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The three-dimensional structure of recombinant nicking endonuclease S11C Cys-free Nt.BspD6I was determined at 1.85 Å resolution. Nickase S11C Cys-free Nt.BspD6I was produced by the reversion back to Cys11 in Cys-free Nt.BspD6I using site-directed mutagenesis. An analysis of the crystal structure of nickase S11C Cys-free Nt.BspD6I demonstrated that the reversion back to Cys11 induces significant conformational changes in the recognition domain of nickase, which are accompanied by changes in its functions, such as a decrease in the affinity to DNA, the loss of the ability to undergo oligomerization, and high activity of restriction endonuclease S11C Cys-free R.BspD6I. [ABSTRACT FROM AUTHOR]

Variation in the composition of different ribosomes, termed ribosome heterogeneity, is a now well established phenomenon. However, the functional implications of this heterogeneity on the regulation of protein synthesis are only now beginning to be revealed. While there are numerous examples of heterogeneous ribosomes, there are comparatively few bona fide specialized ribosomes described. Specialization requires that compositionally distinct ribosomes, through their subtly altered structure, have a functional consequence to the translational output. Even for those examples of ribosome specialization that have been characterized, the precise mechanistic details of how changes in protein and rRNA composition enable the ribosome to regulate translation are still missing. Here, we suggest looking at the evolution of specialization across the tree of life may help reveal central principles of translation regulation. We consider functional and structural studies that have provided insight into the potential mechanisms through which ribosome heterogeneity could affect translation, including through mRNA and open reading frame selectivity, elongation dynamics and post-translational folding. Further, we highlight some of the challenges that must be addressed to show specialization and review the contribution of various models. Several studies are discussed, including recent studies that show how structural insight is starting to shed light on the molecular details of specialization. Finally, we discuss the future of ribosome specialization studies, where advances in technology will likely enable the next wave of research questions. Recent work has helped provide a more comprehensive understanding of how ribosome heterogeneity affects translational control.This article is part of the discussion meeting issue 'Ribosome diversity and its impact on protein synthesis, development and disease'.

The YsxC protein from Staphylococcus aureus is a GTP-binding protein from the TRAFAC superfamily of the TrmE-Era-EngA-EngB-Septin-like GTPase class, EngB family of GTPases. Recent structural and biochemical studies of YsxC function show that it is an integral part of the pathogenic microorganism life cycle, as it is involved in the assembly of the large 50S ribosomal subunit. Structural studies of this protein with its specific functional features make it an attractive target for further development of new selective antimicrobials. In this study, we cloned the ysxC protein gene from S. aureus, overexpressed the protein in E. coli, and subsequently purified and crystallized it. Protein crystals were successfully grown using the vapor diffusion method, yielding diffraction data with a resolution of up to 2 Å. Comparative analysis of the structure of SaYsxC with known three-dimensional structures of homologs from other microorganisms showed the presence of structural differences for the apo form., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Konstantin Usachev reports financial support was provided by Russian Science Foundation. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Ribosome biogenesis is an energy-intense multistep process where even minimal defects can cause severe phenotypes up to cell death. Ribosome assembly is facilitated by biogenesis factors such as ribosome assembly factors. These proteins facilitate the interaction of ribosomal proteins with rRNA and correct rRNA folding. One of these maturation factors is RimP which is required for efficient 16S rRNA processing and 30S ribosomal subunit assembly. Here, we describe the binding mode of Staphylococcus aureus RimP to the small ribosomal subunit and present a 4.2 Å resolution cryo-EM reconstruction of the 30S-RimP complex. Together with the solution structure of RimP solved by NMR spectroscopy and RimP-uS12 complex analysis by EPR, DEER, and SAXS approaches, we show the specificity of RimP binding to the 30S subunit from S. aureus. We believe the results presented in this work will contribute to the understanding of the RimP role in the ribosome assembly mechanism., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Ribosome biogenesis is a complex and highly accurate conservative process of ribosomal subunit maturation followed by association. Subunit maturation comprises sequential stages of ribosomal RNA and proteins' folding, modification and binding, with the involvement of numerous RNAses, helicases, GTPases, chaperones, RNA, protein-modifying enzymes, and assembly factors. One such assembly factor involved in bacterial 30S subunit maturation is ribosomal binding factor A (RbfA). In this study, we present the crystal (determined at 2.2 Å resolution) and NMR structures of RbfA as well as the 2.9 Å resolution cryo-EM reconstruction of the 30S-RbfA complex from Staphylococcus aureus ( S. aureus ). Additionally, we show that the manner of RbfA action on the small ribosomal subunit during its maturation is shared between bacteria and mitochondria. The obtained results clarify the function of RbfA in the 30S maturation process and its role in ribosome functioning in general. Furthermore, given that S. aureus is a serious human pathogen, this study provides an additional prospect to develop antimicrobials targeting bacterial pathogens.

Ribosomal silencing factor S (RsfS) is a conserved protein that plays a role in the mechanisms of ribosome shutdown and cell survival during starvation. Recent studies demonstrated the involvement of RsfS in the biogenesis of the large ribosomal subunit. RsfS binds to the uL14 ribosomal protein on the large ribosomal subunit and prevents its association with the small subunit. Here, we estimated the contribution of RsfS amino acid side chains at the interface between RsfS and uL14 to RsfS anti-association function in Staphylococcus aureus through in vitro experiments: centrifugation in sucrose gradient profiles and an S. aureus cell-free system assay. The detected critical Y98 amino acid on the RsfS surface might become a new potential target for pharmacological drug development and treatment of S. aureus infections.

Staphylococcus aureus is a bacterial pathogen and one of the leading causes of healthcare-acquired infections in the world. The growing antibiotic resistance of S. aureus obliges us to search for new drugs and treatments. As the majority of antibiotics target the ribosome, knowledge of its detailed structure is crucial for drug development. Here, we report the cryo-EM reconstruction at 3.2 Å resolution of the S. aureus ribosome with P-site tRNA, messenger RNA, and 10 RNA modification sites previously not assigned or visualized. The resulting model is the most precise and complete high-resolution structure to date of the S. aureus 70S ribosome with functional ligands., (© 2020 Federation of European Biochemical Societies.)

Elongation factor P (EF-P) is a translation protein factor that plays an important role in specialized translation of consecutive proline amino acid motifs. EF-P is an essential protein for cell fitness in native environmental conditions. It regulates synthesis of proteins involved in bacterial motility, environmental adaptation and bacterial virulence, thus making EF-P a potential drug target. In the present study, we determined the solution and crystal structure of EF-P from the pathogenic bacteria Staphylococcus aureus at 1.48 Å resolution. The structure can serve as a platform for structure-based drug design of novel antibiotics to combat the growing antibiotic resistance of S. aureus.

For the sake of energy preservation, bacteria, upon transition to stationary phase, tone down their protein synthesis. This process is favored by the reversible binding of small stress-induced proteins to the ribosome to prevent unnecessary translation. One example is the conserved bacterial ribosome silencing factor (RsfS) that binds to uL14 protein onto the large ribosomal subunit and prevents its association with the small subunit. Here we describe the binding mode of Staphylococcus aureus RsfS to the large ribosomal subunit and present a 3.2 Å resolution cryo-EM reconstruction of the 50S-RsfS complex together with the crystal structure of uL14-RsfS complex solved at 2.3 Å resolution. The understanding of the detailed landscape of RsfS-uL14 interactions within the ribosome shed light on the mechanism of ribosome shutdown in the human pathogen S. aureus and might deliver a novel target for pharmacological drug development and treatment of bacterial infections.