Recombinant Ribosomal Protein Research Articles

On the pursuit to reconstitute the Escherichia coli ribosome from purified components.

The ribosome, the protein synthesizing machinery composed of dozens of proteins and several ribosomal RNAs (rRNAs), is essential for life. In vitro reconstitution of the ribosome holds significance for understanding biosynthesis, applications in biotechnology and potential contributions to synthetic biology. There is a long history of in vitro reconstitution of bacterial ribosomes, originating in the 1970s when the 30S ribosome of Escherichia coli was reconstituted from the protein and rRNA components prepared from native ribosome. Since then, the reconstitution using in vitro transcribed rRNAs has been established, and more recently, the reconstitution using recombinant ribosomal proteins has also become possible. A recent report by Aoyama et al. (J. Biochem. 2022; 171:227-237), the reconstitution of the 50S ribosome using 33 recombinant ribosomal proteins, is a new leap toward complete reconstitution of the holo ribosome complex from recombinant proteins and in vitro transcribed rRNAs. This commentary also discusses future challenges.

In vitro reconstitution of the Escherichia coli 70S ribosome with a full set of recombinant ribosomal proteins.

Many studies of the reconstitution of the Escherichia coli small ribosomal subunit from its individual molecular parts have been reported, but contrastingly, similar studies of the large ribosomal subunit have not been well performed to date. Here, we describe protocols for preparing the 33 ribosomal proteins of the E. coli 50S subunit and demonstrate successful reconstitution of a functionally active 50S particle that can perform protein synthesis in vitro. We also successfully reconstituted both ribosomal subunits (30S and 50S) and 70S ribosomes using a full set of recombinant ribosomal proteins by integrating our developed method with the previously developed fully recombinant-based integrated synthesis, assembly and translation. The approach described here makes a major contribution to the field of ribosome engineering and could be fundamental to the future studies of ribosome assembly processes.

In vitro reconstitution of functional small ribosomal subunit assembly for comprehensive analysis of ribosomal elements in E. coli

In vitro reconstitution is a powerful tool for investigating ribosome functions and biogenesis, as well as discovering new ribosomal features. In this study, we integrated all of the processes required for Escherichia coli small ribosomal subunit assembly. In our method, termed fully Recombinant-based integrated Synthesis, Assembly, and Translation (R-iSAT), assembly and evaluation of the small ribosomal subunits are coupled with ribosomal RNA (rRNA) synthesis in a reconstituted cell-free protein synthesis system. By changing the components of R-iSAT, including recombinant ribosomal protein composition, we coupled ribosomal assembly with ribosomal protein synthesis, enabling functional synthesis of ribosomal proteins and subsequent subunit assembly. In addition, we assembled and evaluated subunits with mutations in both rRNA and ribosomal proteins. The study demonstrated that our scheme provides new ways to comprehensively analyze any elements of the small ribosomal subunit, with the goal of improving our understanding of ribosomal biogenesis, function, and engineering.

An investigation into the effect of ribosomal protein S15 phosphorylation on its intermolecular interactions by using phosphomimetic mutant.

An investigation using recombinant ribosomal proteins and synthetic peptide models was conducted to uncover the effect of the introduction of a negative charge at the C-terminal tail of ribosomal protein S15. Our results help provide a chemical rationale towards understanding how G2019S LRRK2, a common clinical mutation, causes Parkinson's disease.

Mutations Preventing the Phosphorylation of Human Ribosomal Protein uS15 at Y38 and S48 Reduce the Efficiency of its Transfer into the Nucleolus

The ribosomal protein uS15 is one of the key proteins forming the small (40S) ribosomal subunit structure around the central domain of 18S rRNA. According to a number of proteomic studies based on mass-spectrometry analysis, there are many phosphorylation sites in this protein. However, when the protein uS15 is contained in the 40S subunit, it does not carry such posttranslational modification. In this study, it was found that the cytoplasmic protein kinases of HEK293T cells are able to phosphorylate the human recombinant ribosomal protein uS15 with significantly greater efficiency than that of the nucleus protein kinases. The effect of the amino acid substitutions Y38A and S48A preventing the phosphorylation of the ribosomal protein uS15 at the corresponding sites, on the transport of the recombinant protein uS15 fused to the green fluorescent protein in the nucleolus was studied. It was shown that single mutations at the above sites have little effect on the transport of this protein, whereas double mutation reduces the efficiency of this process by more than a quarter. The findings suggest the importance of phosphorylation of the ribosomal protein uS15 by cytoplasmic protein kinases at several sites, including Y38A and S48A, for its efficient transfer into the nucleolus, where pre-ribosomal subunits are assembled.

Effect of a recombinant ribosomal protein L10a (rRpL10a) on mouse spermatogenesis

Effect of a recombinant ribosomal protein L10a (rRpL10a) on mouse spermatogenesis

Stimulation of ovarian development in white shrimp, Fenneropenaeus merguiensis De Man, with a recombinant ribosomal protein L10a

Abstract Ribosomal protein L10a (RpL10a) is encoded by an essential gene related to the early stages of ovarian development. A recombinant RpL10a protein was produced and evaluated to determine its ability to stimulate ovarian development in the white shrimp Fenneropenaeus merguiensis De Man. The transportation of injected recombinant RpL10a to the ovary was monitored using FITC-conjugated rRpL10a in an in vivo assay. FITC-rRpL10a was imported into the ovarian tissue within 24 h after injection, observed with a fluorescence microscope. The signal was not detected in muscle tissue; thus, muscle was not a target tissue. A dose of 180 μg of rRpL10a/shrimp was effective for inducing the ovaries to reach stages I and II of vitellogenesis within 7 days after the first injection. Observations of the ovarian natural autofluorescence in tissue sections were performed to determine the stage of vitellogenesis in F. merguiensis and were shown to provide the same results as were obtained using sections stained according to the standard hematoxylin and eosin staining procedure, which spent more time. Methyl farnesoate (MF) levels were examined via a HPLC technique in different stages of ovarian development following RpL10a injection, and the observed MF levels indicated that the concentration of MF in the hemolymph increased in stage II of vitellogenesis.

Cross-protective effect of a combined L5 plus L3 Leishmania major ribosomal protein based vaccine combined with a Th1 adjuvant in murine cutaneous and visceral leishmaniasis

BackgroundTwo Leishmania major ribosomal proteins L3 (LmL3) and L5 (LmL5) have been described as protective molecules against cutaneous leishmaniasis due to infection with L. major and Leishmania braziliensis in BALB/c mice when immunized with a Th1 adjuvant (non-methylated CpG-oligodeoxynucleotides; CpG-ODN). In the present study we analyzed the cross-protective efficacy of an LmL3-LmL5-CpG ODN combined vaccine against infection with Leishmania amazonensis and Leishmania chagasi (syn. Leishmania infantum) the etiologic agents of different clinical forms of human leishmaniasis in South America.MethodsThe combined vaccine was administered subcutaneously to BALB/c mice. After immunization the cellular and humoral responses elicited were analyzed. Mice were independently challenged with L. amazonensis and L. chagasi. The size of the cutaneous lesions caused by the infection with the first species, the parasite loads and the immune response in both infection models were analyzed nine weeks after challenge.ResultsMice vaccinated with the combined vaccine showed a Th1-like response against LmL3 and LmL5. Vaccinated mice were able to delay lesion development due to L. amazonensis infection and to control parasite loads in the site of infection. A reduction of the parasite burden in the lymph nodes draining the site of infection and in the liver and spleen was observed in the vaccinated mice after a subcutaneous infection with L. chagasi. In both models of infection, protection was correlated to parasite antigen-specific production of IFN-γ and down-regulation of parasite-mediated IL-4 and IL-10 responses.ConclusionsThe data presented here demonstrate the potential use of L. major L3 and L5 recombinant ribosomal proteins for the development of vaccines against various Leishmania species.

Evaluation of immune responses and analysis of the effect of vaccination of the Leishmania major recombinant ribosomal proteins L3 or L5 in two different murine models of cutaneous leishmaniasis

Four new antigenic proteins located in Leishmania ribosomes have been characterized: S4, S6, L3 and L5. Recombinant versions of the four ribosomal proteins from Leishmania major were recognized by sera from human and canine patients suffering different clinical forms of leishmaniasis. The prophylactic properties of these proteins were first studied in the experimental model of cutaneous leishmaniasis caused by L. major inoculation into BALB/c mice. The administration of two of them, LmL3 or LmL5 combined with CpG-oligodeoxynucleotides (CpG-ODN) was able to protect BALB/c mice against L. major infection. Vaccinated mice showed smaller lesions and parasite burden compared to mice inoculated with vaccine diluent or vaccine adjuvant. Protection was correlated with an antigen-specific increased production of IFN-γ paralleled by a decrease of the antigen-specific IL-10 mediated response in protected mice relative to non-protected controls. Further, it was demonstrated that BALB/c mice vaccinated with recombinant LmL3 or LmL5 plus CpG-ODN were also protected against the development of cutaneous lesions following inoculation of L. braziliensis. Together, data presented here indicate that LmL3 or LmL5 ribosomal proteins combined with Th1 inducing adjuvants, may be relevant components of a vaccine against cutaneous leishmaniasis caused by distinct species.

Identification of Novel RNA-Protein Contact in Complex of Ribosomal Protein S7 and 3’-Terminal Fragment of 16S rRNA in E. coli

For prokaryotes in vitro, 16S rRNA and 20 ribosomal proteins are capable of hierarchical self- assembly yielding a 30S ribosomal subunit. The self-assembly is initiated by interactions between 16S rRNA and three key ribosomal proteins: S4, S8, and S7. These proteins also have a regulatory function in the translation of their polycistronic operons recognizing a specific region of mRNA. Therefore, studying the RNA-protein interactions within binary complexes is obligatory for understanding ribosome biogenesis. The non-conventional RNA-protein contact within the binary complex of recombinant ribosomal protein S7 and its 16S rRNA binding site (236 nucleotides) was identified. UV-induced RNA-protein cross-links revealed that S7 cross-links to nucleotide U1321 of 16S rRNA. The careful consideration of the published RNA- protein cross-links for protein S7 within the 30S subunit and their correlation with the X-ray data for the 30S subunit have been performed. The RNA - protein cross-link within the binary complex identified in this study is not the same as the previously found cross-links for a subunit both in a solution, and in acrystal. The structure of the binary RNA-protein complex formed at the initial steps of self-assembly of the small subunit appears to be rearranged during the formation of the final structure of the subunit.

Binding of the human ribosomal protein S13 to the central domain of 18S rRNA

Human ribosomal protein S13 is a structural element of the small subunit of ribosome. It is a homologue of eubacterial ribosomal protein S15, and, besides, it possesses an extended N-terminal region, characteristic of the S15p family in eukaryotes and archaea. In the present study, we investigated binding of recombinant ribosomal protein S13 and its mutants containing deletions or substitutions of amino acid residues in different regions with an RNA transcript corresponding to a fragment of the central domain of 18S rRNA. We found that replacement of ultra-conservative residues H101 and D108 as well as deletions of either 29 C-terminal or 27 N-terminal residues substantially reduced affinity of the protein to the RNA transcript. Deletion of 54 C-terminal or 80 N-terminal residues completely deprived the protein of binding capacity. Using a footprinting assay, we identified sites in the RNA transcript changing their accessibilities to action of hydroxyl radicals under binding of either full-length protein S13 or its mutant lacking 27 N-terminal residues. It is shown that these sites are located mainly in helix H22 of the 18S rRNA and in the region of its junction with helix H20 and are consistent predominantly with contacts of the rRNA with the conserved part of the protein. We concluded that binding of ribosomal protein S13 to 18S rRNA is provided mainly by conserved motifs of the protein corresponding to those motifs in its eubacterial homologue that are involved in the interaction with 16S rRNA in the 30S subunit. Role of the N-terminal region of the protein in its binding to the central domain of 18S rRNA is discussed.

Isolation of a CK2α Subunit and the Holoenzyme from the Mussel Mytilus galloprovincialis and Construction of the CK2α and CK2β cDNAs

Protein kinase CK2 is a ubiquitous, highly pleiotropic, and constitutively active phosphotransferase that phosphorylates mainly serine and threonine residues. CK2 has been studied and characterized in many organisms, from yeast to mammals. The holoenzyme is generally composed of two catalytic (α and/or α') and two regulatory (β) subunits, forming a differently assembled tetramer. The free and catalytically active α/α' subunits can be present in cells under some circumstances. We present here the isolation of a putative catalytic CK2α subunit and holoenzyme from gills of the mussel Mytilus galloprovincialis capable of phosphorylating the purified recombinant ribosomal protein rMgP1. For further analysis of M. galloprovincialis protein kinase CK2, the cDNA molecules of CK2α and CK2β subunits were constructed and cloned into expression vectors, and the recombinant proteins were purified after expression in Escherichia coli. The recombinant MgCK2β subunit and MgP1 were phosphorylated by the purified recombinant MgCK2α subunit. The mussel enzyme presented features typical for CK2: affinity for GTP, inhibition by both heparin and ATP competitive inhibitors (TBBt, TBBz), and sensitivity towards NaCl. Predicted amino acid sequence comparison showed that the M. galloprovincialis MgCK2α and MgCK2β subunits have similar features to their mammalian orthologs.

Found in Translation?

The netrin receptor DCC regulates local protein translation.

Escheriosome-mediated delivery of recombinant ribosomal L7/L12 protein confers protection against murine brucellosis

Brucellosis is an important zoonotic disease that causes abortion in cattle and undulant fever, arthritis, endocarditis and meningitis in human. In spite of the fact that immunization could be an efficient measure to control brucellosis, not a single ideal vaccine against this important disease has been developed so far. In order to develop an effective vaccine against Brucella abortus ( B. abortus), various protective immunodominant gene/protein products of the pathogen have been studied in combination with different adjuvants. For example, recombinant ribosomal protein L7/L12 (rL7/L12) although an interesting T-cell antigen, normally failed to evoke protective immune response when used in free form. In the present study we have demonstrated that Escherischia coli ( E. coli) lipid liposome (escheriosome)-mediated cytosolic delivery of recombinant rL7/L12 protein can elicit strong immunological responses in the Balb/ c mice. In contrast, egg PC/Chol liposome entrapped rL7/L12, in a manner similar to its free form, was found to impart relatively poor immune response. Furthermore, escheriosome entrapped rL7/L12 protein elicited high IgG2a isotype response suggestive of its relevance in imparting protection against brucellosis in mice. Altogether the present study is a clear indicative of the possible use of escheriosome-based delivery of rL7/L12 protein to induce protective immune responses against experimental murine brucellosis.

Human ribosomal protein S26 suppresses the splicing of its pre-mRNA

Human recombinant ribosomal protein S26 (rpS26) was shown to interact with its pre-mRNA intron I and mRNA fragment. Endogenous rpS26 in HeLa nuclear extract was also found to bind to the intron I, and with a lower extent to the mRNA fragment. The addition of recombinant rpS26 to the nuclear extract increased the binding largely. The in vitro splicing of an RNA that contained exon I, intron I and part of exon II of the rpS26 pre-mRNA yielded conventional and alternative mRNAs. Recombinant rpS26 was found to suppress the formation of both mRNAs. Sites of the pre-mRNA involved in the binding to rpS26 were detected by toe-printing. Nucleotides that caused a stop (pause) of the reverse transcription formed two clusters on the RNA secondary structure. One cluster including A69, A287 and A303 arranged the conventional 3′ site of splicing, another one including A131, A136, G156, A166 and A264 arranged the alternative 3′ site of splicing.

L12 enhances gonococcal transcytosis of polarized Hec1B cells via the lutropin receptor

We previously reported that gonococci convert to a more invasive phenotype (Inv+GC) following contact with cells expressing the lutropin receptor (LHr) and that Inv+GC express a novel adhesin that interacts with LHr. We propose that this adhesion allows Inv+GC to activate LHr and induce gonococcal transcytosis, usurping normal LHr function in fallopian and endometrial epithelium, which is to transport fetal chorionic gonadotropin (hCG) into the mother. Infected polarized Hec1B monolayers, grown on collagen-coated transwells, showed that the passage of GC across the monolayer occurred rapidly, within 30 min, and proceeded at a constant rate with Inv+GC passage three-fold faster than GC grown in tissue culture media alone (Inv−GC). Electron microscopy found that Inv+GC triggered pseudopod formation around the bacterium, with GC found throughout the Hec1B targets within 30 min, while Inv−GC did neither. Pre-treatment of Inv−GC with recombinant ribosomal protein L12, a gonococcal «hCG-like» protein previously shown to increase invasion, also increased Inv−GC transcytosis to the rate of Inv+GC. This enhancement was completely abolished by addition of luteinizing hormone, a cognate ligand of LHr. This is convincing evidence that surface expressed L12 mediates gonococcal invasion and transcytosis via LHr, a mechanism that could be important in the development of invasive gonococcal disease in women.

Efficient reconstitution of functional Escherichia coli 30S ribosomal subunits from a complete set of recombinant small subunit ribosomal proteins.

Previous studies have shown that the 30S ribosomal subunit of Escherichia coli can be reconstituted in vitro from individually purified ribosomal proteins and 16S ribosomal RNA, which were isolated from natural 30S subunits. We have developed a 30S subunit reconstitution system that uses only recombinant ribosomal protein components. The genes encoding E. coli ribosomal proteins S2-S21 were cloned, and all twenty of the individual proteins were overexpressed and purified. Reconstitution, following standard procedures, using the complete set of recombinant proteins and purified 16S ribosomal RNA is highly inefficient. Efficient reconstitution of 30S subunits using these components requires sequential addition of proteins, following either the 30S subunit assembly map (Mizushima & Nomura, 1970, Nature 226:1214-1218; Held et al., 1974, J Biol Chem 249:3103-3111) or following the order of protein assembly predicted from in vitro assembly kinetics (Powers et al., 1993, J MoI Biol 232:362-374). In the first procedure, the proteins were divided into three groups, Group I (S4, S7, S8, S15, S17, and S20), Group II (S5, S6, S9, Sll, S12, S13, S16, S18, and S19), and Group III (S2, S3, S10, S14, and S21), which were sequentially added to 16S rRNA with a 20 min incubation at 42 degrees C following the addition of each group. In the second procedure, the proteins were divided into Group I (S4, S6, S11, S15, S16, S17, S18, and S20), Group II (S7, S8, S9, S13, and S19), Group II' (S5 and S12) and Group III (S2, S3, S10, S14, and S21). Similarly efficient reconstitution is observed whether the proteins are grouped according to the assembly map or according to the results of in vitro 30S subunit assembly kinetics. Although reconstitution of 30S subunits using the recombinant proteins is slightly less efficient than reconstitution using a mixture of total proteins isolated from 30S subunits, it is much more efficient than reconstitution using proteins that were individually isolated from ribosomes. Particles reconstituted from the recombinant proteins sediment at 30S in sucrose gradients, bind tRNA in a template-dependent manner, and associate with 50S subunits to form 70S ribosomes that are active in poly(U)-directed polyphenylalanine synthesis. Both the protein composition and the dimethyl sulfate modification pattern of 16S ribosomal RNA are similar for 30S subunits reconstituted with either recombinant proteins or proteins isolated as a mixture from ribosomal subunits as well as for natural 30S subunits.

Probing the rRNA environment of ribosomal protein S5 across the subunit interface and inside the 30 S subunit using tethered Fe(II)

A newly developed 30 S subunit reconstitution system using a complete set of recombinant proteins was used to study the ribosomal RNA (rRNA) neighborhood of ribosomal protein S5 in 30 S subunits and 70 S ribosomes by directed hydroxyl radical probing. Using three cysteine-containing mutant S5 proteins derivatized with 1-(p -bromoacetamidobenzyl)-Fe(II)-EDTA, we expanded on experiments carried out earlier using a natural protein reconstitution system. Natural 16 S rRNA, Fe(II)-S5, and the other recombinant ribosomal proteins were reconstituted into 30 S subunits. Both 30 S subunits and 70 S ribosomes containing Fe(II)-S5 were purified, and hydroxyl radicals were generated in situ from the tethered Fe(II). In 30 S subunits, 16 S rRNA nucleotides targeted by two positions on S5, C21 and C99, were virtually identical to those observed in the previous work, supporting the validity of the recombinant protein reconstitution system for probing studies. Interestingly, new cleavages were detected using Fe(II)-C129-S5, possibly reflecting incorporation of more derivatized protein into 30 S subunits due to the increased reconstitution efficiency of the recombinant protein system. These newly targeted positions overlap, but are distinct from, those observed using Fe(II) tethered to C21, which is near C129 in the S5 structure.In 70 S ribosomes, the cleavage pattern of 16 S rRNA was very similar to that observed in 30 S subunits for all target sites except for the absence of those at the extreme 5′ end of 16 S rRNA. Additionally, probing of 70 S ribosomes from Fe-C99-S5 results in cleavage of 23 S rRNA in the 1690-1770 region of domain IV. These data provide constraints for the three-dimensional location of nucleotides within domain IV of 23 S ribosomal RNA relative to known features of the 30 S subunit.

Recovery of soluble, active recombinant protein from inclusion bodies.

Recovery of soluble, active recombinant protein from inclusion bodies.

Crystallization and preliminary crystallographic analysis of ribosomal protein S8 fromThermus thermophilus

Crystals have been obtained for recombinant ribosomal protein S8 from Thermus thermophilus produced by Escherichia coli. The protein crystals have been grown in 40 mM potassium phosphate buffer (pH 6.0) in hanging drops equilibrated against saturated ammonium sulfate (unbuffered) with 2-methyl-2,4-pentandiol (v/v). The crystals belong to the space group P4(1(3)) 2(1)2 with cell parameters a = b = 67.65 A, c = 171.12 A. They diffract x-rays to 2.9 A resolution.