Midterm #2: Translation

Question 1

Translation Overview

Answer 1

• Proteins are synthesized by ribonucleprotein complexes called ribosomes from mRNA
• Genetic code: relationship between the sequence of bases in DNA, it’s mRNA transcript and the sequence of amino acids in a protein
• U replaces T in RNA
• A codon is the three nucleotide bases in an mRNA transcript

Question 2

The Genetic Code

Answer 2

• Each codon codes for a single, specific amino acid
• Many amino acids are coded for by more than a single codon
  
  • The genetic code is degenerate
• Three codons code for the termination of protein synthesis and are called stop codons

Question 3

Point Mutations

Answer 3

• Changing a single nucleotide in the sequence results in a point mutation
• Silent mutation: the same amino acid specified
• Missense mutation: a different amino acid is specified
• Nonsense mutation: turns an amino acid codon into a stop codon
• Read-though mutation: turns a stop codon into an amino acid codon

Question 4

Errors in the code: Frameshift mutation

Answer 4

• Addition or deletion of a single base in the mRNA transcript
• Changes the identities of all subsequent residues in the protein

Question 5

Chemistry of Amide Bond Formation: A condensation reaction

Answer 5

• Energetically unfavorable reaction, delta G is greater than 0
• Energy barrier overcome by activating the amino acid
• The activated intermediate is an aminoacyl-tRNA

Question 6

Amino Acid Activation

Answer 6

• AMP and PPi as by-products
• Activated for polymerization into polypeptide chain by enzymes called aminoacyl-tRNA synthetases
• Attach a specific tRNA molecule to the carboxylic functional group
• Note that 2 high-energy bonds are hydrolyzed to activate each amino acid

Question 7

Ribosomes: The Engines of Translation

Answer 7

• Very large (~2700 kDa)
• Ribonucleoprotein proteins of approximately 200 angstroms
• Large 50S subunit and small 30S subunit
• Are approximately 20,000 ribosomes in an E. coli

Question 8

Ribosomes have 3 functionally distinct tRNA-binding sites

Answer 8

• Aminoacyl (A) site: accommodates the incoming aminoacyl-tRNA
• Peptidyl (P) site accommodates the peptidyl-tRNA (nascent polypeptide)
• Exit (E) site accommodates a deacylated tRNA that is about to exit the ribosome

Question 9

Initiation of Translation in Prokaryotes: Shine-Delgarno Sequence and anti-codon

Answer 9

• Shine Delgarno Sequence:
  
  • purine rich
  • Defines the initiation codon in prokaryotic mRNA
  • protein synthesis initiates at a downstream AUG codon
  • Recognized by the 16s RNA (part of the 30s which is part of the 70s RNA)
• The anti-codon of tRNA^fMet recognizes an AUG codon
  
  • Protein synthesis always starts at and AUG (met) codon, but not all AUG initiate translation
  • during elongation, AUG is recognized by tRNA^Met

Question 10

Initiation Complex in Prokaryotes

Answer 10

• Initiation complex is composed of the small ribosomal subunit, mRNA, and fMet-tRNA^fMet
• Ribsomes assembly requires proteins called initiation factors
• The Shine-Delgarno sequence in the mRNA base pairs with the 16S rRNA in the small (30s) ribosome subunit
• Note that the only thing required to initiate translation of prokaryotic mRNA is the Shine-Delgarno sequence. If a single mRNA strand has 3 S-D sequenuences, 3 protiens will be translated.
  
  • **polycistronic genes **

Question 11

Initiation of Tranlation in Prokaryotes

Answer 11

• AUG codon placed in the P site where fMet-tRNA^fMet enters
  
  • represents the “nascent” peptide chain
• The 50s subunit binds the mRNA*fMet-tRNA^fMet*30s subunit complex
  
  • poised to bind a second aminoacyl-tRNA in the A site
• Note that GTP hydrolysis by IF2 is required to assemble the functional ribosome

Question 12

Polypeptide Elongation

Answer 12

• activated tRNA is delivered to the A site by elongation factor EF-Tu
• if the tRNA anticodon is NOT properly paired, EF-Tu•GTP and the aminoacyl-tRNA dissociate from the ribosome.
• If the tRNA anticodon is properly paired with the A-site codon, EF-Tu hydrolyzes it bound GTP and dissociates from the ribosome.
  
  • powered by GTP hydrolysis
  • fidelity of bond formation is 10^-4 errors/codon

Question 13

Peptidyl Transferase

Answer 13

• peptidyl transferase activity of 50s ribosomal subunit catalyzes a trans-peptidation reaction
  
  • catalyzed by the 23s rRNA- a ribozyme (ie: an RNA with catalytic activity)
• free amino group of aminoacyl-tRNA at the A-site attacks the activated carbonyl of the amino acid at the P site
• Lengthens nascent peptide by one amino acid
• Note that the nascent peptide chain grows in the N→C direction

Question 14

Translocation

Answer 14

• The ribosome slides one codon to the right (towards the 3’ end) on the mRNA
  
  • powered by GTP hydrolysis
• places peptidyl-tRNA in the P-site and the uncharged tRNA in the E site (dissociates)
• the ribosome•mRNA•peptidyl-tRNA complex is now ready for EF-Tu to deliver the next aminoacyl-tRNA to the A-site and subsequent peptide bond formation

Question 15

Polyribosome

Answer 15

• Because mRNA is syntheized in the 5’→3’ direction, and because ribosomes read mRNA in the 5’→3’ direction, prokaryotic ribosomes can commence translation as soon as mRNA emerges from RNA polymerase
• A second ribosome can bind to an mRNA as soon as the first one has cleared the initiation site
  
  • multiple ribosomes to a single mRNA transcript gives rise to a polyribosome

Question 16

Termintation of Translation

Answer 16

• There are no tRNA molecules that recognize the codons UAA, UGA, or UAG.
• These are STOP codons that are recognized by release factors
  
  • bind to A site and trigger hydrolysis of the protein from the tRNA, which leads to dissociation of the ribosome
• Dissassembly requires one GTP

Question 17

Eukaryotic Protein Synthesis

Answer 17

• ​Ribsomes are larger (4200 kDa vs. 2700 kDa)
• No Shine-Delgarno sequence
  
  • 5’-cap plays an important role in the initation of protein synthesis
• Therefore, mRNA contains a single start site and are monocistronic “one gene, one protein”
• the initator tRNA in euk is tRNA^Met rather than tRNA^fMet

Question 18

Aminoglycosides

Answer 18

• Ex: gentamycin, kanamycin, neomycin
• MOA:
  
  • Inhibit fMet-tRNAfMet binding
  • Impair proofreading

Question 19

Tetracyclines

Answer 19

• Ex: tetracycline, doxycycline, minocycline
• MOA:
  
  • Inhibit aminoacyl-tRNA binding to the A-site

Question 20

Chloramphenicol

Answer 20

MOA: Inhibits peptidyltransferase activity

Question 21

Macrolide antibiotics

Answer 21

• Ex: erythromycin, azithromycin
• MOA:
  
  • Inhibit translocation by binding to the P-site

Question 22

Puromycin

Answer 22

MOA: Mimics 3’-end of aminoacyl-tRNA – causes premature termination in eukaryotes and prokaryotes

Question 23

Aminoglycosides bind to the A site

Answer 23

• Note the 2 bases flipped out of the helix
  
  • indicates that the correct tRNA is in the A site
• Aminoglycoside lock the A site into this conformation
  
  • preventing recognition of a mismatch and impair proofreading