Is RsfS a Hibernation Factor or a Ribosome Biogenesis Factor?

Solving the structures of bacterial, archaeal, and eukaryotic ribosomes by crystallography and cryo-electron microscopy has given an impetus for studying intracellular regulatory proteins affecting various stages of protein translation. Among them are ribosome hibernation factors, which have been actively investigated during the last decade. These factors are involved in the regulation of protein biosynthesis under stressful conditions. The main role of hibernation factors is the reduction of energy consumption for protein biosynthesis and preservation of existing functional ribosomes from degradation, which increases cell survival under unfavorable conditions. Despite a broad interest in this topic, only a few articles have been published on the ribosomal silencing factor S (RsfS). According to the results of these studies, RsfS can be assigned to the group of hibernation factors. However, recent structural studies of the 50S ribosomal subunit maturation demonstrated that RsfS has the features inherent to biogenesis factors for example, ability to bind to the immature ribosomal subunit (similar to the RsfS mitochondrial ortholog MALSU1, mitochondrial assembly of ribosomal large subunit 1). In this review, we summarized the information on the function and structural features RsfS, as well as compared RsfS with MALSU1 in order to answer the emerging question on whether RsfS is a hibernation factor or a ribosome biogenesis factor. We believe that this review might promote future studies of the RsfS-involving molecular mechanisms, which so far remain completely unknown.

The key role of E418 carboxyl group in the formation of Nt.BspD6I nickase active site: Structural and functional properties of Nt.BspD6I E418A mutant.

The mutated nickase Nt.BspD6I E418A has been obtained by site-directed mutagenesis. The purified protein has been crystallized, and its spatial structure has been determined at 2.45 Å resolution. An analysis of the crystal structures of the wild-type and mutated nickase have shown that the elimination of a carboxyl group due to the E418A mutation initiates marked conformational changes in both the N-terminal recognition domain and the C-terminal catalytic domain of nickase and insignificantly affects its linker domain. This is supported by changes in the functional properties of mutated nickase: an increase in the oligomerization capacity in the presence of a substrate, a reduction in the capacity to bind a substrate, and complete loss of catalytic activity.

Dimerization of long hibernation promoting factor from Staphylococcus aureus: Structural analysis and biochemical characterization.

Staphylococcus aureus hibernation promoting factor (SaHPF) is responsible for the formation of 100S ribosome dimers, which in turn help this pathogen to reduce energy spent under unfavorable conditions. Ribosome dimer formation strongly depends on the dimerization of the C-terminal domain of SaHPF (CTDSaHPF). In this study, we solved the crystal structure of CTDSaHPF at 1.6 Šresolution and obtained a precise arrangement of the dimer interface. Residues Phe160, Val162, Thr171, Ile173, Tyr175, Ile185 andThr187 in the dimer interface of SaHPF protein were mutated and the effects were analyzed for the formation of 100S disomes of ribosomes isolated from S. aureus. It was shown that substitution of any of single residues Phe160, Val162, Ile173, Tyr175 and Ile185 in the SaHPF homodimer interface abolished the ribosome dimerization in vitro.

Structural analysis of cysteine-free Nt.BspD6 nicking endonuclease and its functional features.

Nicking endonuclease Nt.BspD6I (Nt.BspD6I) is the large subunit of the heterodimeric restriction endonuclease R.BspD6I. It recognizes the short specific DNA sequence 5´'- GAGTC and cleaves only the top strand in dsDNA at a distance of four nucleotides downstream the recognition site toward the 3´'-terminus. A mechanism of interaction of this protein with DNA is still unknown. Here we report the crystal structure of Cysteine-free Nt.BspD6I, with four cysteine residues (11, 160, 508, 578) substituted by serine, which was determined with a resolution of 1.93 Å. A comparative structural analysis showed that the substitution of cysteine residues induced marked conformational changes in the N-terminal recognition and the C-terminal cleavage domains. As a result of this changes were formed three new hydrogen bonds and the electrostatic field in these regions changed compared with wild type Nt.BspD6I. The substitution of cysteine residues did not alter the nicking function of Cysteine-free Nt.BspD6I but caused change in the activity of Cysteine-free heterodimeric restriction endonuclease R.BspD6I due to a change in the interaction between its large and small subunits. The results obtained contribute to the identification of factors influencing the interactions of subunits in the heterodimeric restriction enzyme R.BspD6I.

Backbone and side chain NMR assignments for the ribosome maturation factor P (RimP) from Staphylococcus aureus.

The ribosomal maturation factor (RimP) is a 17.7 kDa protein and is the assembly factor of the 30S subunit. RimP is essential for efficient processing of 16S rRNA and maturation (assembly) of the 30S ribosome. It was suggested that RimP takes part in stabilization of the central pseudoknot at the early stages of the 30S subunit maturation, and this process may occur before the head domain assembly and later stages of the 30S assembly, but the mechanism of this interaction is still not fully understood. Here we report the assignment of the 1H, 13C and 15N chemical shift in the backbone and side chains of RimP from Staphylococcus aureus. Analysis of chemical shifts of the main chain using TALOS + suggests that the RimP contains eight ß-strands and three α-helices with the topology α1-ß1-ß2-α2- ß3- α3- ß4- ß5- ß6- ß7- ß8. Structural studies of RimP and its complex with the ribosome by integrated structural biology approaches (NMR spectroscopy, X-ray diffraction analysis and cryoelectron microscopy) will allow further screening of highly selective inhibitors of the translation of S. aureus.