Translation and the Genetic Code

Central Dogma

  • Central Dogma: DNA -> mRNA -> Protein

Lecture Overview

  • The genetic code
  • Degeneracy
  • Wobble
  • RNA molecules involved in translation
    • mRNA – Codons and reading frames
    • tRNAs – Structure, anticodons & codons, and how they’re charged with amino acids
    • rRNA and ribosomes

RNA Molecules and Their Functions

  • RNA molecules are encoded by genes through transcription.
  • Different types of RNA serve various roles:
    • mRNA: Carries the genetic code for protein synthesis; produced by structural genes and translated into proteins.
    • rRNAs: Form the structural and catalytic core of ribosomes.
    • tRNA: Adaptor molecules that match amino acids to the coding RNA sequence.
    • Special Function RNAs (snRNAs, microRNAs, siRNAs): Involved in various regulatory processes.

Translation

  • Translation: The synthesis of a polypeptide chain (protein) according to the sequence of mRNA bases.
  • Only RNA from protein-coding genes is translated.
  • tRNA form the adaptor molecules that match amino acids to the coding RNA sequence
  • Ribosomes (made up of rRNA and proteins) are the site of synthesis

Cracking the Genetic Code

  • There are 4 nucleotide bases: A, C, G, and U.
  • There are 20 possible amino acids.
  • Using single bases: 4 ≠ 20 (insufficient)
  • Using pairs of bases: 42=164^2 = 16 which is < 20 (insufficient)
  • Using triplets of bases: 43=644^3 = 64 which is > 20 (more than enough)

Genetic Code: Codons and Redundancy

  • Four bases (C, U, A, G) in a combination of 3.
  • 43=644^3 = 64 codons.
  • Example: CAG codes for glutamine (Gln).
  • Only 20 different amino acids, resulting in "redundancy" or "degeneracy" of the genetic code.
  • The degeneracy isn't uniform.
    • One codon for Tryptophan.
    • Six codons for Leucine.

Features of the Genetic Code

  • The code is written in the 5’ → 3’ direction.
  • The third base is less important in binding to tRNAs.
  • The first codon establishes the reading frame.
  • 61/64 codons encode amino acids.
  • There are three termination (‘stop’) codons: UAA, UGA, UAG.
  • AUG = the initiation (‘start’) codon (as well as coding for methionine).

Evolution of the Genetic Code

  • The genetic code is almost universal.
  • Vertebrate mitochondrial code is slightly different.
    • UGA encodes Trp (instead of STOP).
    • AGA/AGG encodes STOP (instead of Arg).
  • Mitochondria encode their own tRNAs, use 22 instead of 32.
  • Some bacteria can use alternative start codons (Ile/Val/Leu).

Reading Frames

  • A single mRNA molecule has three potential reading frames.
  • Start and stop codons are crucial for identifying the correct reading frame.

Prokaryotic mRNA

  • Key regions of a single prokaryotic gene:
    • Promoter region
    • 5’ untranslated region
    • Initiation site of transcription
    • Protein coding region of the gene
    • 3’ untranslated region
    • Terminator

Prokaryotic mRNA Components

The mRNA of a prokaryotic gene contains:

  • 5’ and 3’ untranslated regions.
  • Shine-Dalgarno sequence – a ribosome binding site (RBS).
  • Start codon.
  • Open reading frame.
  • Stop codon.

Shine-Dalgarno Sequence

  • 8-12 nucleotides upstream of AUG start.
  • Facilitates binding of ribosome to mRNA.
  • Serves as an initiation sequence for translation.
  • Complimentary to the 3' end of 16S rRNA.

Polycistronic mRNA

  • Many prokaryotic mRNAs are polycistronic.
  • Several genes are transcribed as a single mRNA – organized into an operon.
  • Each transcribed gene sequence will have a Shine-Dalgarno sequence, Start codon, and Stop codon.

Eukaryotic mRNA

  • Eukaryotic mRNAs are usually monocistronic.
  • No Shine-Dalgarno sequence.
  • 5’ cap and poly(A) tail bind to initiation factors to assemble the ribosome.
  • The complete ribosome "scans" mRNA for the first available start codon.

Codon Wobble

  • There are at least 32 tRNAs required to translate all 61 codons.
  • Crick’s Wobble hypothesis: Lack of specificity at the third (degenerate) position of the codon = first base of the anticodon (tRNA side).
  • Codon: anti-codon matches are not perfect.

Transfer RNAs

  • Transfer RNAs = adapters between codons and amino acids.
  • Single-stranded, self-complimentary.
  • ‘Cloverleaf’ structure in 2D.
  • ‘Twisted L’ structure in 3D.

General tRNA Characteristics

Each tRNA molecule:

  • Has an anticodon that matches to a mRNA codon.
  • Occurs via complementary base pairing.
  • Most begin (5’ end) with a G.
  • All end in ‘CCA’ (site of amino acid attachment).
  • Have modified bases (e.g., pseudouridine (); 5,6-dihydrouridine (D)).
  • Conserved bases are recognized by aminoacyl-tRNA synthetases.
  • Py = pyrimidine; Pu = purine.

tRNAs Role in Translation

  • Amino acids are attached to the tRNA molecule by enzymes called aminoacyl-tRNA synthetases.
  • Each aminoacyl-tRNA synthetase enzyme is specific for a single amino acid.
  • Attachment of the amino acid to the tRNA requires ATP.
  • Divided into two classes (I and II) that differ slightly in their mechanism.
  • Reaction catalysed: Amino acid + tRNA + ATP Mg2+Mg^{2+} aminoacyl tRNA + AMP + PPi

Activation of Amino Acids by ATP

  • In the first step, an aminoacyl adenylate (aminoacyl-AMP) is formed in the enzyme's active site.
  • Carboxyl of amino acid attacks the α phosphorus of ATP, forming 5’ aminoacyl adenylate (aminoacyl-AMP).

Aminoacyl Group Transfer to tRNA

  • Depending on the class of aminoacyl-tRNA synthetase:
    • Class I: The aminoacyl group is transferred to the 2’ OH of the terminal A residue, releasing AMP. Transesterification then moves the aminoacyl group to the 3’ OH.
    • Class II: The aminoacyl group is directly transferred to the 3’ OH, releasing AMP.

Aminoacyl-tRNAs and Ester Linkage

  • The result is an aminoacyl-tRNA with an ester linkage.
  • Has a high negative free energy of hydrolysis (like ATP).
  • Prepares the amino acid for peptide bond formation in the ribosome.

Wobble Base Pairing

  • The ‘wobble’ base of a tRNA anticodon can form non-canonical base pairs with the mRNA codons.
  • The base ‘wobbles’ from its normal position to facilitate these hydrogen bondings.

Wobble Mechanism

  • Allows a single tRNA to recognize multiple codons.
  • Example: Phe tRNA: AAG can base pair with UUC or UUU.

Inosine in tRNAs

  • Some tRNAs contain the nucleotide inosine, which can H-bond with U, C, and A.
  • This allows for several codons to specify a single amino acid.
  • Example: in yeast, the tRNAArg has the anticodon 3’-GCI-5’ recognizes the codons (5’ to 3’) CGA, CGU and CGC.

Wobble Benefits

  • 32 tRNAs are required to translate all 61 codons (31 to encode the amino acids, 1 for initiation).
  • Allows for faster dissociation of tRNA from mRNA and thus faster protein synthesis.
  • Minimizes the damage that can be caused by a misreading of the code.

Redundancy Beyond Wobble

  • Leucine and arginine have 6 codons each.
  • Different tRNA molecules are required for codons that differ in the first or second base.
  • Example: E. coli tRNALeu (UAG) vs. tRNALeu (UAA).

Genetic Code Mutation Resistance

  • Degenerate code allows for mutations to occur, but still code for the same amino acid (silent mutation).
  • Mutation in the first base of a codon usually produces a conservative substitution (e.g., GUU to AUU results in Val to Ile).
  • Though different, these are biochemically similar.

Ribosomes Composition and Function

  • Ribosomes ‘supervise’ the interactions between tRNA and mRNA.
  • Bacterial ribosomes contain roughly 65% rRNA and 35% protein.

Ribosome Structure

  • Bacterial ribosomes are composed of two unequal subunits.
  • Named after their sedimentation coefficients.
  • Prokaryotic ribosome = 30S + 50S = 70S.
  • Eukaryotic ribosome = 40S + 60S = 80S.
  • Many copies in the cell (e.g., an E. coli cell has about 15,000 ribosomes = 25% dry mass of cell).

Ribosomes and Protein Synthesis

  • The ribosomes function in positioning all relevant molecules for translation to occur.
  • Positions the mRNA such that tRNAs can bind to the appropriate codons, bringing the amino acids specified by the mRNA.
  • Positions the aminoacyl tRNAs so that peptide bond formation can occur.
  • Translocates along the mRNA molecule to continuously add amino acids to the growing peptide.

tRNA Binding Sites

Ribosomes have 3 sites for tRNAs:

  1. A-site (aminoacyl site): The entry point for charged tRNAs.
  2. P-site (peptidyl site): Holds the tRNA attached to the growing peptide chain.
  3. E-site (exit site): Holds the deacylated tRNA before it leaves.

Summary

  • mRNA is translated into protein according to the genetic code (61 encoding amino acids; 4 bases = 64 possible codons; 3 stop codons; recognised by (min) 31 tRNAs).
  • Degeneracy ‘Wobble’ Faster & resistant to mutation
  • tRNAs are single stranded multi-hair-pinned ‘cloverleaf’ RNA molecules
    • Possess an anticodon that binds to codons in mRNA
    • Match amino acids to the appropriate mRNA codons at the ribosomes
    • Are ‘charged’ with amino acids by specific aminoacyl-tRNA synthetases
  • Ribosomes are large multi-protein and rRNA complexes,
    • Form the site of translation by bringing all molecules involved into proximity
    • Possess three sites for tRNAs that facilitate protein synthesis