The elongation factor Tu.kirromycin complex has two binding sites for tRNA molecules.

Area of Science:

• Molecular Biology
• Biochemistry
• Protein-Nucleic Acid Interactions

Background:

• Elongation factor Tu (EF-Tu) is crucial for polypeptide chain synthesis.
• The antibiotic kirromycin modulates EF-Tu function.
• Understanding EF-Tu's interaction with tRNA and kirromycin is key to deciphering translation regulation.

Purpose of the Study:

• To investigate the effect of kirromycin on EF-Tu's interaction with tRNA.
• To identify conformational changes in EF-Tu induced by kirromycin and tRNA binding.
• To map the specific modification sites on EF-Tu.

Main Methods:

• Protein modification assays using N-tosyl-L-phenylalanine chloromethylketone (TPCK) and N-ethylmaleimide (NEM).
• Titration experiments with varying concentrations of aminoacyl-tRNA and uncharged tRNA.
• Peptide mapping of modified EF-Tu using [14C]TPCK labeling.

Main Results:

• Kirromycin protects EF-Tu from TPCK modification.
• Kirromycin induces an additional tRNA binding site on EF-Tu, capable of binding various tRNA forms.
• NEM modification confirms this additional binding site on EF-Tu.GTP.kirromycin.
• TPCK modification consistently targets cysteine-81 on EF-Tu, irrespective of kirromycin or tRNA presence.

Conclusions:

• Kirromycin binding to EF-Tu.GDP creates a novel tRNA binding site.
• This kirromycin-induced site accommodates different tRNA species, influencing EF-Tu's conformation.
• Cysteine-81 is the primary TPCK modification site, unaffected by kirromycin or tRNA binding.