Crystal structure of the human CCA-adding enzyme: insights into template-independent polymerization.
All tRNA molecules carry the invariant sequence CCA at their 3'-terminus for amino acid attachment. The post-transcriptional addition of CCA is carried out by ATP(CTP):tRNA nucleotidyltransferase, also called CCase. This enzyme catalyses a unique template-independent but sequence-specific nucleotide polymerization reaction. In order to reveal the molecular mechanism of this activity, we solved the crystal structure of human CCase by single isomorphous replacement. The structure reveals a four domain architecture with a cluster of conserved residues forming a positively charged cleft between the first two domains. Structural homology of the N-terminal CCase domain to other nucleotidyltransferases could be exploited for modeling a tRNA-substrate complex. The model places the tRNA 3'-end into the N-terminal nucleotidyltransferase site, close to a patch of conserved residues that provide the binding sites for CTP and ATP. Based on our results, we introduce a corkscrew model for CCA addition that includes a fixed active site and a traveling tRNA-binding region formed by flexible parts of the protein.
Augustin
Martin A
MA
Max-Planck-Institut für Biochemie, Abteilung Strukturforschung, Am Klopferspitz 18A, D-82152 Martinsried, Germany. augustin@biochem.mpg.de
Reichert
Andreas S
AS
Betat
Heike
H
Huber
Robert
R
Mörl
Mario
M
Steegborn
Clemens
C
D000595:Amino Acid Sequence; D020134:Catalytic Domain; D017124:Conserved Sequence; D018360:Crystallography, X-Ray; D019281:Dimerization; D006801:Humans; D066298:In Vitro Techniques; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D012316:RNA Nucleotidyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012323:RNA Processing, Post-Transcriptional; D012343:RNA, Transfer / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D055672:Static Electricity; D013379:Substrate Specificity
D011994:Recombinant Proteins; D012343:RNA, Transfer; D012316:RNA Nucleotidyltransferases; C023198:tRNA nucleotidyltransferase