Lecture 18: Translation and Polarity

Lecture 18: Translation Termination and Polarity

Overview of Translation Termination

  • Concept: Translation termination is the final step of translation where it is critical to know when to stop.

  • Key Element: The termination signal is a stop codon.

    • Stop Codons: Three main stop codons are recognized:

    • UAG

    • UAA

    • UGA

  • Interaction with tRNA: Normally, no tRNAs can interact with these stop codons, except in rare cases involving nonstandard amino acids like cysteine or pyrolysin.

Release Factors

  • Definition: Release factors are proteins that resemble tRNA in structure and occupy the A site in the ribosome during termination.

  • Types of Release Factors:

    • Release Factor 1 (RF1): Recognizes UAG and UAA codons.

    • Release Factor 2 (RF2): Recognizes UGA and UAA codons.

  • Function:

    • The binding of release factors to the A site triggers the disassembly of the ribosomal complex, releasing the peptide chain and mRNA.

Frequency of Stop Codon Usage

  • Statistical Usage:

    • UAA: Used 64% of the time

    • UGA: Used 29% of the time

    • UAG: Used 7% of the time

Synthetic Biology Applications

  • Concept: Synthetic biology involves engineering biological systems to produce desired outcomes.

  • Example: Engineering of E. coli strains to accept nonnatural amino acids by converting UAG to another stop codon, thereby repurposing UAG as a coding codon.

    • Mechanism: The absence of RF1 (no UAG present) allows RF2 to recognize both UAA and UGA, enabling the use of UAG for coding nonstandard amino acids.

Stalled Ribosomes

  • Issue: Ribosomes can become stalled if they encounter a situation where they cannot reach a stop codon, wasting cellular energy.

  • Cause of Stalling:

    • Short half-lives of mRNA lead to endonuclease cleavage which separates the translation start information from the stop codon.

  • Resolution Mechanism: tRNA-mRNA (tmRNA) functions to rescue stalled ribosomes.

    • Definitions: tmRNA has properties of both tRNA and mRNA; it rescues stalled ribosomes and tags incomplete polypeptides for degradation.

    • Process:

    1. tmRNA binds to the open A site of a stalled ribosome.

    2. It provides a template for further translation until it reaches its own stop codon.

    3. The completed chain is tagged by the tmRNA before degradation by proteases, such as split protease.

Polarity and Its Implications

  • Definition: Polarity refers to how mutations in upstream genes can affect the expression of downstream genes, particularly in operons.

  • Operational Structure:

    • Operons consist of a promoter, multiple genes, and a transcriptional terminator.

    • A mutation in an upstream gene (like insertion by a transposon) can disrupt downstream transcription if terminator elements are introduced.

  • Mechanism Example: Factor-dependent transcription termination may lead to polarity effects if the ribosome translates upstream genes while transcription continues.

    • Case: A stop codon mutation in gene A stops translation, leading to reduced expression of gene B due to lack of ribosomal protection.

Translational Coupling

  • Concept: The translation of an upstream gene can significantly affect the translation of a downstream gene either inducing or inhibiting it.

  • Example Mechanism:

    1. Messenger RNA structure with ribosomal binding site obscured by a stem loop configuration disables translation of gene B initially.

    2. As the upstream gene (gene A) is translated, the ribosome disrupts the stem loop, allowing the downstream ribosomal binding site to be exposed, facilitating the translation of gene B.

    3. A nonsense mutation upstream would prevent the ribosome from reaching the stop codon of gene A, resulting in no disruption of the stem loop, thereby inhibiting expression of gene B.

Conclusion

  • Wrap-Up: The lecture concludes with a transition to discussions on protein processing and mutations. Students are encouraged to consolidate their knowledge of translation, termination, and the implications of genetic mutations in operons.