The L7Ae protein binds to two kink-turns in the Pyrococcus furiosus RNase P RNA.

Stella M Lai,Venkat Gopalan,Mark P Foster,Lien B Lai

doi:10.1093/nar/gku994

Abstract

The RNA-binding protein L7Ae, known for its role in translation (as part of ribosomes) and RNA modification (as part of sn/oRNPs), has also been identified as a subunit of archaeal RNase P, a ribonucleoprotein complex that employs an RNA catalyst for the Mg2+-dependent 5′ maturation of tRNAs. To better understand the assembly and catalysis of archaeal RNase P, we used a site-specific hydroxyl radical-mediated footprinting strategy to pinpoint the binding sites of Pyrococcus furiosus (Pfu) L7Ae on its cognate RNase P RNA (RPR). L7Ae derivatives with single-Cys substitutions at residues in the predicted RNA-binding interface (K42C/C71V, R46C/C71V, V95C/C71V) were modified with an iron complex of EDTA-2-aminoethyl 2-pyridyl disulfide. Upon addition of hydrogen peroxide and ascorbate, these L7Ae-tethered nucleases were expected to cleave the RPR at nucleotides proximal to the EDTA-Fe–modified residues. Indeed, footprinting experiments with an enzyme assembled with the Pfu RPR and five protein cofactors (POP5, RPP21, RPP29, RPP30 and L7Ae–EDTA-Fe) revealed specific RNA cleavages, localizing the binding sites of L7Ae to the RPR's catalytic and specificity domains. These results support the presence of two kink-turns, the structural motifs recognized by L7Ae, in distinct functional domains of the RPR and suggest testable mechanisms by which L7Ae contributes to RNase P catalysis.

Highlights

The maturation of nascent tRNAs requires multiple processing and modification steps
While some minor thematic variations exist, the characteristic protein-RNA interface seen in these structures reveals positions in Pyrococcus furiosus (Pfu) L7Ae that, upon mutagenesis to Cys and modification with EPD-Fe, should reliably report on L7Ae binding sites in the Pfu RNase P RNA (RPR)
In the P12 and P16 regions of the Pfu RPR, there are two similar sets of L7Ae footprints that are consistent with this binding interface: K42C– and R46C–EDTA-Fe led to cleavages at and 3 to the putative GA bps while V95C–EDTA-Fe resulted in hits 5 to the GA bps, including the 3-nt bulge

Summary

Introduction

The maturation of nascent tRNAs requires multiple processing and modification steps. The Mg2+-dependent removal of the 5 leader of precursor tRNAs (pre-tRNAs) is catalyzed by RNase P, an enzyme remarkable for the diversity of its subunit composition [1,2,3]. A catalytic RNase P RNA (RPR) is aided by a single RNase P protein (RPP) in bacteria and by as many as 10 RPPs in eukaryotes [1,2,3]; archaeal RNase P is intermediate in complexity, with one RPR and up to five RPPs [1,2,3]. This compositional variability in RNase P provides a model system for understanding how the function of an evolutionarily conserved ribozyme might be fine-tuned by different suites of protein cofactors. To uncover the function of these protein subunits, archaeal RNase P has been used as an experimental proxy for the eukaryotic variant, which has not been reconstituted in vitro

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Conclusion