Structural basis for template-independent RNA polymerization.
The 3'-terminal CCA nucleotide sequence (positions 74-76) of transfer RNA is essential for amino acid attachment and interaction with the ribosome during protein synthesis. The CCA sequence is synthesized de novo and/or repaired by a template-independent RNA polymerase, 'CCA-adding enzyme', using CTP and ATP as substrates. Despite structural and biochemical studies, the mechanism by which the CCA-adding enzyme synthesizes the defined sequence without a nucleic acid template remains elusive. Here we present the crystal structure of Aquifex aeolicus CCA-adding enzyme, bound to a primer tRNA lacking the terminal adenosine and an incoming ATP analogue, at 2.8 A resolution. The enzyme enfolds the acceptor T helix of the tRNA molecule. In the catalytic pocket, C75 is adjacent to ATP, and their base moieties are stacked. The complementary pocket for recognizing C74-C75 of tRNA forms a 'protein template' for the penultimate two nucleotides, mimicking the nucleotide template used by template-dependent polymerases. These results are supported by systematic analyses of mutants. Our structure represents the 'pre-insertion' stage of selecting the incoming nucleotide and provides the structural basis for the mechanism underlying template-independent RNA polymerization.
Tomita
Kozo
K
Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology, 1-1-1, Higashi, Tsukuba-shi, Ibaragi 305-8666, Japan.
Fukai
Shuya
S
Ishitani
Ryuichiro
R
Ueda
Takuya
T
Takeuchi
Nono
N
Vassylyev
Dmitry G
DG
Nureki
Osamu
O
D000249:Adenosine Monophosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D001419:Bacteria / Q000201:enzymology* / Q000235:genetics; D001665:Binding Sites; D001704:Biopolymers / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D003460:Crystallization; D018360:Crystallography, X-Ray; D003570:Cytidine Triphosphate / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000378:metabolism; D001411:Geobacillus stearothermophilus / Q000201:enzymology; D008958:Models, Molecular; D011487:Protein Conformation; D012313:RNA / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D012316:RNA Nucleotidyltransferases / Q000737:chemistry* / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013698:Templates, Genetic
D001704:Biopolymers; C034477:RNA primers; D000249:Adenosine Monophosphate; D012313:RNA; D003570:Cytidine Triphosphate; D000255:Adenosine Triphosphate; D012316:RNA Nucleotidyltransferases; C023198:tRNA nucleotidyltransferase; D012321:DNA-Directed RNA Polymerases