RNase P is the essential activity removing 5′-leader sequences from transfer RNA precursors. RNase P was always associated with ribonucleoprotein complexes before the discovery of protein-only RNase P enzymes called PRORPs (PROteinaceous RNase P) in eukaryotes. Here we provide biophysical and functional data to understand the mode of action of PRORP enzymes. Activity assays and footprinting experiments show that the anticodon domain of transfer RNA is dispensable, whereas individual residues in D and TψC loops are essential for PRORP function. PRORP proteins are characterized in solution and a molecular envelope is derived from small-angle X-ray scattering. Conserved residues are shown to be involved in the binding of one zinc atom to PRORP. These results facilitate the elaboration of a model of the PRORP/transfer RNA interaction. The comparison with the ribonucleoprotein RNase P/transfer RNA complex suggests that transfer RNA recognition by PRORP proteins is similar to that by ribonucleoprotein RNase P.

The non-coding RNA 7SK is the scaffold for a small nuclear ribonucleoprotein (7SKsnRNP) which regulates the function of the positive transcription elongation factor P-TEFb in the control of RNA polymerase II elongation in metazoans. The La-related protein LARP7 is a component of the 7SKsnRNP required for stability and function of the RNA. To address the function of LARP7 we determined the crystal structure of its La module, which binds a stretch of uridines at the 3′-end of 7SK. The structure shows that the penultimate uridine is tethered by the two domains, the La-motif and the RNA-recognition motif (RRM1), and reveals that the RRM1 is significantly smaller and more exposed than in the La protein. Sequence analysis suggests that this impacts interaction with 7SK. Binding assays, footprinting and small-angle scattering experiments show that a second RRM domain located at the C-terminus binds the apical loop of the 3′ hairpin of 7SK, while the N-terminal domains bind at its foot. Our results suggest that LARP7 uses both its N- and C-terminal domains to stabilize 7SK in a closed structure, which forms by joining conserved sequences at the 5′-end with the foot of the 3′ hairpin and has thus functional implications.

Translation initiation factor 2 (IF2) promotes 30S initiation complex (IC) formation and 50S subunit joining, which produces the 70S IC. The architecture of full-length IF2, determined by small angle X-ray diffraction and cryo electron microscopy, reveals a more extended conformation of IF2 in solution and on the ribosome than in the crystal. The N-terminal domain is only partially visible in the 30S IC, but in the 70S IC, it stabilizes interactions between IF2 and the L7/ L12 stalk of the 50S, and on its deletion, proper N-formyl-methionyl (fMet)-tRNA fMet positioning and efficient transpeptidation are affected. Accordingly, fast kinetics and single-molecule fluorescence data indicate that the N terminus promotes 70S IC formation by stabilizing the productive sampling of the 50S subunit during 30S IC joining. Together, our data highlight the dynamics of IF2-dependent ribosomal subunit joining and the role played by the N terminus of IF2 in this process.protein synthesis | integrated structural biology I n bacteria, three translation initiation factors (IF1, IF2, and IF3) participate in the first phase of protein synthesis during which the 30S subunit binds the initiator tRNA to form codonanticodon interactions with the mRNA. The 30S initiation complex (IC) thus formed is converted into a 70S IC by joining of the 50S ribosomal subunit (1, 2). The IFs enhance the rate of ICs formation and ensure translation accuracy. Within this process, IF2 plays a key role by promoting the recruitment and stabilization of the N-formyl-methionyl(fMet)-tRNA fMet on the 30S IC in the peptidyl/initiator (P/I) site of the small ribosomal subunit with the help of initiation factors IF1 and IF3 (3-6). IF2 mediates subunit joining on which its GTPase activity is stimulated, leading to the formation of a 70S IC in a conformation that is productive in the first transpeptidation reaction (7). Snapshots of this process, visualized by cryo electron microscopy (cryo-EM) (3,(8)(9)(10), have localized IF2 on the ribosome and provided structural insights into 30S and 70S IC's.Bacterial IF2 is a multidomain protein (Figs. S1 and S2) for which the overall architecture is unknown. Unlike 70S-interacting elongation GTPases, IF2 contains a specific N-terminal (N) domain that is variable in sequence (in Escherichia coli, two copies of it comprise the bona fide N domain and the G1 domain). Furthermore, it contains a highly conserved C-terminal region with a central core comprising domains G [GTP/GDP (G) binding domain, G2] and G3 (II), and a C-terminal part consisting of domain C1 and of the initiator tRNA-binding domain C2. Recently, the crystal structure of the core part (1-363) of Thermus thermophilus IF2 was determined in different functional states (apo-protein, GTP, and GDP forms) (11). These structures gave important insights into the mechanism of nucleotide binding and GTP hydrolysis and also reveal significant structural differences with a related archaeal and eukaryotic protein a/eIF5B (11). However, because the entire C-terminal region was ...