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Related Experiment Videos

Poly(C) synthesis by class I and class II CCA-adding enzymes.

Malini Seth1, David L Thurlow, Ya-Ming Hou

  • 1Department of Biochemistry and Molecular Pharmacology, Thomas Jefferson University, 233 South 10th Street, Philadelphia, Pennsylvania 19107, USA.

Biochemistry
|April 3, 2002
PubMed
Summary

Both classes of CCA-adding enzymes synthesize poly(C) and CCA, revealing conserved nucleotide addition strategies despite sequence differences. This suggests a common mechanism for CCA specificity in tRNA 3' end synthesis.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Enzymology

Background:

  • CCA-adding enzymes (ATP(CTP):tRNA nucleotidyl transferases) add the conserved CCA sequence to tRNA 3' ends.
  • These enzymes are classified into two distinct families: Class I and Class II.
  • Class II enzymes, like the one from Escherichia coli, can synthesize poly(C) from CTP alone and CCA when ATP is present.

Purpose of the Study:

  • To investigate if Class I CCA-adding enzymes also exhibit poly(C) synthesis activity.
  • To compare the catalytic mechanisms and nucleotide addition strategies between Class I and Class II CCA-adding enzymes.
  • To understand the conserved mechanisms underlying CCA sequence specificity.

Main Methods:

  • Enzymatic assays using purified Class I enzymes from Sulfolobus shibatae and Methanococcus jannaschii.

Related Experiment Videos

  • Kinetic analysis of nucleotide incorporation in the presence and absence of ATP.
  • Site-directed mutagenesis to identify key residues involved in substrate discrimination.

    • Main Results:

    • Both archaeal Class I CCA-adding enzymes (S. shibatae and M. jannaschii) were shown to synthesize poly(C).
    • These Class I enzymes displayed kinetic responses to ATP similar to the Class II E. coli enzyme.
    • A mutation in the E. coli enzyme was identified that affects A76 addition but not poly(C) synthesis.
    • Enzymes from both classes demonstrated 3' to 5' exonucleolytic processing activity.

    Conclusions:

    • Despite significant sequence divergence, Class I and Class II CCA-adding enzymes share common nucleotide addition strategies.
    • Conserved mechanisms likely contribute to the specificity of CCA synthesis.
    • The same kinetic strategy is employed by the E. coli enzyme for discriminating poly(C) from CCA synthesis.