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

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.

Figure 1.

Residues selected for Cys substitution in Pfu L7Ae. (A) Tertiary structure of Pfu L7Ae in complex with an RNA [PDB: 3NVI (60)]. The positions where single-Cys residues were introduced are highlighted; this color scheme is used throughout the paper. Inset shows a standard K-turn motif, with canonical (C) and non-canonical (NC) helices shown (28); the two sheared G•A bps as well as the 3-nt bulge are color-coded in the inset and the crystal structure. (B) Amino acid sequence of Pfu L7Ae with its secondary structural elements indicated. Mutation of the native Cys to Val is noted while asterisks mark sites of single-Cys substitution and subsequent EPD-Fe modification.

Figure 2.

Mapping OH^•-mediated cleavages of L7Ae–EDTA-Fe derivatives in the context of the 5-RPP enzyme on the P12 region of the Pfu RPR. RNA cleavage products were reverse transcribed using 5′-³²P-PfuRPR-2R. U, unmodified; M, EDTA-Fe–modified.

Figure 3.

L7Ae binding sites on the Pfu RPR with and without other RPPs (POP5•RPP30 and RPP21•RPP29). RNA cleavage products were reverse transcribed using 5′-³²P-PfuRPRj15/2-R. U, unmodified; M, EDTA-Fe–modified.

Figure 4.

(A) The OH^•-mediated cleavages of Pfu RPR promoted by Pfu L7Ae K42C–, R46C– and V95C–EDTA-Fe (in the absence or presence of other RPPs) are summarized on the secondary structure using filled triangles, squares and stars, respectively, and the same color scheme as in Figure 1. Overlapping K42C and R46C footprints are indicated using purple hexagons. Open triangles, hexagons and stars denote weak footprints in the P5, P6, L8 and P12 regions (see text for additional details). The catalytic (C) and substrate specificity (S) domains are also marked. The secondary structure of the Pfu RPR was modeled using as template a bacterial RPR whose high-resolution tertiary structure is available (Supplementary Figure S6). (B) Putative K-turns in the P12 and P16–17 regions of the RPR from Pfu (secondary structures shown) and its close relatives. In addition to our footprinting data, the assignment of K-turns in the Pfu RPR is supported by sequence alignment of P12 and P16 regions from six other euryarchaeal type A RPRs belonging to Thermococcales and Methanobacteriales. In the top panel, the sequence alignment also includes two RPRs from Methanococcales to show broader conservation of the P12 K-turn in type A and M RPRs. Mfo, Methanobacter formicicum; Msm, Methanobrevibacter smithii; Tce, Thermococcus celer; Tko, Thermococcus kodakaraensis.