Highly conserved modified nucleosides influence Mg2+-dependent tRNA folding.

Area of Science:

• Molecular Biology
• Biochemistry
• Structural Biology

Background:

• Transfer RNA (tRNA) tertiary structure is crucial for its function.
• The TPsiC and D domains of tRNA interact through complex folding.
• The role of conserved nucleoside modifications in the TPsiC domain (rT54, Psi55, m5C49) in this folding remains unclear.

Purpose of the Study:

• To investigate the role of specific TPsiC domain nucleoside modifications in tRNA folding.
• To quantify the effect of these modifications on the association between tRNA T-half and D-half molecules.

Main Methods:

• Utilized a native polyacrylamide gel mobility shift assay to study the association of modified and unmodified yeast tRNA(Phe) T-half molecules with the D-half molecule.
• Mg2+ was employed as a necessary cofactor for complex formation.
• Lead cleavage patterns and nucleoside substitutions were used to assess global conformation.

Main Results:

• The presence of modified nucleosides (rT54, m5C49, Psi55) in the T-half molecule significantly decreased the dissociation constants (Kds) for binding to the D-half molecule compared to the unmodified T-half.
• Dissociation constants for modified T-halves ranged from 6 to 14 microM, while the unmodified T-half showed a Kd of 40 microM.
• Global folding patterns of both modified and unmodified complexes were comparable to native tRNA(Phe).

Conclusions:

• Conserved nucleoside modifications in the tRNA TPsiC domain enhance the affinity between the T-half and D-half molecules.
• These modifications stabilize the tRNA complex without altering its global conformation.
• The findings suggest an altered folding pathway or enhanced complex stability due to these modifications.