• Home
  • Search Results
  • Bacillus subtilis RNA deprotection enzyme RppH recognizes guanosine in the second position of its substrates.

Bacillus subtilis RNA deprotection enzyme RppH recognizes guanosine in the second position of its substrates.

Proceedings of the National Academy of Sciences of the United States of America (2013-04-24)
Jérémie Piton, Valéry Larue, Yann Thillier, Audrey Dorléans, Olivier Pellegrini, Inés Li de la Sierra-Gallay, Jean-Jacques Vasseur, Françoise Debart, Carine Tisné, Ciarán Condon
ABSTRACT

The initiation of mRNA degradation often requires deprotection of its 5' end. In eukaryotes, the 5'-methylguanosine (cap) structure is principally removed by the Nudix family decapping enzyme Dcp2, yielding a 5'-monophosphorylated RNA that is a substrate for 5' exoribonucleases. In bacteria, the 5'-triphosphate group of primary transcripts is also converted to a 5' monophosphate by a Nudix protein called RNA pyrophosphohydrolase (RppH), allowing access to both endo- and 5' exoribonucleases. Here we present the crystal structures of Bacillus subtilis RppH (BsRppH) bound to GTP and to a triphosphorylated dinucleotide RNA. In contrast to Bdellovibrio bacteriovorus RppH, which recognizes the first nucleotide of its RNA targets, the B. subtilis enzyme has a binding pocket that prefers guanosine residues in the second position of its substrates. The identification of sequence specificity for RppH in an internal position was a highly unexpected result. NMR chemical shift mapping in solution shows that at least three nucleotides are required for unambiguous binding of RNA. Biochemical assays of BsRppH on RNA substrates with single-base-mutation changes in the first four nucleotides confirm the importance of guanosine in position two for optimal enzyme activity. Our experiments highlight important structural and functional differences between BsRppH and the RNA deprotection enzymes of distantly related bacteria.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Guanosine, ≥98%
Sigma-Aldrich
Guanosine, BioReagent, suitable for cell culture
Sigma-Aldrich
Guanosine, ≥97.0% (HPLC)