PMC

NMR spectra of Mth Rpp29. (a) ¹⁵N HSQC spectrum with backbone amide assignments indicated. (b) Heteronuclear {¹H}-¹⁵N NOE data of Mth Rpp29 indicates the N- and C-terminal residues are highly flexible. The trypsin-resistant core consists of residues 6–85.

In vitro reconstitution of Mth RNase P activity. (a) Sequence and phylogenetically predicted secondary structure of the Mth RNase P RNA subunit (). (b) Reconstitution experiment shows Mth Rpp29 is essential for effective Mth RNase P activity against E. coli ptRNA^Tyr. The negative control is the substrate alone; positive control is the substrate incubated with E. coli RNase P; Pfu Pop5 was used in place of Mth Pop5.

Sequence alignment of Mth Rpp29 with Rpp29 sequences from Archaea and Eukarya. Alignment was generated with clustalw () and colored according to similarity by using a Risler scoring matrix (); shaded residues indicate identity, and boxed residues indicate a global similarity score >0.7. Secondary structural features observed in the structure ensemble are indicated. Arrows indicate the core of the protein protected from trypsin digestion as identified by electrospray-MS (9,114 Da expected; 9,115 Da observed).

Mth Rpp29 solution structure. (a) Ensemble of 20 low-energy structures of Mth Rpp29 superposed on the backbone heavy atoms of residues 12–82; for clarity, only the main-chain atoms (N, C^α, and C′) of the structured core residues are shown. Strands are blue, turns and coil regions are gray, and the C-terminal helix is red. (b) Close-up showing a potential salt bridge between the conserved Lys-56 and Glu-37 residues; hydrophobic residues in the core are green. (c) Surface representation illustrates several potential surface salt bridges. The side chains of Lys and Arg residues are blue, Asp and Glu are red, and hydrophobic residues are green. Images were rendered with molmol ().

Identification of protein–RNA contacts by ¹H-¹⁵N correlated NMR of the 113-kDa Mth Rpp29 –RNA complex. (a) Overlay of a portion of the HSQC spectra of free Mth Rpp29 (black) and the 1:1 complex between the 11-kDa (93 residues) protein and 102-kDa (314 nt) Mth P RNA (800 MHz, 50°C, 50 mM phosphate, 400 mM KCl). (b) Weighted average amide proton and nitrogen weighted-average shift perturbations () are mapped onto the ribbon diagram of the Rpp29 OB-fold by using a linear color ramp from gray (no change) to red [Δ_av(NH) = 0.15]. Residues in green are those for which the effect of RNA binding could not be assessed.