title: An inhibitory C-terminal region dictates the specificity of A-adding enzymes.
abstract: For efficient aminoacylation, tRNAs carry the conserved 3'-terminal sequence C-C-A, which is synthesized by highly specific tRNA nucleotidyltransferases (CCA-adding enzymes). In several prokaryotes, this function is accomplished by separate enzymes for CC- and A-addition. As A-adding enzymes carry an N-terminal catalytic core identical to that of CCA-adding enzymes, it is unclear why their activity is restricted. Here, it is shown that C-terminal deletion variants of A-adding enzymes acquire full and precise CCA-incorporating activity. The deleted region seems to be responsible for tRNA primer selection, restricting the enzyme's specificity to tRNAs ending with CC. The data suggest that A-adding enzymes carry an intrinsic CCA-adding activity that can be reactivated by the introduction of deletions in the C-terminal domain. Furthermore, a unique subtype of CCA-adding enzymes could be identified that evolved out of A-adding enzymes, suggesting that mutations and deletions in nucleotidyltransferases can lead to altered and even more complex activities, as a simple A-incorporation is converted into sequence-specific addition of C and A residues. Such activity-modifying events may have had an important role in the evolution of tRNA nucleotidyltransferases.
authors.affiliation: Institute for Biochemistry, University of Leipzig, Brüderstrasse 34, 04103 Leipzig, Germany.
authors.forename: Sandy
authors.initials: S
authors.lastname: Tretbar
authors.forename: Anne
authors.initials: A
authors.lastname: Neuenfeldt
authors.forename: Heike
authors.initials: H
authors.lastname: Betat
authors.forename: Mario
authors.initials: M
authors.lastname: Mörl
mesh_terms: D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology; D003001:Cloning, Molecular; D016000:Cluster Analysis; D019295:Computational Biology; D017931:DNA Primers / Q000235:genetics; D034301:Deinococcus / Q000201:enzymology*; D019143:Evolution, Molecular; D008969:Molecular Sequence Data; D010802:Phylogeny; D012316:RNA Nucleotidyltransferases / Q000235:genetics* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics; D016415:Sequence Alignment; D013045:Species Specificity; D016963:Thermus thermophilus / Q000201:enzymology; D046249:Transfer RNA Aminoacylation / Q000235:genetics* / Q000502:physiology
chemical_list: D017931:DNA Primers; D011993:Recombinant Fusion Proteins; C440000:A adding enzyme; D012316:RNA Nucleotidyltransferases