Exchange of regions between bacterial poly(A) polymerase and the CCA-adding enzyme generates altered specificities.
Bacterial poly(A) polymerases (PAP) and tRNA nucleotidyltransferases are highly similar in sequence but display different activities: whereas tRNA nucleotidyltransferase catalyzes the addition of CCA to 3' ends of tRNAs, PAP adds poly(A) tails to a variety of transcripts. Using domain substitution experiments, we show that these enzymes follow a modular concept: exchange of N- and C-terminal regions leads to chimeric enzymes with unexpected activities, indicating that tRNA nucleotidyltransferase carries an "anchor domain" in the C-terminal section that restricts polymerization to three nucleotides. A 27 amino acid region was identified that determines whether poly(A) or CCA is synthesized by the enzyme chimeras. Sequence alignments suggest that the catalytic cores of both enzymes carry identical components involved in nucleotide recognition and incorporation. This seems to be the prerequisite for the observed reprogramming of the catalytic center of PAP to incorporate a sequence of defined length and composition instead of long stretches of A residues.
Max-Planck-Institute for Evolutionary Anthropology, Deutscher Platz 6, D-04103 Leipzig, Germany.
Heike
H
Betat
Christiane
C
Rammelt
Georges
G
Martin
Mario
M
Mörl
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D020134:Catalytic Domain; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology / Q000378:metabolism; D019143:Evolution, Molecular; D007700:Kinetics; D008969:Molecular Sequence Data; D011062:Polynucleotide Adenylyltransferase / Q000737:chemistry* / Q000378:metabolism; D012316:RNA Nucleotidyltransferases / Q000737:chemistry* / Q000378:metabolism
D001426:Bacterial Proteins; D012316:RNA Nucleotidyltransferases; C023198:tRNA nucleotidyltransferase; D011062:Polynucleotide Adenylyltransferase