include?

Question 20

HCV IRES

Answer 20

• recognized specifically by the small ribosomal subunit / eukaryotic initiation factor (eIF3)
• interactions allow cap-indepedent initiation of viral protein synthesis through + scaffold eIFG

Question 21

regulation of eukaryotic translation (EIF2B)

Answer 21

• EIF2-GTP–> binds the initiator tRNA and forms a complex with the 40S r subunit
• Under stress: (oxidative, temperature), nutrient deprivation, lack of heme (reticulocytes), double-stranded RNA (induces production of interferon) etc, which stimulates production of eIF2B (kinase), eIF2 has 100X more affinity to it
• eIF2B phosphorylates it EIF2-GDP, inhibiting protein synthesis

Question 22

RNA interference / post transcriptional gene regulation in lower eukaryotes

Answer 22

dsDNA–(Dicer; RNAse III-like enzyme)–>

siRNAs (2 unpaired nucleotides at 3’ ends–>

RISC (RNA-induced silencing complex, contains an endonuclease activity)–>

activated RISC–> (antisense strand remains associated with RISC) target mRNA–>

Argonaute (slicer) is the enzyme within RISC responsible for mRNA degradation

Question 23

microRNAs

Answer 23

• Naturally occurring silencing RNAs in mammalian cells synthesized as longer hairpin precursors in the nucleus by Pol II
• Processed by Drosha to form pre-miRNA in nucleus (transported to cytoplasm and processed by Dicer; RISC retains antisense strand)
• RISC bind to mRNA 3’ untranslated regions (imperfect pairing) and inhibit translation
• May also lead to mRNA degradation (perfect pairing)