Single protein omission reconstitution studies of tetracycline binding to the 30S subunit of Escherichia coli ribosomes

Buck, Melissa A.; Cooperman, Barry S.

doi:10.1021/bi00474a024

Cited by 53 publications

(22 citation statements)

References 34 publications

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“…The complete suppression of the partition between the head and body is in agreement with immunoelectron microscopic localization of S3 and S2 (41). A comparison of the results of sequential protein addition and protein omission experiments revealed that the association of S3 and S2 depends on prior binding of S14 and S10 (7,43). Protein-protein crosslinks (20) observed in the 30S subunit between S3-S10 and S3-S4 suggest that S3 may bridge the 3' and 5' domains.…”

Section: Resultssupporting

confidence: 75%

“…Further studies provided evidence that S5 association stimulates the binding of S12 (7,36,40). Single protein omission experiments established that the lack of S5 has a significant effect on the sedimentation coefficient of core particles (43), which may be due to the absence of merging of the central and 5' domains. Interestingly, S5 and S12 are part of the domain where EF-Tu and EF-G interact with the 30S subunit (44,45).…”

Section: Resultsmentioning

confidence: 98%

“…Thus, the merging of the head and body involves the interactions of S3 with S10 and S4, and of S2 with S8, S5, and S3. The 30S particles assembled in the absence ofprotein S5 are deficient in protein S2 and partially deficient in protein S3 (7,43). Thus, proteins S3 and S2 may be filling up the regions between S10 and S4 and S5 and S8, respectively, leading to the formation of the compact 30S particle.…”

Section: Resultsmentioning

confidence: 99%

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Assembly of the Escherichia coli 30S ribosomal subunit reveals protein-dependent folding of the 16S rRNA domains.

Mandiyan

Tumminia

Wall

et al. 1991

Proc. Natl. Acad. Sci. U.S.A.

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Protein-nucleic acid interactions involved in the assembly process of the Escherichia coli 30S ribosomal subunit were quantitatively analyzed by high-resolution scanning transmission electron microscopy. The in vitro reconstituted ribonucleoprotein (core) particles were characterized by their morphology, mass, and radii of gyration. During the assembly of the 30S subunit, the 16S rRNA underwent significant conformational changes that were governed by the cooperative interactions of the ribosomal proteins. The sequential association of the first 12 proteins with the 16S rRNA resulted in the formation of core particles containing up to three mass centers at distinct stages of the assembly process. These globular mass centers may correspond to the three major domains (5', central, and 3') of the 16S rRNA. Through the subsequent interactions of the late assembly proteins with the 16S rRNA, two of the three domains merge, yielding the basic structural traits ofthe native 30S subunit. The fine morphological features of the native 30S subunit became distinctly resolved only after the addition of the full complement of proteins. The fully reconstituted 30S subunits are active in polyphenylalanine synthesis assays. Visualiztion of the assembly mechanism of the E. coli 30S ribosomal subunit revealed domain-specific folding of the 16S rRNA through the formation bf distinct intermediate core particles hitherto not observed.Ribosomes are ubiquitous macromolecular assemblies of proteins and ribonucleic acids involved in protein biosynthesis. This bipartite cellular organelle is composed of a large and a small subunit. The structure-function relationship of the small (30S) ribosomal subunit ofEscherichia coli has been extensively studied. This subunit is actively involved in the initiation of protein synthesis by its interactions with initiation factors, messenger RNAs, transfer RNAs, and the large (50S) ribosomal subunit (1-4). The 30S subunit possesses characteristic structural features known as the head, body, cleft, and platform (5, 6) and is composed of a single 16S rRNA molecule and 21 different ribosomal proteins (S1-S21). Several ribosomal proteins interact with the 16S rRNA through a sequential and cooperative process as revealed from the in vitro assembly of the 30S subunit (7-9). Results obtained from these studies led to the classification of the proteins as primary binding proteins, which bind directly to the 16S rRNA, and secondary binding proteins, which require the previous association of the primary binding proteins. Apart from being the structural base for protein interactions, the 16S rRNA has an established role in protein biosynthesis (10-14). However, the role of the ribosomal proteins in ribosome structure and function remains unclear.Scanning transmission electron microscopy (STEM) was used to quantitatively evaluate the conformational changes induced in the 16S rRNA molecule by its interactions with the ribosomal proteins under nondenaturing conditions (15). Unlike other physical techniques tha...

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Section: Resultssupporting

confidence: 75%

Section: Resultsmentioning

confidence: 98%

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Assembly of the Escherichia coli 30S ribosomal subunit reveals protein-dependent folding of the 16S rRNA domains.

Mandiyan

Tumminia

Wall

et al. 1991

Proc. Natl. Acad. Sci. U.S.A.

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“…Underexpression of gyrB sensitizes B. cenocepacia (this study) and S. aureus (35) to tetracycline. Despite the ribosome being the primary target of tetracycline (40), tetracycline can also can bind and introduce damage to DNA, which is enhanced by the formation of metal complexes (41,42). GyrB depletion also sensitized B. cenocepacia to colistin, although the effect was observed only with colistin at the IC 30 .…”

Section: Discussionmentioning

confidence: 99%

Competitive Growth Enhances Conditional Growth Mutant Sensitivity to Antibiotics and Exposes a Two-Component System as an Emerging Antibacterial Target in Burkholderia cenocepacia

Gislason

Choy

Bloodworth

et al. 2017

Antimicrob Agents Chemother

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Chemogenetic approaches to profile an antibiotic mode of action are based on detecting differential sensitivities of engineered bacterial strains in which the antibacterial target (usually encoded by an essential gene) or an associated process is regulated. We previously developed an essential-gene knockdown mutant library in the multidrug-resistant Burkholderia cenocepacia by transposon delivery of a rhamnose-inducible promoter. In this work, we used Illumina sequencing of multiplex-PCR-amplified transposon junctions to track individual mutants during pooled growth in the presence of antibiotics. We found that competition from nontarget mutants magnified the hypersensitivity of a clone underexpressing gyrB to novobiocin by 8-fold compared with hypersensitivity measured during clonal growth. Additional profiling of various antibiotics against a pilot library representing most categories of essential genes revealed a two-component system with unknown function, which, upon depletion of the response regulator, sensitized B. cenocepacia to novobiocin, ciprofloxacin, tetracycline, chloramphenicol, kanamycin, meropenem, and carbonyl cyanide 3-chlorophenylhydrazone, but not to colistin, hydrogen peroxide, and dimethyl sulfoxide. We named the gene cluster esaSR for enhanced sensitivity to antibiotics sensor and response regulator. Mutational analysis and efflux activity assays revealed that while esaS is not essential and is involved in antibiotic-induced efflux, esaR is an essential gene and regulates efflux independently of antibiotic-mediated induction. Furthermore, microscopic analysis of cells stained with propidium iodide provided evidence that depletion of EsaR has a profound effect on the integrity of cell membranes. In summary, we unraveled a previously uncharacterized two-component system that can be targeted to reduce antibiotic resistance in B. cenocepacia.

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“…Binding of the drug to the ribosome prevents the a�achment of the aminoacyl-tRNA to the "A site" of the ribosome. Tetracyclines bind directly to the 30S-subunit protein S7 (Goldman et al, 1983), other ribosomal proteins (S3, S14, and S19) are also involved (Franklin, 1966;Buck and Cooperman, 1990). Some bases in the 16S-rRNA, e.g.…”

Section: Mode Of Actionmentioning

confidence: 99%

Tetracyclines in veterinary medicine and bacterial resistance to them

Michalova¹,

PNovotna²,

Schlegelová³

2004

Vet. Med.

121

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Since their discovery in 1945, tetracyclines have been used extensively in the therapy and prophylaxis of infectious diseases and as growth promoters. These wide applications have led to the equally fast spread of tetracycline resistant strains of gram-positive and gram-negative bacterial genera, including strains belonging to pathogenic as well as nonpathogenic species. Nonpathogenic bacteria could act as a reservoir of resistance determinants, which can be disseminated by horizontal transfer into pathogens. More than thirty different tetracycline resistance genes have been characterized. They encode two major mechanisms of resistance: 1 -active efflux of the antibiotic, and 2 -protection of ribosomes. Further mechanisms of tetracycline resistance include enzymatic inactivation of antibiotic, permeability barriers, mutations or multidrug transporter systems. Effective horizontal spread is favoured by the location of tetracycline resistance genes on mobile genetic elements such as plasmids and transposons. Their exchange, enhanced by the use of tetracyclines, is observed between bacteria of the same or different species and genera as well. Thus, questions of reevaluating and global reducing of tetracyclines in human and animal healthcare and food production are extensively discussed.

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Single protein omission reconstitution studies of tetracycline binding to the 30S subunit of Escherichia coli ribosomes

Cited by 53 publications

References 34 publications

Assembly of the Escherichia coli 30S ribosomal subunit reveals protein-dependent folding of the 16S rRNA domains.

Assembly of the Escherichia coli 30S ribosomal subunit reveals protein-dependent folding of the 16S rRNA domains.

Competitive Growth Enhances Conditional Growth Mutant Sensitivity to Antibiotics and Exposes a Two-Component System as an Emerging Antibacterial Target in Burkholderia cenocepacia

Tetracyclines in veterinary medicine and bacterial resistance to them

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