Recombinant Legionella pneumophila Probable tRNA threonylcarbamoyladenosine biosynthesis protein Gcp (gcp)

Basic Research Questions

• What is the function of tRNA threonylcarbamoyladenosine biosynthesis protein Gcp in Legionella pneumophila?

  The Gcp protein (also known as TsaD) is a critical enzyme involved in the biosynthesis of threonylcarbamoyladenosine (t6A), a universal tRNA modification found in nearly all tRNAs that decode ANN codons . In L. pneumophila, this protein is part of a conserved pathway responsible for the modification of tRNAs, which is essential for accurate translation. The t6A modification occurs at position 37 adjacent to the anticodon of tRNAs, playing a crucial role in maintaining translational fidelity .

  The biosynthesis pathway includes several steps:

  • Formation of threonylcarbamoyl-adenylate (TC-AMP) intermediate

  • Transfer of the threonylcarbamoyl moiety to the tRNA

  • Completion of the t6A modification

• How is recombinant L. pneumophila Gcp typically expressed and purified for research?

  Recombinant L. pneumophila Gcp is typically expressed using heterologous expression systems. Based on available protocols :

  Expression systems:

  • Yeast expression systems

  • E. coli expression systems

  Purification process:

  1. Cell lysis under appropriate buffer conditions

  2. Initial purification using affinity chromatography (commonly His-tag based systems)

  3. Secondary purification steps may include ion exchange or gel filtration chromatography

  4. Quality control by SDS-PAGE (>85% purity is typically achievable)

  Storage conditions:

  • Lyophilized form can be stored at -20°C/-80°C for up to 12 months

  • Liquid form typically maintains stability for 6 months at -20°C/-80°C

  • Recommended to avoid repeated freeze-thaw cycles

• What are the implications of t6A modification absence in L. pneumophila?

  While the specific consequences of t6A absence in L. pneumophila haven't been fully characterized, studies in other bacteria provide valuable insights :

  • Translation efficiency: Absence of t6A modification can lead to misreading of codons and reduced translation fidelity

  • Virulence: May impact production of virulence factors required for pathogenesis

  • Stress response: In some bacteria like D. radiodurans, t6A deficiency induces proteotoxic stress responses

  • Bacterial survival: In many prokaryotes, t6A synthesis genes are essential, though this essentiality varies across bacterial species

  Understanding the specific role of t6A in L. pneumophila could provide insights into potential therapeutic targets, as the bacterium is the causative agent of Legionnaires' disease, a severe form of pneumonia .

Advanced Research Questions