Midterm #2: Translation Flashcards Preview

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Flashcards in Midterm #2: Translation Deck (23):
1

Translation Overview

  • 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 

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2

The Genetic Code

  • 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

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3

Point Mutations

  • 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 

4

Errors in the code: Frameshift mutation 

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

5

Chemistry of Amide Bond Formation: A condensation reaction

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

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Amino Acid Activation

  • 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

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Ribosomes: The Engines of Translation

  • 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 

8

Ribosomes have 3 functionally distinct tRNA-binding sites

  • 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

9

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

  • 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 tRNAfMet 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 tRNAMet

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10

Initiation Complex in Prokaryotes

  • Initiation complex is composed of the small ribosomal subunit, mRNA, and fMet-tRNAfMet
  • 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 

11

Initiation of Tranlation in Prokaryotes

  • AUG codon placed in the P site where fMet-tRNAfMet enters
    • represents the "nascent" peptide chain
  • The 50s subunit binds the mRNA*fMet-tRNAfMet*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 

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12

Polypeptide Elongation

  • 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

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13

Peptidyl Transferase

  • 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

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14

Translocation

  • 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 

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15

Polyribosome

  • 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

16

Termintation of Translation

  • 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

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17

Eukaryotic Protein Synthesis

  • ​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 tRNAMet rather than tRNAfMet

18

Aminoglycosides

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

19

Tetracyclines

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

20

Chloramphenicol 

MOA: Inhibits peptidyltransferase activity 

21

Macrolide antibiotics

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

22

Puromycin 

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

23

Aminoglycosides bind to the A site

  • 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 

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