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.

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.

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.

Solution structure of the N-terminal domain of the Staphylococcus aureus hibernation promoting factor.

Staphylococcus aureus hibernation promoting factor (SaHPF) is a 22,2 kDa protein which plays a crucial role in 100S Staphylococcus aureus ribosome formation during stress. SaHPF consists of N-terminal domain (NTD) that prevents proteins synthesis by binding to the 30S subunit at the P- and A-sites, connected through a flexible linker with a C-terminal domain (CTD) that keeps ribosomes in 100S form via homodimerization. Recently obtained 100S ribosome structure of S. aureus by cryo-EM shown that SaHPF-NTD bound to the ribosome active sites, however due to the absence of SaHPF-NTD structure it was modeled by homology with the E. coli hibernation factors HPF and YfiA. In present paper we have determined the solution structure of SaHPF-NTD by high-resolution NMR spectroscopy which allows us to increase structural knowledge about HPF structure from S. aureus.

Backbone and side chain NMR assignments for the ribosome binding factor A (RbfA) from Staphylococcus aureus.

Ribosome binding factor A (RbfA) is a 14.9 kDa adaptive protein of cold shock, which is important for bacterial growth at low temperatures. RbfA can bind to the free 30S ribosomal subunit and interacts with the 5'-terminal helix (helix I) of 16S rRNA. RbfA is important for the efficient processing of 16S rRNA and for the maturation (assembly) of 30S ribosomal subunits. Here we report backbone and side chains 1H, 13C and 15N chemical shift assignments of RbfA from Staphylococcus aureus. Analysis of the backbone chemical shifts by TALOS+ suggests that RbfA contains four α-helixes and three ß-strands with α1-ß1-ß2-α2-α3-ß3-α4 topology. Secondary structure of RbfA have KH-domain fold topology with ßααß subunit which is characterized by a helix-kink-helix motif in which the GxxG sequence is replaced by a conserved AxG sequence, where an Ala residue at position 70 forming an interhelical kink. The solution of the structure of this protein factor and its complex with the ribosome by NMR spectroscopy, X-ray diffraction analysis and cryo-electron microscopy will allow further development of highly selective substances for slowing or completely stopping the translation of the pathogenic bacterium S. aureus, which will interfere with the synthesis and isolation of its pathogenicity factors.

Backbone and side chain NMR assignments for the ribosome Elongation Factor P (EF-P) from Staphylococcus aureus.

Elongation Factor P (EF-P) is a 20.5 kDa protein that provides specialized translation of special stalling amino acid motifs. Proteins with stalling motifs are often involved in various processes, including stress resistance and virulence. Thus it has been shown that the virulent properties of microorganisms can be significantly reduced if the work of EF-P is disrupted. In order to elucidate the structure, dynamics and function of EF-P from Staphylococcus aureus (S. aureus), here we report backbone and side chains 1H, 13C and 15N chemical shift assignments of EF-P. Analysis of the backbone chemical shifts by TALOS+ suggests that EF-P contains 1 α-helix and 13 ß-strands (ß1-ß2-ß3-ß4-ß5-ß6-ß7-α1-ß8-ß9-ß10-ß11-ß12-ß13). The solution of the structure of this protein by NMR and X-ray diffraction analysis, as well as the structure of the ribosome complex by cryo-electron microscopy, will allow further screening of highly selective inhibitors of the translation of the pathogenic bacterium S. aureus. Here we report the almost complete 1H, 13C, 15N backbone and side chain NMR assignment of a 20.5 kDa EF-P.

NMR assignments of the N-terminal domain of Staphylococcus aureus hibernation promoting factor (SaHPF).

Staphylococcus aureus: hibernation-promoting factor (SaHPF) is a 22.2 kDa stationary-phase protein that binds to the ribosome and turns it to the inactive form favoring survival under stress. Sequence analysis has shown that this protein is combination of two homolog proteins obtained in Escherichia coli-ribosome hibernation promoting factor (HPF) (11,000 Da) and ribosome modulation factor RMF (6500 Da). Binding site of E. coli HPF on the ribosome have been shown by X-ray study of Thermus thermophilus ribosome complex. Hence, recent studies reported that the interface is markedly different between 100S from S. aureus and E. coli. Cryo-electron microscopy structure of 100S S. aureus ribosomes reveal that the SaHPF-NTD binds to the 30S subunit as observed for shorter variants of HPF in other species and the C-terminal domain (CTD) protrudes out of each ribosome in order to mediate dimerization. SaHPF-NTD binds to the small subunit similarly to its homologs EcHPF, EcYfiA, and a plastid-specific YfiA. Furthermore, upon binding to the small subunit, the SaHPF-NTD occludes several antibiotic binding sites at the A site (hygromycin B, tetracycline), P site (edeine) and E site (pactamycin, kasugamycin). In order to elucidate the structure, dynamics and function of SaHPF-NTD from S. aureus, here we report the backbone and side chain resonance assignments for SaHPF-NTD. Analysis of the backbone chemical shifts by TALOS+ suggests that SaHPF-NTD contains two α-helices and four ß-strands (ß1-α1-ß2-ß3-ß4-α2 topology). Investigating the long-term survival of S. aureus and other bacteria under antibiotic pressure could lead to advances in antibiotherapy.

Amicoumacin A induces cancer cell death by targeting the eukaryotic ribosome.

Amicoumacin A is an antibiotic that was recently shown to target bacterial ribosomes. It affects translocation and provides an additional contact interface between the ribosomal RNA and mRNA. The binding site of amicoumacin A is formed by universally conserved nucleotides of rRNA. In this work, we showed that amicoumacin A inhibits translation in yeast and mammalian systems by affecting translation elongation. We determined the structure of the amicoumacin A complex with yeast ribosomes at a resolution of 3.1 Å. Toxicity measurement demonstrated that human cancer cell lines are more susceptible to the inhibition by this compound as compared to non-cancerous ones. This might be used as a starting point to develop amicoumacin A derivatives with clinical value.

Ribosomal position and contacts of mRNA in eukaryotic translation initiation complexes.

The position of mRNA on 40S ribosomal subunits in eukaryotic initiation complexes was determined by UV crosslinking using mRNAs containing uniquely positioned 4-thiouridines. Crosslinking of mRNA positions (+)11 to ribosomal protein (rp) rpS2(S5p) and rpS3(S3p), and (+)9-(+)11 and (+)8-(+)9 to h18 and h34 of 18S rRNA, respectively, indicated that mRNA enters the mRNA-binding channel through the same layers of rRNA and proteins as in prokaryotes. Upstream of the P-site, the proximity of positions (-)3/(-)4 to rpS5(S7p) and h23b, (-)6/(-)7 to rpS14(S11p), and (-)8-(-)11 to the 3'-terminus of 18S rRNA (mRNA/rRNA elements forming the bacterial Shine-Dalgarno duplex) also resembles elements of the bacterial mRNA path. In addition to these striking parallels, differences between mRNA paths included the proximity in eukaryotic initiation complexes of positions (+)7/(+)8 to the central region of h28, (+)4/(+)5 to rpS15(S19p), and (-)6 and (-)7/(-)10 to eukaryote-specific rpS26 and rpS28, respectively. Moreover, we previously determined that eukaryotic initiation factor2alpha (eIF2alpha) contacts position (-)3, and now report that eIF3 interacts with positions (-)8-(-)17, forming an extension of the mRNA-binding channel that likely contributes to unique aspects of eukaryotic initiation.

The fidelity of translation initiation: reciprocal activities of eIF1, IF3 and YciH.

Eukaryotic initiation factor eIF1 and the functional C-terminal domain of prokaryotic initiation factor IF3 maintain the fidelity of initiation codon selection in eukaryotes and prokaryotes, respectively, and bind to the same regions of small ribosomal subunits, between the platform and initiator tRNA. Here we report that these nonhomologous factors can bind to the same regions of heterologous subunits and perform their functions in heterologous systems in a reciprocal manner, discriminating against the formation of initiation complexes containing codon-anticodon mismatches. We also show that like IF3, eIF1 can influence initiator tRNA selection, which occurs at the stage of ribosomal subunit joining after eIF5-induced hydrolysis of eIF2-bound GTP. The mechanisms of initiation codon and initiator tRNA selection in prokaryotes and eukaryotes are therefore unexpectedly conserved and likely involve related conformational changes induced in the small ribosomal subunit by factor binding. YciH, a prokaryotic eIF1 homologue, could perform some of IF3's functions, which justifies the possibility that YciH and eIF1 might have a common evolutionary origin as initiation factors, and that IF3 functionally replaced YciH in prokaryotes.

Translocation of tRNA during protein synthesis.

Coupled translocation of tRNA and mRNA in the ribosome during protein synthesis is one of the most challenging and intriguing problems in the field of translation. We highlight several key questions regarding the mechanism of translocation, and discuss possible mechanistic models in light of the recent crystal structures of the ribosome and its subunits.