A model for C74 addition by CCA-adding enzymes: C74 addition, like C75 and A76 addition, does not involve tRNA translocation.
The CCA-adding enzyme adds CCA to the 3'-end of tRNA one nucleotide at a time, using CTP and ATP as substrates. We found previously that tRNA does not rotate or translocate on the enzyme during the addition of C75 and A76. We therefore predicted that the growing 3'-end of tRNA must, upon addition of each nucleotide, refold to reposition the new 3'-hydroxyl equivalently relative to the solitary nucleotidyltransferase motif. Cocrystal structures of the class I archaeal Archaeoglobus fulgidus enzyme, poised for addition of C75 and A76, confirmed this prediction. We have also demonstrated that an evolutionarily flexible beta-turn facilitates progressive refolding of the 3'-terminal C74 and C75 residues during C75 and A76 addition. Although useful cocrystals corresponding to C74 addition have not yet been obtained, we now show experimentally that tRNA does not rotate or translocate during C74 addition. We therefore propose, based on the existing A. fulgidus cocrystal structures, that the same flexible beta-turn functions as a wedge between the discriminator base (N73) and the terminal base pair of the acceptor stem, unstacking and repositioning N73 to attack the incoming CTP. Thus a single flexible beta-turn would orchestrate consecutive addition of all three nucleotides without significant movement of the tRNA on the enzyme surface.
Cho
Hyundae D
HD
Department of Biochemistry, School of Medicine, University of Washington, Seattle, Washington 98195-7350, USA.
Chen
Yu
Y
Varani
Gabriele
G
Weiner
Alan M
AM
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry* / Q000378:metabolism*; D019611:Archaeoglobus fulgidus / Q000235:genetics* / Q000502:physiology; D001483:Base Sequence; D003460:Crystallization; D003570:Cytidine Triphosphate / Q000378:metabolism; D019143:Evolution, Molecular; D008954:Models, Biological; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009711:Nucleotides; D011487:Protein Conformation; D012316:RNA Nucleotidyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012343:RNA, Transfer / Q000737:chemistry / Q000378:metabolism*
D019843:Archaeal Proteins; D009711:Nucleotides; D003570:Cytidine Triphosphate; D000255:Adenosine Triphosphate; D012343:RNA, Transfer; D012316:RNA Nucleotidyltransferases; C023198:tRNA nucleotidyltransferase