Binding Induced RNA Conformational Changes Control Substrate Recognition and Catalysis by the Thiostrepton Resistance Methyltransferase (Tsr)

Emily G. Kuiper,Graeme L. Conn

doi:10.1074/jbc.m114.574780

Abstract

Ribosomal RNA (rRNA) post-transcriptional 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 23 S 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 enzyme domains 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 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, which 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.

Highlights

Methylation of Ribosomal RNA (rRNA) is a common resistance mechanism in antibiotic-producing bacteria
The Thiostrepton-resistance methyltransferase (Tsr) NTDs Aid in rRNA Binding and Are Necessary for Catalysis—We predicted that the Tsr NTD and carboxyl-terminal domain (CTD) domains might form stable and correctly folded proteins in isolation as homologous proteins are known, e.g. L30e and TrmL, and inspection of the Tsr NTD-CTD interface revealed it to be comprised primarily of polar and charged amino acids with almost no hydrophobic residues that would be exposed to solvent
To define the contribution of each Tsr domain to rRNA substrate recognition and catalysis, proteins corresponding to FL-Tsr (Fig. 1A), the NTD (Tsr-NTD; amino acids 1–101), and CTD (Tsr-CTD; amino acids 106 –269) were expressed, purified, and their binding and catalytic activities quantified

Summary

Background

Methylation of rRNA is a common resistance mechanism in antibiotic-producing bacteria. We demonstrate that specific RNA recognition by Tsr involves docking of its CTD on the A1067 target loop, followed by engagement of one or both NTDs in a process that drives specific RNA conformational changes at a site distant from the target loop This RNA structural change is an essential step for catalysis and may form part of a pre-catalytic recognition signal from the N-terminal RNA recognition domain. These studies reveal new mechanistic details of the intricate process of specific substrate recognition by Tsr and suggest a direct role for the RNA substrate in control of catalysis

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION