The role of tightly bound ATP in Escherichia coli tRNA nucleotidyltransferase.
BACKGROUND
The CCA-adding enzyme [ATP(CTP): tRNA nucleotidyltransferase (EC. 2.7.7.25)] catalyses the addition of the conserved CCA sequence to the 3'-terminus of tRNAs. All CCA-adding enzymes are classified into the nucleotidyltransferase superfamily. In the absence of ATP, the Escherichia coli CCA-adding enzyme displays anomalous poly(C) polymerase activity.


RESULTS
We show that CCA-adding enzyme over-expressed in E. coli exists in an ATP-bound form. The affinities of ATP and CTP towards the enzyme were estimated by several methods, and the dissociation constants for ATP and CTP were determined to be 6.3 and 188 microM, respectively. AMP-incorporation terminated the nucleotidyltransferase reaction, while in the absence of ATP, the enzyme continued poly(C) polymerization. In the case of a tRNA substrate with a mutation in the T-loop region, normal CC was added at a much slower rate compared with the wild-type, but anomalous poly(C) polymerization occurred at the same rate as in the wild-type.


CONCLUSIONS
Based on the findings outlined above, we concluded that the E. coli CCA-adding enzyme possesses at least two distinct nucleotide binding sites, one responsible for ATP binding and the other(s) for CTP binding. The addition of ATP from the tight ATP binding site terminates nucleotide incorporation, thus limiting poly(C) polymerization to CCA. It is also suggested that during anomalous poly(C) polymerization, tRNA translocates from the tRNA binding site upon the third C addition.
Tomari
Y
Y
Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-8656, Japan.
Suzuki
T
T
Watanabe
K
K
Ueda
T
T
D000255:Adenosine Triphosphate / Q000378:metabolism*; D001665:Binding Sites; D002850:Chromatography, Gel; D003570:Cytidine Triphosphate / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology / Q000378:metabolism*; D008954:Models, Biological; D011066:Poly C / Q000378:metabolism; D011485:Protein Binding; D012316:RNA Nucleotidyltransferases / Q000378:metabolism*; D012343:RNA, Transfer / Q000378:metabolism*
D011066:Poly C; D003570:Cytidine Triphosphate; D000255:Adenosine Triphosphate; D012343:RNA, Transfer; D012316:RNA Nucleotidyltransferases; C023198:tRNA nucleotidyltransferase