Johans Anteckningar

Translation Undertexter

Hello, my name is Anna Lee and in today’s lecture I’ll be talking about protein biosynthesis or translation.

In previous lectures you have learned that during transcription the RNA polymerase transcribes genes into the messenger RNA or mRNA. In this lecture we’ll cover how the cell translates the mRNA information into proteins.

The main learning goals of this lecture are:

  • explaining how nucleic acid information is translated into amino acid sequence
  • defining the role of aminoacyl tRNA synthetases in this process
  • understanding the features of the genetic code
  • understanding the structure and function of tRNA
  • understanding the structure and function of ribosomes
  • understanding the steps of protein synthesis
  • understanding how antibiotics and toxins block protein biosynthesis

Before we start with the details of translation, consider the question: why is it important to understand the mechanisms of translation?

Reasons:

  • inhibition of translation of specific mRNAs can lead to diseases
    • example: fragile X mental retardation syndrome
  • translation is targeted by bacterial toxins
    • example: diphtheria toxin
  • translation is a major target of antibiotics
    • examples: streptomycin, tetracycline
  • new antibiotics are being developed that target bacterial ribosomes

Protein biosynthesis is called translation because:

  • the four-letter nucleic acid alphabet is translated into the 20-letter amino acid alphabet
  • this conversion makes translation more complex than replication or transcription

General principles:

  • mRNA is decoded 5′→3′
  • protein is synthesized from amino (N) to carboxyl (C) terminus
  • amino acids are added sequentially to the C-terminus

Translation requires:

  • a codon (three RNA bases)
  • a tRNA acting as an adaptor
  • correct codon–anticodon recognition

tRNA molecules:

Common features:

  • contain 7–15 modified bases
  • secondary structure resembles a cloverleaf
  • five major regions:
    • 3′ CCA acceptor stem
    • TΨC loop
    • extra arm
    • anticodon loop
    • DHU loop
  • 3D structure is L-shaped
    • anticodon is positioned ~90° from the 3′ CCA end
    • the CCA region can undergo conformational changes

The genetic code:

Key characteristics:

  • codons are groups of three nucleotides
  • the code is read 5′→3′
  • non-overlapping
  • no punctuation
  • degenerate (most amino acids have multiple codons)
  • three stop codons

The wobble effect:

  • some tRNAs recognize more than one codon
  • the third codon base is less strictly recognized
  • wobble depends on the first base of the anticodon

Aminoacyl tRNA synthetases:

Functions:

  • attach amino acids to the correct tRNAs
  • establish the genetic code
  • activate amino acids with ATP
  • form an ester bond with the 3′ end of tRNA

Fidelity mechanisms:

  • activation site
  • editing (proofreading) site

Example: threonine vs valine vs serine

  • the enzyme’s zinc-dependent binding site excludes valine
  • serine can bind but is removed in the editing site

tRNA recognition:

  • often involves acceptor stem and anticodon loop
  • sometimes additional structural features

Ribosomes:

In bacteria:

  • 70S ribosome
    • 50S large subunit (23S rRNA, 5S rRNA, proteins L1–L34)
    • 30S small subunit (16S rRNA, proteins S1–S21)

Functional sites:

  • A site (aminoacyl)
  • P site (peptidyl)
  • E site (exit)

Translation stages:

  • initiation
  • elongation
  • translocation
  • termination

Initiation in bacteria:

Requirements:

  • start codon (usually AUG)
  • Shine–Dalgarno sequence upstream
    • base-pairs with 16S rRNA
    • positions start codon in P site

Initiator:

  • formyl-methionine (fMet)
  • delivered by fMet-tRNAᶠᴹᵉᵗ

Initiation factors:

  • IF1 and IF3 bind the 30S subunit to prevent premature 50S binding
  • IF2-GTP recruits fMet-tRNAᶠᴹᵉᵗ
  • GTP hydrolysis allows 50S binding and forms 70S initiation complex

This step is a major regulatory point and is targeted by antibiotics such as streptomycin.

Elongation:

  • EF-Tu-GTP delivers aminoacyl tRNA to the A site
  • correct codon recognition → GTP hydrolysis → EF-Tu-GDP release
  • EF-Ts regenerates EF-Tu-GTP

Peptide bond formation:

  • catalyzed by the 23S rRNA peptidyl transferase center
  • the amino group in the A site attacks the ester bond in the P site

Translocation:

  • driven by EF-G-GTP
  • tRNAs shift A→P and P→E
  • empty tRNA exits

Termination:

  • no tRNAs recognize stop codons
  • RF1 or RF2 bind stop codons in the A site
  • RF3-GTP triggers release
  • the polypeptide is freed from the P-site tRNA

Coupling in bacteria:

  • transcription and translation occur simultaneously
  • multiple ribosomes can translate the same mRNA (polysomes)

Eukaryotic translation:

Differences:

  • ribosomes are larger (80S = 60S + 40S)
  • initiating amino acid is methionine (not fMet)
  • no Shine–Dalgarno sequence
  • the start codon is identified as the first AUG near the 5′ end
  • eukaryotic mRNA is processed before translation
  • mRNA circularizes via 5′ cap–poly(A) interactions

Drugs and toxins affecting translation:

Examples:

  • streptomycin: inhibits initiation in bacteria
  • tetracycline: blocks aminoacyl tRNA binding
  • cycloheximide: inhibits eukaryotic elongation
  • puromycin: chain terminator in both systems
  • diphtheria toxin: blocks eukaryotic translocation

This lecture covered:

  • molecules involved in translation
  • features of the genetic code
  • ribosome structure
  • steps of translation
  • differences between bacterial and eukaryotic translation
  • drugs and toxins that block protein biosynthesis

If you have any questions, feel free to contact me via email or in Canvas.