Mercury(II) binding to s4U in E. coli tRNA(Val)

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• Transfer RNA (tRNA) plays a crucial role in protein synthesis.
• The 4-thiouridine (s(4)U) base is a modified nucleotide found in tRNA.
• Understanding the accessibility of modified bases in tRNA is vital for elucidating its function.

Purpose of the Study:

• To investigate the accessibility of the s(4)U base in native tRNA(Val) from E. coli.
• To determine the binding characteristics of various mercurial compounds to the s(4)U base.
• To explore the influence of tRNA tertiary structure on s(4)U accessibility.

Main Methods:

• Monitoring the binding of different mercurial compounds (e.g., HgBr2, HgCl2, CH3HgOAc, CH3HgCl, PCMB) to E. coli tRNA(Val).
• Utilizing equilibrium dialysis with 14C-labeled PCMB to quantify binding.
• Disrupting tRNA tertiary structure by removing Mg(2+) ions and heating to 40°C.

Main Results:

• The relative binding order of mercurials suggests steric factors influence binding to the sulfur atom of s(4)U.
• Para-chloromercuri-benzoate (PCMB) did not bind to native tRNA(Val) under physiological conditions.
• PCMB binding occurred only after disruption of tRNA tertiary structure, with one binding site and an association constant of 9 x 10^4 M^-1.

Conclusions:

• The accessibility of the s(4)U base in tRNA(Val) is significantly restricted by its tertiary structure.
• Mercurial binding studies provide insights into the structural constraints and accessibility of modified tRNA bases.
• These findings contribute to understanding tRNA structure-function relationships in E. coli.