Chapter 15: Translation of mRNA

translation

• produces polypeptide using the information in mRNA

Beadle and Tatum’s Experiments

• Had many different mutants grow on media

  • Minimal media has no additional amino acids - only wild type present b/c can make its own amino acids/proteins 

  • Added O-acteylhomoserine → produces mutant #1 (no need for enzyme 1)

  • Cystathionine media → 2 mutants (no enzyme 1 and 2) 

  • Mutated enzymes don’t grow 

• One gene-one enzyme hypothesis

One gene-one enzyme hypothesis

a single gene controlled the synthesis of a single enzyme 

2nd experiment

• Added cell-free translation system into 20 tubes

• Added random mRNA molecules made from 70% G and 30% U 

• Added one radiolabeled amino acid (different for each tube) and 19 other non-labeled amino acids 

• Allow translation to occur

• Precipitate polypeptides and catch them on a filter 

• Calculate percentage of radioactivity in total amount of protein

Nirenberg and Leder’s Triplet RNAs

• Discovered that RNA triplets (short RNAs containing 3 nucleotides) could stimulate ribosomes to bind a tRNA 

• Different RNA triplets could cause binding of different tRNAs and their associated amino acids 

  • Ex: CCC triplet caused binding of proline 

Summary of Genetic Code Experiments 

• Cell-free translation systems were used to…
  

1. Find correlation between amount of radioactive amino acid that was incorporated into total protein with predicted codons formed 

2. Find correlation between amount of radioactive amino acid that was incorporated into total protein when copolymers were translated (predicted codons were fewer than in first experiment) 

3. Determine that triplet RNAs were sufficient for a charged tRNA to bind to the ribosome 

Synonymous codons

specify the same amino acid

wobble base

Most times the third base is the degenerate base

codon bias

• occurs when a species has an enrichment of one codon that encodes for a particular amino acid 

  • Can impact rates of translation 

  • Which codon is present in mRNA is not always random 

adaptor hypothesis

an adaptor molecule connects the codon sequence with the amino acid 

tRNA function

During mRNA-tRNA recognition, the anticodon in tRNA binds to a complementary codon in mRNA

tRNAs share common structural features

• 3 stem-loop structures 

• Variable regions 

• An acceptor stem with a 3’ single-stranded region that binds to an amino acid 

• tRNAs commonly contain modified nucleotides 

charging of tRNAs

• Amino acid and ATP bind to aminoacyl0tRNA synthetase, PPi is released 

• A specific tRNA binds to an enzyme, the amino acid covalently bonds to tRNA through an ester bond and AMP is released 

• Charged tRNA is released 

aminoacyl-tRNA synthetases

Enzymes that attach amino acids to tRNAs

wobble rules

Francis Crick, The first 2 positions pair strictly according to the AU/GC rule 

• However the 3rd position can tolerate certain types of mismatches 

• Isoacceptor tRNAs have different anticodon sequences but can recognize the same codon 

Peptidyl site (P site)

where tRNA attached to the polypeptide resides 

Aminoacyl site (A site)

where the new tRNA charged with an amino acid resides

Exit site (E site)

where the uncharged tRNA exits the ribosome 

Shine-Dalgarno sequence

• binding of mRNA to the 30S subunit is facilitated by a ribosomal-binding site 

  • Complementary to a sequence in the 16S rRNA

  • Process allows the mRNA to be positioned in the ribosome correctly to start translation at the start codon 

• in bacteria

Differences in Initiation in Eukaryotes 

1. Initiation factors are named eukaryotic Initiation Factors (eIF)

2. The mRNA is positioned on the small subunit by a consensus sequence upstream of the start codon → Kozak’s rules

3. Initiator tRNA is designated tRNA(met) 

   1. Carries methionine not formylmethionine 

Consensus sequence

for optimal start codon recognition 

Kozak’s rules

rules for optimal translation

imitation in eukaryotes

1. An initiation factor complex binds to the 5’ cap  (m7G) in mRNA

2. mRNA binds to a complex consisting of the 40S subunit with tRNA(met) and other initiation factors 

3. Entire assembly moves along the mRNA scanning for the right start codon (determined by Kozak’s rules) 

4. Once it finds this AUG, the 40S small subunit binds to it

5. The 60S large subunit joins

6. This forms the 80S initiation complex 

release factors

recognizes stop codons

bacteria release factors

• RF1 recognizes UAA and UAG

• RF2 recognizes UAA and UGA

• RF3 binds GTP and helps facilitate the termination process

eukaryotic release factors

• eRF1 recognizes all 3 stop codons

• eRF3 is needed for termination 

Polyribosomes/polysomes

transcripts with multiple ribosomes translating simultaneously