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The CCA-adding enzyme has a single active site

D Yue¹, A M Weiner, N Maizels

¹Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, Connecticut 06520-8024, USA.

The Journal of Biological Chemistry

|October 29, 1998

Summary

The CCA-adding enzyme uses a single active site to add both C and A nucleotides to tRNA. Specificity is achieved through collaborative templating as the tRNA refolds during the addition process.

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Area of Science:

Biochemistry
Molecular Biology
Enzymology

Background:

The CCA-adding enzyme (tRNA nucleotidyltransferase) is crucial for tRNA synthesis and repair.
This enzyme belongs to the nucleotidyltransferase superfamily, sharing a conserved active-site motif across all domains of life.

Purpose of the Study:

To investigate the catalytic mechanism of the CCA-adding enzyme.
To determine if a single active site is responsible for adding both C and A nucleotides.
To elucidate the mechanism of nucleotide addition specificity.

Main Methods:

Site-directed mutagenesis of key residues (Asp-53, Asp-55, Asp-106, Glu-173, Asp-215) in the Sulfolobus shibatae CCA-adding enzyme.
Assessing the effect of mutations on the addition of C and A nucleotides to tRNA.

Main Results:

Mutations at Asp-53 or Asp-55 abolished the addition of both C and A, confirming a single active site.
Mutations at Asp-106 selectively impaired A addition, but not C addition.
Previous findings showed the tRNA acceptor stem remains fixed during catalysis.

Conclusions:

The CCA-adding enzyme utilizes a single active site for the sequential addition of C and A nucleotides.
Nucleotide addition specificity is governed by a collaborative templating mechanism involving progressive refolding of the tRNA 3'-end.
This mechanism ensures accurate CCA sequence synthesis essential for tRNA function.

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