"Binding Induced RNA Conformational Changes Control Substrate Recognition and Catalysis by the Thiostrepton-resistance Methyltransferase (Tsr)."

Kuiper EG, Conn GL...



Published 2014-08-01 in J Biol Chem .

Pubmed ID: 25086036
DOI identifier: -

Abstract:
Ribosomal RNA (rRNA) posttranscriptional modifications are essential for ribosome maturation, translational fidelity and are one mechanism used by both antibiotic-producing and pathogenic bacteria to resist the effects of antibiotics that target the ribosome. The thiostrepton producer Streptomyces azureus prevents self-intoxication by expressing the thiostrepton-resistance methyltransferase (Tsr), which methylates the 2'-hydroxyl of 23S rRNA nucleotide adenosine 1067 within the thiostrepton binding site. Tsr is a homodimer with each protomer containing an L30e-like amino-terminal domain (NTD) and a SPOUT methyltransferase family catalytic carboxyl-terminal domain (CTD). We show that both domains of the enzyme are required for high affinity RNA substrate binding. The Tsr-CTD has intrinsic, weak RNA affinity that is necessary to direct the specific high-affinity Tsr-RNA interaction via the NTDs, which have no detectable RNA affinity in isolation. RNA structure probing experiments identify the Tsr footprint on the RNA and structural changes in the substrate, induced specifically upon NTD binding, that are necessary for catalysis by the CTD. Additionally, we identify a key amino acid in each domain responsible for CTD-RNA binding and the observed NTD-dependent RNA structural changes. These studies allow us to develop a model for Tsr-RNA interaction in which the coordinated substrate recognition of each Tsr structural domain is an obligatory pre-catalytic recognition event. Our findings underscore the complexity of substrate recognition by RNA modification enzymes and the potential for direct involvement of the RNA substrate in controlling the process of its modification.


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Last modification of this entry: Aug. 14, 2014