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J. Biol. Chem., Vol. 280, Issue 10, 9555-9566, March 11, 2005

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Archaeal CCA-adding Enzymes

CENTRAL ROLE OF A HIGHLY CONSERVED -TURN MOTIF IN RNA POLYMERIZATION WITHOUT TRANSLOCATION^*

HyunDae D. Cho, Christophe L. Verlinde, and Alan M. Weiner

From the Department of Biochemistry, School of Medicine, University of Washington, Seattle, Washington 98195-7350

The CCA-adding enzyme (tRNA nucleotidyltransferase) builds and repairs the 3' end of tRNA. A single active site adds both CTP and ATP, but the enzyme has no nucleic acid template, and tRNA does not translocate or rotate during C75 and A76 addition. We modeled the structure of the class I archaeal Sulfolobus shibatae CCA-adding enzyme on eukaryotic poly(A) polymerase and mutated residues in the vicinity of the active site. We found mutations that specifically affected C74, C75, or A76 addition, as well as mutations that progressively impaired addition of CCA. Many of these mutations clustered in an evolutionarily versatile -turn located between strands 3 and 4 of the nucleotidyltransferase domain. Our mutational analysis confirms and extends recent crystallographic studies of the highly homologous Archaeoglobus fulgidus enzyme. We suggest that the unusual phenotypes of the -turn mutants reflect the consecutive conformations assumed by the -turn as it presents the discriminator base N73, then C74, and finally C75 to the active site without translocation or rotation of the tRNA acceptor stem. We also suggest that -turn mutants can affect nucleotide selection because the growing 3' end of tRNA must be properly positioned to serve as part of the ribonucleoprotein template that selects the incoming nucleotide.

Received for publication, November 8, 2004

^* This work was supported by National Institutes of Health Grant GM59804 (to A. M. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains additional text and Figs. S1-S5.

To whom correspondence should be addressed. Tel.: 206-543-1768; Fax: 206-685-9231; E-mail: amweiner{at}u.washington.edu.

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This article has been cited by other articles:

				H. D. Cho, V. D. Sood, D. Baker, and A. M. Weiner On the role of a conserved, potentially helix-breaking residue in the tRNA-binding {alpha}-helix of archaeal CCA-adding enzymes RNA, July 1, 2008; 14(7): 1284 - 1289. [Abstract] [Full Text] [PDF]

				H. D. Cho, C. L. M. J. Verlinde, and A. M. Weiner Reengineering CCA-adding enzymes to function as (U,G)- or dCdCdA-adding enzymes or poly(C,A) and poly(U,G) polymerases PNAS, January 2, 2007; 104(1): 54 - 59. [Abstract] [Full Text] [PDF]

				H. D. Cho, Y. Chen, G. Varani, and A. M. Weiner A Model for C74 Addition by CCA-adding Enzymes: C74 ADDITION, LIKE C75 AND A76 ADDITION, DOES NOT INVOLVE tRNA TRANSLOCATION J. Biol. Chem., April 7, 2006; 281(14): 9801 - 9811. [Abstract] [Full Text] [PDF]