The acidic ribosomal phosphoprotein of eukaryotes and its relationship to ribosomal proteins L7 and L12 of Escherichia coli.
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ABSTRACT:
SUBMITTER: Leader DP
PROVIDER: S-EPMC1186266 | biostudies-other | 1978 Nov
REPOSITORIES: biostudies-other
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Project description:For Escherichia coli growing rapidly in rich medium at 37 °C, the doubling time can be as short as ~20 min and the average rate of translation (ktrl) can be as fast as ~20 amino acids/s. For slower growth arising from poor nutrient quality or from higher growth osmolality, ktrl decreases significantly. In earlier work from the Hwa lab, a simplified Michaelis-Menten model suggested that the decrease in ktrl arises from a shortage of ternary complexes (TCs) under nutrient limitation and from slower diffusion of TCs under high growth osmolality. Here we present a single-molecule tracking study of the diffusion of EF-Tu in E. coli growing with doubling times in the range 62-190 min at 37 °C due to nutrient limitation, high growth osmolality, or both. The diffusive properties of EF-Tu remain quantitatively indistinguishable across all growth conditions studied. Dissection of the total population into ribosome-bound and free sub-populations, combined with copy number estimates for EF-Tu and ribosomes, indicates that in all cases ~3.7 EF-Tu copies are bound on average to each translating 70S ribosome. Thus, the four L7/L12 binding sites adjacent to the ribosomal A-site in E. coli are essentially saturated with TCs in all conditions, facilitating rapid testing of aminoacyl-tRNAs for a codon match. Evidently, the average translation rate is not limited by either the supply of cognate TCs under nutrient limitation or by the diffusion of free TCs at high osmolality. Some other step or steps must be rate limiting for translation in slow growth.
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Project description:Inspection of the structure of the C-terminal domain of ribosomal protein L7/L12 (1) reveals a helix-turn-helix motif similar to the one found in many DNA-binding regulatory proteins (2-5). The 19 alpha-carbon atoms of the L7/L12 alpha-helices superimpose on the DNA binding helices of CAP and cro with root-mean-square distances between corresponding alpha carbons of 1.45 and 1.55 A, respectively. These helices in L7/L12 are within a patch of highly conserved residues on the surface of L7/L12 whose role is as yet uncertain. We raise the possibility that they may constitute a binding site for nucleic acids, most probably RNA. Consistent with this hypothesis are calculations of the electrostatic charge potential surrounding the protein, which show a region of positive potential centered on the first of these helices.
| S-EPMC317856 | biostudies-other
Project description:BackgroundWe generated novel, effective candidate vaccine against Brucella abortus based on recombinant influenza viruses expressing the Brucella ribosomal protein L7/L12 or outer membrane protein (Omp)-16 from the NS1 open reading frame. The main purpose of this work was to evaluate the safety, immunogenicity and protectiveness of vaccine candidate in laboratory animals.Methods and resultsFour recombinant influenza A viral constructs of the subtypes Н5N1 or H1N1 expressing the Brucella proteins L7/L12 or Omp16 were obtained by a reverse genetics method: Flu-NS1-124-L7/L12-H5N1, Flu-NS1-124-Omp16-H5N1, Flu-NS1-124-L7/L12-H1N1 and Flu-NS1-124-Omp16-H1N1. Despite of substantial modification of NS1 gene, all constructs replicated well and were retain their Brucella inserts over five passages in embryonated chicken eggs (CE). Administration of the mono- or bivalent vaccine formulation via prime-boost intranasal (i.n.), conjunctival (c.) or subcutaneous (s.c.) immunization was safe in mice; no deaths, body weight loss or pathomorphological changes were observed over 56 days. Moreover, guinea pigs vaccinated i.n. with vaccine vectors did not shed the vaccine viruses through their upper respiratory tract after the prime and booster vaccination. These findings confirmed the replication-deficient phenotype of viral vectors. The highest antibody response to Brucella antigen was obtained with constructs expressing L7/L12 (ELISA, GMT 242.5-735.0); whereas the highest T-cell immune response- with construct expressing Omp16 (ELISPOT, 337 ± 52-651 ± 45 spots/4×105cells), which was comparable (P > 0.05) to the response induced by the commercial vaccine B. abortus 19. Interestingly, c. immunization appeared to be optimal for eliciting T-cell immune response. In guinea pigs, the highest protective efficacy after challenge with B. abortus 544 was achieved with Omp16 expressing constructs in both monovalent or bivalent vaccine formulations; protective efficacy was comparable to those induced by a commercial live B. abortus 19 vaccine.ConclusionThus, influenza vectors expressing Brucella protective antigens can be developed as novel influenza vectored vaccine against B. abortus infection.
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Project description:Helix 42 of Domain II of Escherichia coli 23S ribosomal RNA underlies the L7/L12 stalk in the ribosome and may be significant in positioning this feature relative to the rest of the 50S ribosomal subunit. Unlike the Haloarcula marismortui and Deinococcus radiodurans examples, the lower portion of helix 42 in E.coli contains two consecutive G*A oppositions with both adenines on the same side of the stem. Herein, the structure of an analog of positions 1037-1043 and 1112-1118 in the helix 42 region is reported. NMR spectra and structure calculations support a cis Watson-Crick/Watson-Crick (cis W.C.) G*A conformation for the tandem (G*A)2 in the analog and a minimally perturbed helical duplex stem. Mg2+ titration studies imply that the cis W.C. geometry of the tandem (G*A)2 probably allows O6 of G20 and N1 of A4 to coordinate with a Mg2+ ion as indicated by the largest chemical shift changes associated with the imino group of G20 and the H8 of G20 and A4. A cross-strand bridging Mg2+ coordination has also been found in a different sequence context in the crystal structure of H.marismortui 23S rRNA, and therefore it may be a rare but general motif in Mg2+ coordination.
| S-EPMC1143578 | biostudies-literature
Project description:Protein phosphorylation is a central biological mechanism for cellular adaptation to environmental changes. Dysregulation of phosphorylation signaling is implicated in a wide variety of diseases. Thus, the ability to detect and quantify protein phosphorylation is highly desirable for both diagnostic and research applications. Here we present a general strategy for detecting phosphopeptide-protein interactions in Escherichia coli. We first redesign a model tetratricopeptide repeat (TPR) protein to recognize phosphoserine in a sequence-specific fashion and characterize the interaction with its target phosphopeptide in vitro. We then combine in vivo site-specific incorporation of phosphoserine with split mCherry assembly to observe the designed phosphopeptide-protein interaction specificity in E. coli. This in vivo strategy for detecting and characterizing phosphopeptide-protein interactions has numerous potential applications for the study of natural interactions and the design of novel ones.
| S-EPMC4245168 | biostudies-literature
Project description:Ribosomes are the sophisticated machinery that is responsible for protein synthesis in a cell. Recently, quantitative mass spectrometry (qMS) have been successfully applied for understanding the dynamics of protein complexes. Here, we developed a highly specific and reproducible method to quantify all ribosomal proteins (r-proteins) by combining selected reaction monitoring (SRM) and isotope labeling. We optimized the SRM methods using purified ribosomes and Escherichia coli lysates and verified this approach as detecting 41 of the 54 r-proteins separately synthesized in E. coli S30 extracts. The SRM methods will enable us to utilize qMS as a highly specific analytical tool in the research of E. coli ribosomes, and this methodology have potential to accelerate the understanding of ribosome biogenesis, function, and the development of engineered ribosomes with additional functions.
| S-EPMC7735604 | biostudies-literature
Project description:Brucella, a genus of bacteria that causes brucellosis, infects and threatens domestic animals, and humans in endemic areas. Presently, some live attenuated vaccines of Brucella are used to immunize livestock; however, these vaccines are pathogenic to humans, can provoke abortion when administered to pregnant livestock, and induce antibodies in vaccinated livestock that affect the diagnosis of field infection. It is, therefore, very important for improving the safety and immune protection effects of Brucella vaccine. Currently, recombinant protein-based subunit vaccines are considered promising safe and effective alternatives against brucellosis. Here, we separately expressed the recombinant Omp10-Omp28-L7/L12 proteins of Brucella using eukaryotic and prokaryotic expression systems, which were then used as immunogens for evaluating their immune responses. Taishan Pinus massoniana pollen polysaccharides (TPPPS), an already verified natural adjuvant, was utilized to evaluate the immune conditioning effect on the recombinant proteins. Antibody levels, spleen lymphocyte proliferation, percentages of CD4+ and CD8+ T cells, and cytokine secretion in mice were examined after three successive immunizations. The protective effects against Brucella challenge were also evaluated in mice, and used a live vaccine as a positive control. The results indicated that the immune responses of the recombinant Omp10-Omp28-L7/L12 protein groups were significantly higher than those of the PBS control group. The recombinant Omp10-Omp28-L7/L12 protein expressed in Pichia pastoris (P. pastoris) exhibited a slightly higher expression level and immunogenicity than that expressed in Escherichia coli (E. coli), and the Omp10-Omp28-L7/L12 (P. pastoris) + TPPPS group provided the most pronounced immune effect. The protective results showed that the recombinant Omp10-Omp28-L7/L12 proteins expressed in the two expression systems had significantly better protective effects against Brucella melitensis challenge compared with the negative control, and the addition of TPPPS adjuvant could significantly improve the protective effects of subunit vaccines. However, we also noticed that all of the evaluated subunit vaccines induced less protection than the B. melitensis M5 live vaccine. These results indicate that the combination of recombinant Omp10-Omp28-L7/L12 antigen and TPPPS adjuvant shows potential as an effective brucellosis subunit vaccine, and P. pastoris is a preferred expression system to prepare this recombinant subunit antigen.
| S-EPMC7531237 | biostudies-literature
Project description:Ribosomal protein L7/L12 is associated with translation initiation, elongation, and termination by the 70S ribosome. The guanosine 5' triphosphate hydrolase (GTPase) activity of elongation factor G (EF-G) requires the presence of L7/L12, which is critical for ribosomal translocation. Here, we have developed new methods for the complete depletion of L7/L12 from Escherichia coli 70S ribosomes to analyze the effect of L7/L12 on the activities of the GTPase factors EF-G, RF3, IF2, and LepA. Upon removal of L7/L12 from ribosomes, the GTPase activities of EF-G, RF3, and IF2 decreased to basal levels, while the activity of LepA decreased marginally. Upon reconstitution of ribosomes with recombinant L12, the GTPase activities of all GTPases returned to full activity. Moreover, ribosome binding assays indicated that EF-G, RF3, and IF2 require L7/L12 for stable binding in the GTP state, and LepA retained > 50% binding. Lastly, an EF-G?G' truncation mutant possessed ribosome-dependent GTPase activity, which was insensitive to L7/L12. Our results indicate that L7/L12 is required for stable binding of ribosome-dependent GTPases that harbor direct interactions to the L7/L12 C-terminal domains, either through a G' domain (EF-G, RF3) or a unique N-terminal domain (IF2). Furthermore, we hypothesize this interaction is concomitant with counterclockwise ribosomal intersubunit rotation, which is required for translocation, initiation, and post-termination.
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Project description:The Brucella abortus L7/L12 ribosomal gene was amplified by PCR and subcloned into the prokaryotic expression vector pMAL-c2. Escherichia coli DH5 alpha was transformed with the pMAL-L7/L12 construct, and gene expression was induced by IPTG (isopropyl-beta-D-thiogalactopyranoside). The resulting fusion protein was purified by affinity chromatography and confirmed by Western blot (immunoblot) analysis using an anti-maltose-binding protein antibody. Additionally, purified recombinant L7/L12 protein induced T-lymphocyte proliferation of B. abortus-primed bovine peripheral blood mononuclear cells. Phenotypic analysis of the proliferating cell population demonstrated an increase in the percentage of CD4+ T lymphocytes when peripheral blood mononuclear cells were cultured with recombinant L7/L12 compared with cells cultured in medium alone. Subcloning and expression of a B. abortus gene encoding a previously demonstrated immunodominant protein for bovine lymphocytes are important steps in selecting Brucella proteins that have potential as a component of a genetically engineered candidate vaccine.
| S-EPMC303250 | biostudies-other
Project description:RPLP1 is one of acidic ribosomal phosphoproteins encoded by RPLP1 gene, which plays an important role in the elongation step of protein synthesis. The cDNA of RPLP1 was cloned successfully for the first time from the Giant Panda (Ailuropoda melanoleuca) using RT-PCR technology, which was also sequenced, analyzed preliminarily and expressed in E.coli. The cDNA fragment cloned is 449bp in size, containing an open reading frame of 344bp encoding 114 amino acids. Alignment analysis indicated that the nucleotide sequence and the deduced amino acid sequence are highly conserved to other five species studied, including Homo sapiens, Mus musculus, Rattus norvegicus, Bos Taurus and Sus scrofa. The homologies for nucleotide sequences of Giant Panda PPLP1 to that of these species are 92.4%, 89.8%, 89.0%, 91.3% and 87.5%, while the homologies for amino acid sequences are 96.5%, 94.7%, 95.6%, 96.5% and 88.6%. Topology prediction showed there are three Casein kinase II phosphorylation sites and two N-myristoylation sites in the RPLP1 protein of the Giant Panda (Ailuropoda melanoleuca). The RPLP1 gene was overexpressed in E. coli and the result indicated that RPLP1 fusion with the N-terminally His-tagged form gave rise to the accumulation of an expected 18kDa polypeptide, which was in accordance with the predicted protein and could also be used to purify the protein and study its function.
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