scholarly journals Regulation of Ribosomal Protein Synthesis in Mycobacteria: The Autogenous Control of rpsO

2021 ◽  
Vol 22 (18) ◽  
pp. 9679
Author(s):  
Leonid V. Aseev ◽  
Ludmila S. Koledinskaya ◽  
Oksana S. Bychenko ◽  
Irina V. Boni
Keyword(s):  
Protein Synthesis ◽  
Ribosomal Protein ◽  
Shuttle Vector ◽  
Ectopic Expression ◽  
Egfp Expression ◽  
Reporter System ◽  
Fluorescent Reporter ◽  
E Coli ◽  
Ribosomal Protein Synthesis

The autogenous regulation of ribosomal protein (r-protein) synthesis plays a key role in maintaining the stoichiometry of ribosomal components in bacteria. In this work, taking the rpsO gene as a classic example, we addressed for the first time the in vivo regulation of r-protein synthesis in the mycobacteria M. smegmatis (Msm) and M. tuberculosis (Mtb). We used a strategy based on chromosomally integrated reporters under the control of the rpsO regulatory regions and the ectopic expression of Msm S15 to measure its impact on the reporter expression. Because the use of E. coli as a host appeared inefficient, a fluorescent reporter system was developed by inserting Msm or Mtb rpsO-egfp fusions into the Msm chromosome and expressing Msm S15 or E. coli S15 in trans from a novel replicative shuttle vector, pAMYC. The results of the eGFP expression measurements in Msm cells provided evidence that the rpsO gene in Msm and Mtb was feedback-regulated at the translation level. The mutagenic analysis showed that the folding of Msm rpsO 5′UTR in a pseudoknot appeared crucial for repression by both Msm S15 and E. coli S15, thus indicating a striking resemblance of the rpsO feedback control in mycobacteria and in E. coli.

scholarly journals Regulation of ribosomal protein synthesis in mycobacteria: autogenous control of rpsO

2021 ◽  
Author(s):  
Leonid V Aseev ◽  
Ludmila S Koledinskaya ◽  
Oksana S Bychenko ◽  
Irina V Boni
Keyword(s):  
Protein Synthesis ◽  
Ribosomal Protein ◽  
Ribosome Biogenesis ◽  
Bacterial Species ◽  
Shuttle Vector ◽  
Gc Content ◽  
E Coli ◽  
Autogenous Control ◽  
Autogenous Regulation

ABSTRACTAutogenous regulation of ribosomal protein (r-protein) synthesis plays a key role in maintaining the stoichiometry of ribosomal components in bacteria. Our main goal was to develop techniques for investigating the r-protein synthesis regulation in mycobacteria, Gram-positive organisms with a high GC-content, which has never been addressed. We started with the rpsO gene known to be autoregulated by its product, r-protein S15, in a broad range of bacterial species. To study the in vivo regulation of rpsO from Mycobacterium smegmatis (Msm), we first applied an approach based on chromosomally integrated Msm rpsO’-’lacZ reporters by using E. coli as a surrogate host. The β-galactosidase assay has shown that mycobacterial rpsO expression is feedback regulated at the translation level in the presence of Msm S15 in trans, like in E. coli. Next, to overcome difficulties caused by the inefficiency of mycobacterial gene expression in E. coli, we created a fluorescent reporter system based on M. smegmatis. To this end, the integrative shuttle plasmid pMV306 was modified to provide insertion of the Msm or Mtb (M. tuberculosis) rpsO-egfp reporters into the Msm chromosome, and a novel E. coli-mycobacteria replicative shuttle vector, pAMYC, a derivative of pACYC184, was built. Analysis of the eGFP expression in the presence of the pAMYC derivative expressing Msm rpsO vs an empty vector confirms the autogenous regulation of the rpsO gene in mycobacteria. Additionally, we have revealed that the mycobacterial rpsO core promoters are rather weak and require upstream activating elements to enhance their strength.IMPORTANCEBacterial ribosomes are targets for a majority of as-yet reported antibiotics, hence ribosome biogenesis and its regulation are central for development of new antimicrobials. One of the key mechanisms regulating ribosome biogenesis in bacteria is the autogenous control of r-protein synthesis, which has been so far explored for E. coli and Bacillus spp. but not yet for mycobacteria. Here, we describe experimental approaches for in vivo analysis of mechanisms regulating r-protein synthesis in mycobacteria, including M. tuberculosis, and show, for the first time, that the autogenous control at the translation level is really functioning in these microorganisms. The developed system paves the way for studying various regulatory circuits involving proteins or sRNAs as mRNA- targeting trans-regulators in mycobacteria as well as in other actinobacterial species.

scholarly journals Expression of Staphylococcus aureusClumping Factor A in Lactococcus lactis subsp.cremoris Using a New Shuttle Vector

2000 ◽  
Vol 68 (6) ◽  
pp. 3516-3522 ◽  
Author(s):  
Yok-Ai Que ◽  
Jacques-Antoine Haefliger ◽  
Patrick Francioli ◽  
Philippe Moreillon
Keyword(s):  
Lactococcus Lactis ◽  
Cell Walls ◽  
Solid Phase ◽  
Shuttle Vector ◽  
Luciferase Reporter ◽  
Reporter System ◽  
Western Blots ◽  
E Coli ◽  
Pathogenic Factors

ABSTRACT Staphylococcus aureus harbors redundant adhesins mediating tissue colonization and infection. To evaluate their intrinsic role outside of the staphylococcal background, a system was designed to express them in Lactococcus lactis subsp.cremoris 1363. This bacterium is devoid of virulence factors and has a known genetic background. A new Escherichia coli-L. lactis shuttle and expression vector was constructed for this purpose. First, the high-copy-number lactococcal plasmid pIL253 was equipped with the oriColE1 origin, generating pOri253 that could replicate in E. coli. Second, the lactococcal promoters P23 or P59 were inserted at one end of the pOri253 multicloning site. Gene expression was assessed by a luciferase reporter system. The plasmid carrying P23 (named pOri23) expressed luciferase constitutively at a level 10,000 times greater than did the P59-containing plasmid. Transcription was absent in E. coli. The staphylococcal clumping factor A (clfA) gene was cloned into pOri23 and used as a model system. Lactococci carrying pOri23-clfA produced an unaltered and functional 130-kDa ClfA protein attached to their cell walls. This was indicated both by the presence of the protein in Western blots of solubilized cell walls and by the ability of ClfA-positive lactococci to clump in the presence of plasma. ClfA-positive lactococci had clumping titers (titer of 4,112) similar to those of S. aureus Newman in soluble fibrinogen and bound equally well to solid-phase fibrinogen. These experiments provide a new way to study individual staphylococcal pathogenic factors and might complement both classical knockout mutagenesis and modern in vivo expression technology and signature tag mutagenesis.

scholarly journals The Reverse Side of a Coin: “Factor-Free” Ribosomal Protein Synthesis In Vitro is a Consequence of the In Vivo Proofreading Mechanism

Biomolecules ◽  
2019 ◽  
Vol 9 (10) ◽  
pp. 588
Author(s):  
Finkelstein
Keyword(s):  
Quality Control ◽  
Protein Synthesis ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Ribosomal Protein ◽  
Elongation Factor ◽  
Ribosomal Protein Synthesis

This paper elucidates a close connection between two well-known facts that until now have seemed independent: (i) the quality control (“proofreading”) of the emerging amino acid sequence, occurring during the normal, elongation-factor-dependent ribosomal biosynthesis, which is performed by removing those Aa-tRNAs (aminoacyl tRNAs) whose anticodons are not complementary to the exhibited mRNA codons, and (ii) the in vitro discovered existence of the factor-free ribosomal synthesis of polypeptides. It is shown that a biological role of proofreading is played by a process that is exactly opposite to the step of factor-free binding of Aa-tRNA to the ribosome-exposed mRNA: a factor-free removal of that Aa-tRNA whose anticodon is not complementary to the ribosome-exhibited mRNA codon.

scholarly journals The Escherichia coli mqsR and ygiT Genes Encode a New Toxin-Antitoxin Pair

2010 ◽  
Vol 192 (11) ◽  
pp. 2908-2919 ◽  
Author(s):  
Villu Kasari ◽  
Kristi Kurg ◽  
Tõnu Margus ◽  
Tanel Tenson ◽  
Niilo Kaldalu
Keyword(s):  
Escherichia Coli ◽  
Protein Synthesis ◽  
Growth Inhibition ◽  
Protein Complexes ◽  
Ectopic Expression ◽  
E Coli ◽  
New Family ◽  
Single Operon ◽  
Growing Population

ABSTRACT Toxin-antitoxin (TA) systems are plasmid- or chromosome-encoded protein complexes composed of a stable toxin and a short-lived inhibitor of the toxin. In cultures of Escherichia coli, transcription of toxin-antitoxin genes was induced in a nondividing subpopulation of bacteria that was tolerant to bactericidal antibiotics. Along with transcription of known toxin-antitoxin operons, transcription of mqsR and ygiT, two adjacent genes with multiple TA-like features, was induced in this cell population. Here we show that mqsR and ygiT encode a toxin-antitoxin system belonging to a completely new family which is represented in several groups of bacteria. The mqsR gene encodes a toxin, and ectopic expression of this gene inhibits growth and induces rapid shutdown of protein synthesis in vivo. ygiT encodes an antitoxin, which protects cells from the effects of MqsR. These two genes constitute a single operon which is transcriptionally repressed by the product of ygiT. We confirmed that transcription of this operon is induced in the ampicillin-tolerant fraction of a growing population of E. coli and in response to activation of the HipA toxin. Expression of the MqsR toxin does not kill bacteria but causes reversible growth inhibition and elongation of cells.

Processing of the precursor to a chloroplast ribosomal protein made in the cytosol occurs in two steps, one of which depends on a protein made in the chloroplast

1985 ◽  
Vol 5 (5) ◽  
pp. 1093-1099
Author(s):  
R J Schmidt ◽  
N W Gillham ◽  
J E Boynton
Keyword(s):  
Molecular Weight ◽  
Protein Synthesis ◽  
Ribosomal Protein ◽  
Apparent Molecular Weight ◽  
Protein L ◽  
Mature Form ◽  
Chloroplast Protein Synthesis ◽  
Made In

In pulse-chase experiments in which log-phase cells of Chlamydomonas reinhardtii were labeled in vivo for 5 min with H2(35)SO4, fluorographs of immunoprecipitates from whole cell extracts revealed that chloroplast ribosomal proteins L-2, L-6, L-21, and L-29, which are made in the cytosol and imported, appeared in their mature forms. However, in the case of chloroplast ribosomal protein L-18, which is also made in the cytoplasm and imported, a prominent precursor with an apparent molecular weight of 17,000 was found at the end of a 5-min pulse. This precursor was processed to its mature size (apparent molecular weight of 15,500) within the first 5 min of the subsequent chase. As determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the precursor to L-18 formed in vivo was 1.5 kilodaltons smaller than the primary product detected in translations of Chlamydomonas polyadenylated RNA in vitro. Upon a 10-min incubation with a postribosomal supernatant from Chlamydomonas, the 18,500-dalton precursor detected in vitro could be partially converted into a polypeptide that comigrated with the 17,000-dalton precursor detected in extracts of cells labeled in vivo. Under conditions in which the total amounts of chloroplast proteins had been reduced and cells were made to synthesize ribosomes rapidly, the apparent half-life of the 17,000-dalton precursor was extended over that seen in log-phase cells. When chloroplast protein synthesis was inhibited with lincomycin for 3 h before labeling under these conditions, the 17,000-dalton L-18 precursor but not the mature form was found, and the precursor was slowly degraded during a 60-min chase. When cells were placed in the dark for 3 h before labeling, processing of this precursor to the mature form appeared unaffected, but the chloroplast-synthesized ribosomal protein L-26 was detected, indicating that chloroplast protein synthesis was still occurring. We interpret these results to indicate that the maturation of protein L-18 in vivo involves at least two processing steps, one of which depends on a protein made on chloroplast ribosomes.

Relationship between glutamine concentration and protein synthesis in rat skeletal muscle

1988 ◽  
Vol 255 (2) ◽  
pp. E166-E172 ◽  
Author(s):  
M. M. Jepson ◽  
P. C. Bates ◽  
P. Broadbent ◽  
J. M. Pell ◽  
D. J. Millward
Keyword(s):  
Protein Synthesis ◽  
Protein Deficiency ◽  
Synthesis Rate ◽  
Protein Synthesis Rate ◽  
Glutamine Concentration ◽  
E Coli ◽  
Protein Group ◽  
Highly Correlated ◽  
Rna Concentration

Muscle glutamine concentration ([GLN]) and protein synthesis rate (Ks) have been examined in vivo in well-fed, protein-deficient, starved, and endotoxemic rats. With protein deficiency (8 or 5% casein diet), [GLN] fell from 7.70 to 5.58 and 3.56 mmol/kg in the 8 and 5% diet groups, with Ks falling from 15.42 to 9.1 and 6.84%/day. Three-day starvation reduced [GLN] and Ks to 2.38 mmol/kg and 5.6%/day, respectively. In all these groups food intakes and insulin were generally well maintained (except in the starved group), whereas free 3,5,3'-triiodothyronine (T3) was depressed in the starved and 5% protein group. The E. coli lipopolysaccharide endotoxin (3 mg/kg) reduced [GLN] to 5.85 and 4.72 mmol/kg and Ks to 10.5 and 9.10%/day in two well-fed groups. Insulin levels were increased, and free T3 levels fell. Combined protein deficiency and endotoxemia further reduced [GLN] and Ks to 1.88 mmol/kg and 4.01%/day, respectively, in the 5% protein rats. Changes in both ribosomal activity (KRNA) and concentration (RNA/protein) contributed to the fall in Ks in malnutrition and endotoxemia, although reductions in the RNA concentration were most marked with protein deficiency and reductions in the KRNA dominated the response to the endotoxin. The changes in [GLN] and Ks were highly correlated as were [GLN] and both KRNA and the RNA concentration, and these relationships were unique to glutamine. These relationships could reflect sensitivity of glutamine transport and protein synthesis to the same regulatory influences, and the particular roles of insulin and T3 are discussed, as well as any direct influence of glutamine on protein synthesis.

Translational regulation of ribosomal protein synthesis during xenopus development

1989 ◽  
Vol 27 ◽  
pp. 219
Author(s):  
B. Cardinali ◽  
C. Bagni ◽  
F. Amaldi ◽  
N. Campioni ◽  
P. Mariottini ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Ribosomal Protein ◽  
Translational Regulation ◽  
Xenopus Development ◽  
Ribosomal Protein Synthesis
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