Abstract
The CCA-adding enzyme ATP(CTP):tRNA nucleotidyltransferase builds and repairs the 3'-terminal CCA sequence of tRNA. Although this unusual RNA polymerase has no nucleic acid template, it can construct the CCA sequence one nucleotide at a time using CTP and ATP as substrates. We found previously that tRNA does not translocate along the enzyme during CCA addition (Yue, D., Weiner, A. M., and Maizels, N. (1998) J. Biol. Chem. 273, 29693-29700) and that a single nucleotidyltransferase motif adds all three nucleotides (Shi, P.-Y., Maizels, N., and Weiner, A. M. (1998) EMBO J. 17, 3197-3206). Intriguingly, the CCA-adding enzyme from the archaeon Sulfolobus shibatae is a homodimer that forms a tetramer upon binding two tRNAs. We therefore asked whether the active form of the S. shibatae enzyme might have two quasi-equivalent active sites, one adding CTP and the other ATP. Using an intersubunit complementation approach, we demonstrate that the dimer is active and that a single catalytically active subunit can carry out all three steps of CCA addition. We also locate one UV light-induced tRNA cross-link on the enzyme structure and provide evidence suggesting the location of another. Our data rule out shuttling models in which the 3'-end of the tRNA shuttles from one quasi-equivalent active site to another, demonstrate that tRNA-induced tetramerization is not required for CCA addition, and support a role for the tail domain of the enzyme in tRNA binding.
Highlights
Using an intersubunit complementation approach, we demonstrate that the dimer is active and that a single catalytically active subunit can carry out all three steps of CCA addition
Soaking the class II B. stearothermophilus apoprotein crystals in CTP or ATP yielded cocrystals exhibiting a single nucleotide binding site that could function as a protein template by recognizing both CTP and ATP; this protein template, consisting of aspartate (Asp-154) and arginine (Arg-157) residues contributed by the neck domain, appears to “base pair” with the Watson-Crick face of both nucleotides (20); the class I archaeal A. fulgidus enzyme apparently binds nucleotides nonspecifically, at least in the absence of tRNA substrates (18)
Model proposes that the growing 3Ј-end of the tRNA is progressively sequestered within a pocket near the active site; progressive packing of this pocket would create a binding site for the incoming nucleotide, and CCA synthesis would cease when the pocket was full (16, 28)
Summary
Using the wild type S. shibatae CCA-adding enzyme as well as three different mutants that block the addition of C74 (Y95V), the addition of A76 (H93V), or the addition of both CTP and ATP (K153A), we generated heterodimers by the reassortment of tagged and untagged subunits under partially denaturing conditions. To test whether a single wild type subunit in a heterodimer can add CCA, we took advantage of Ni-NTA HisSorb plates (Qiagen) to immobilize histidine-tagged dimers in the absence of tRNA, preventing tRNA-induced tetramer formation when tRNA substrate is added.
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