Abstracts (poster) - Wissenschaft Online

Andrea Just, Falk Butter, Esther Lizano, Michelle Trenkmann, Tony Heitkam, Heike
Betat, Mario Mörl
The function of two conserved elements in the bacterial
Poly(A)Polymerase and CCA-adding enzyme
Bacterial Poly(A)polymerases (PAP) and CCA-adding enzymes are both members of the
polymerase β superfamily. Although catalyzing different reactions (PAP adds poly(A) tails
to RNA 3’-ends and CCA-adding enzyme synthesizes the CCA triplet at the tRNA 3’-end),
they show a high sequence similarity. Here, two conserved structural elements of both
enzymes from E. coli were investigated.
The first element – the highly conserved amino acid templating region (EDxxR-motif) –
selects in the CCA-adding enzyme the nucleotide to be incorporated. In PAP, however,
the function of this element is unclear. Therefore, individual amino acid exchanges were
introduced and the resulting proteins tested for activity. Our results indicate that the
EDxxR motif of PAP is essential for ATP specificity of its nucleotide binding pocket,
whereas the CCA-adding enzyme can tolerate mutations in this motif and has a “backup”
mechanism that enables the mutant enzyme to synthesize the CCA triplet.
In the second analyzed region – a flexible loop in the catalytic head domain of both
enzymes – amino acid replacements as well as reciprocal exchanges and deletions were
introduced and tested. The resulting enzyme variants show that this flexible region is an
essential element for the terminal A-addition catalyzed by the CCA-adding enzyme,
whereas it is dispensable in PAP.
Our results indicate how these regions in the enzymes contribute to an effective and
accurate catalysis. Furthermore, it seems that PAP, probably having a structure highly
similar to the CCA-adding enzyme, is fixed in a conformational state that restricts its
nucleotide specificity towards ATP.
contact:
Andrea Just
Universität Leipzig Fakultät für Biowissenschaften
Biochemie
justa@uni-leipzig.de
Brüderstr. 34
04103 Leipzig (Deutschland)