Structural basis for sulfur relay to RNA mediated by heterohexameric TusBCD complex

Area of Science:

• Biochemistry
• Structural Biology
• Molecular Biology

Background:

• Uridine at position 34 of specific tRNAs (Lys, Glu, Gln) is modified to 2-thiouridine (s2U).
• This modification is vital for accurate codon recognition and aminoacyl-tRNA synthetase interaction.
• The Escherichia coli tusABCDE genes are known to be involved in the s2U modification pathway.

Purpose of the Study:

• To elucidate the structural basis of the sulfur transfer mechanism in 2-thiouridine (s2U) modification.
• To identify the catalytic residues involved in sulfur transfer mediated by the TusBCD complex.

Main Methods:

• X-ray crystallography was used to determine the 2.15 angstroms crystal structure of the E. coli TusBCD complex.
• Structure-based sequence alignment was performed to identify potential active site residues.
• In vivo mutant analyses were conducted to validate the function of identified cysteine residues.

Main Results:

• The crystal structure revealed the TusBCD complex forms a heterohexamer, a dimer of a heterotrimer.
• Two putative active site cysteine residues, Cys79 (TusC) and Cys78 (TusD), were identified.
• In vivo studies confirmed that only Cys78 in the TusD subunit is essential for sulfur transfer in s2U modification.

Conclusions:

• The TusBCD complex acts as a sulfur transfer mediator in the s2U modification pathway.
• Cys78 of the TusD subunit is the catalytic residue responsible for sulfur transfer.
• A sulfur relay mechanism involving a persulfide intermediate on TusD is proposed.