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Nucleic Acids Res. 2014 Dec 1;42(21):13328-38. doi: 10.1093/nar/gku994. Epub 2014 Oct 31.

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

Author information

1
Department of Chemistry & Biochemistry, Center for RNA Biology, The Ohio State University, Columbus, OH 43210, USA.
2
Department of Chemistry & Biochemistry, Center for RNA Biology, The Ohio State University, Columbus, OH 43210, USA gopalan.5@osu.edu.

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 Mg(2+)-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.

PMID:
25361963
PMCID:
PMC4245976
DOI:
10.1093/nar/gku994
[Indexed for MEDLINE]
Free PMC Article

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