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.