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Unusual synthesis by the Escherichia coli CCA-adding enzyme

Published online by Cambridge University Press:  01 July 2000

YA-MING HOU
Affiliation:
Department of Biochemistry and Molecular Pharmacology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA
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Abstract

The tRNA 3′ end contains the conserved CCA sequence at the 74–76 positions. The CCA sequence is synthesized and maintained by the CCA-adding enzymes. The specificity of the Escherichia coli enzyme at each of the 74–76 positions was investigated using synthetic minihelix substrates that contain permuted 3′ ends. Results here indicate that the enzyme has the ability to synthesize unusual 3′ ends. When incubated with CTP alone, the enzyme catalyzed the addition of C74, C75, C76, and multiple Cs. Although the addition of C74 and C75 was as expected, that of C76 and multiple Cs was not. In particular, the addition of C76 generated CCC, which would have conflicted with the biological role of the enzyme. However, the presence of ATP prevented the synthesis of CCC and completely switched the specificity to CCA. The presence of ATP also had an inhibitory effect on the synthesis of multiple Cs. Thus, the E. coli CCA enzyme can be a poly(C) polymerase but its synthesis of poly(C) is regulated by the presence of ATP. These features led to a model of CCA synthesis that is independent of a nucleic acid template. The synthesis of poly(C) by the CCA-adding enzyme is reminiscent of that of poly(A) by poly(A) polymerase and it provides a functional rationale for the close sequence relationship between these two enzymes in the family of nucleotidyltransferases.

Type
Research Article
Copyright
2000 RNA Society

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