scholarly journals A NEW 3-D RECONSTRUCTION SCHEME APPLIED TO THE 50S RIBOSOMAL SUBUNIT OF E. COLI

1986 ◽  
Vol 141 (1) ◽  
pp. RP1-RP2 ◽  
Author(s):  
M. Radermacher ◽  
T. Wagenknecht ◽  
A. Verschoor ◽  
J. Frank
Keyword(s):  
Ribosomal Subunit ◽  
E Coli ◽  
Reconstruction Scheme

3D reconstruction of the 50s ribosomal subunit of escherichia coli

1986 ◽  
Vol 44 ◽  
pp. 40-41
Author(s):  
M. Radermacher ◽  
T. Wagenknecht ◽  
A. Verschoor ◽  
J. Frank
Keyword(s):  
3D Reconstruction ◽  
3D Structure ◽  
Three Dimensional ◽  
Ribosomal Subunit ◽  
Main Body ◽  
Large Ribosomal Subunit ◽  
E Coli ◽  
Reconstruction Scheme ◽  
Particle Preparation ◽  
Electron Microscopical

The three-dimensional (3D) structure of the large ribosomal subunit from E. coli was determined from micrographs of a negatively stained 50S particle preparation using our new reconstruction scheme. The 50S subunit occurs in electron microscopical preparations mainly in the crown-view orientation with the interface side of the main body situated parallel to the specimen plane, but in random in plane orientations. An image of such a specimen tilted by a large tilt angle, which inherently contains a conical tilt series of the particle, was used to calculate a 3D reconstruction.

Protein-induced conformational changes in 16S rRNA during assembly of the Escherichia Coli 30S ribosomal subunit: A STEM study

1987 ◽  
Vol 45 ◽  
pp. 910-911
Author(s):  
M. Boublik ◽  
V. Mandiyan ◽  
J.F. Hainfeld ◽  
J.S. Wall
Keyword(s):  
16S Rrna ◽  
Conformational Changes ◽  
Ribosomal Proteins ◽  
Structural Changes ◽  
Ribosomal Subunit ◽  
Compact Structure ◽  
E Coli ◽  
Freeze Dried ◽  
16S Rna ◽  
30S Subunit

The aim of this study is to understand the mechanism of 16S rRNA folding into the compact structure of the small 30S subunit of E. coli ribosome. The assembly of the 30S E. coli ribosomal subunit is a sequence of specific interactions of 16S rRNA with 21 ribosomal proteins (S1-S21). Using dedicated high resolution STEM we have monitored structural changes induced in 16S rRNA by the proteins S4, S8, S15 and S20 which are involved in the initial steps of 30S subunit assembly. S4 is the first protein to bind directly and stoichiometrically to 16S rRNA. Direct binding also occurs individually between 16S RNA and S8 and S15. However, binding of S20 requires the presence of S4 and S8. The RNA-protein complexes are prepared by the standard reconstitution procedure, dialyzed against 60 mM KCl, 2 mM Mg(OAc)2, 10 mM-Hepes-KOH pH 7.5 (Buffer A), freeze-dried and observed unstained in dark field at -160°.

scholarly journals A comparison of the unfolding and dissociation of the large ribosome subunits from Rhodopseudomonas·spheroides N.C.I.B. 8253 and Escherichia coli M.R.E. 600

1973 ◽  
Vol 133 (4) ◽  
pp. 739-747 ◽  
Author(s):  
A. Robinson ◽  
J. Sykes
Keyword(s):  
Escherichia Coli ◽  
Protein Interactions ◽  
Ribosomal Proteins ◽  
Ribosomal Subunit ◽  
23S Rrna ◽  
Core Particle ◽  
Protein Loss ◽  
E Coli ◽  
Rrna Species ◽  
Rhodopseudomonas Spheroides

1. The behaviour of the large ribosomal subunit from Rhodopseudomonas spheroides (45S) has been compared with the 50S ribosome from Escherichia coli M.R.E. 600 (and E. coli M.R.E. 162) during unfolding by removal of Mg2+ and detachment of ribosomal proteins by high univalent cation concentrations. The extent to which these processes are reversible with these ribosomes has also been examined. 2. The R. spheroides 45S ribosome unfolds relatively slowly but then gives rise directly to two ribonucleoprotein particles (16.6S and 13.7S); the former contains the intact primary structure of the 16.25S rRNA species and the latter the 15.00S rRNA species of the original ribosome. No detectable protein loss occurs during unfolding. The E. coli ribosome unfolds via a series of discrete intermediates to a single, unfolded ribonucleoprotein unit (19.1S) containing the 23S rRNA and all the protein of the original ribosome. 3. The two unfolded R. spheroides ribonucleoproteins did not recombine when the original conditions were restored but each simply assumed a more compact configuration. Similar treatments reversed the unfolding of the E. coli 50S ribosomes; replacement of Mg2+ caused the refolding of the initial products of unfolding and in the presence of Ni2+ the completely unfolded species (19.1S) again sedimented at the same rate as the original ribosomes (44S). 4. Ribosomal proteins (25%) were dissociated from R. spheroides 45S ribosomes by dialysis against a solution with a Na+/Mg2+ ratio of 250:1. During this process two core particles were formed (21.2S and 14.2S) and the primary structures of the two original rRNA species were conserved. This dissociation was not reversed. With E. coli 50S approximately 15% of the original ribosomal protein was dissociated, a single 37.6S core particle was formed, the 23S rRNA remained intact and the ribosomal proteins would reassociate with the core particle to give a 50S ribosome. 5. The ribonuclease activities in R. spheroides 45S and E. coli M.R.E. 600 and E. coli M.R.E. 162 50S ribosomes are compared. 6. The observations concerning unfolding and dissociation are consistent with previous reports showing the unusual rRNA complement of the mature R. spheroides 45S ribosome and show the dependence of these events upon the rRNA and the importance of protein–protein interactions in the structure of the R. spheroides ribosome.

scholarly journals Studies of the Interaction of Escherichia coli YjeQ with the Ribosome In Vitro

2004 ◽  
Vol 186 (5) ◽  
pp. 1381-1387 ◽  
Author(s):  
Denis M. Daigle ◽  
Eric D. Brown
Keyword(s):  
Escherichia Coli ◽  
Ribosomal Subunit ◽  
Gtp Hydrolysis ◽  
E Coli ◽  
Cell Extracts ◽  
30S Subunit ◽  
Ob Fold ◽  
Nucleotide Binding Proteins ◽  
Dependent Interaction

ABSTRACT Escherichia coli YjeQ represents a conserved group of bacteria-specific nucleotide-binding proteins of unknown physiological function that have been shown to be essential to the growth of E. coli and Bacillus subtilis. The protein has previously been characterized as possessing a slow steady-state GTP hydrolysis activity (8 h−1) (D. M. Daigle, L. Rossi, A. M. Berghuis, L. Aravind, E. V. Koonin, and E. D. Brown, Biochemistry 41: 11109-11117, 2002). In the work reported here, YjeQ from E. coli was found to copurify with ribosomes from cell extracts. The copy number of the protein per cell was nevertheless low relative to the number of ribosomes (ratio of YjeQ copies to ribosomes, 1:200). In vitro, recombinant YjeQ protein interacted strongly with the 30S ribosomal subunit, and the stringency of that interaction, revealed with salt washes, was highest in the presence of the nonhydrolyzable GTP analog 5′-guanylylimidodiphosphate (GMP-PNP). Likewise, association with the 30S subunit resulted in a 160-fold stimulation of YjeQ GTPase activity, which reached a maximum with stoichiometric amounts of ribosomes. N-terminal truncation variants of YjeQ revealed that the predicted OB-fold region was essential for ribosome binding and GTPase stimulation, and they showed that an N-terminal peptide (amino acids 1 to 20 in YjeQ) was necessary for the GMP-PNP-dependent interaction of YjeQ with the 30S subunit. Taken together, these data indicate that the YjeQ protein participates in a guanine nucleotide-dependent interaction with the ribosome and implicate this conserved, essential GTPase as a novel factor in ribosome function.

scholarly journals Resistance mutations generate divergent antibiotic susceptibility profiles against translation inhibitors

2016 ◽  
Vol 113 (29) ◽  
pp. 8188-8193 ◽  
Author(s):  
Alexis I. Cocozaki ◽  
Roger B. Altman ◽  
Jian Huang ◽  
Ed T. Buurman ◽  
Steven L. Kazmirski ◽  
...  
Keyword(s):  
Binding Site ◽  
Single Molecule ◽  
Antibiotic Susceptibility ◽  
Ribosomal Subunit ◽  
Structural Defects ◽  
Resistance Mutations ◽  
Antibiotic Resistant ◽  
E Coli ◽  
X Ray Crystallography ◽  
Susceptibility Profiles

Mutations conferring resistance to translation inhibitors often alter the structure of rRNA. Reduced susceptibility to distinct structural antibiotic classes may, therefore, emerge when a common ribosomal binding site is perturbed, which significantly reduces the clinical utility of these agents. The translation inhibitors negamycin and tetracycline interfere with tRNA binding to the aminoacyl-tRNA site on the small 30S ribosomal subunit. However, two negamycin resistance mutations display unexpected differential antibiotic susceptibility profiles. Mutant U1060A in 16S Escherichia coli rRNA is resistant to both antibiotics, whereas mutant U1052G is simultaneously resistant to negamycin and hypersusceptible to tetracycline. Using a combination of microbiological, biochemical, single-molecule fluorescence transfer experiments, and X-ray crystallography, we define the specific structural defects in the U1052G mutant 70S E. coli ribosome that explain its divergent negamycin and tetracycline susceptibility profiles. Unexpectedly, the U1052G mutant ribosome possesses a second tetracycline binding site that correlates with its hypersusceptibility. The creation of a previously unidentified antibiotic binding site raises the prospect of identifying similar phenomena in antibiotic-resistant pathogens in the future.

scholarly journals An O-Phosphotransferase Catalyzes Phosphorylation of Hygromycin A in the Antibiotic-Producing Organism Streptomyces hygroscopicus

2008 ◽  
Vol 52 (10) ◽  
pp. 3580-3588 ◽  
Author(s):  
Vidya Dhote ◽  
Shuchi Gupta ◽  
Kevin A. Reynolds
Keyword(s):  
Ribosomal Subunit ◽  
Biosynthetic Gene Cluster ◽  
Open Reading Frames ◽  
Streptomyces Hygroscopicus ◽  
Gram Negative Bacteria ◽  
E Coli ◽  
Naturally Occurring ◽  
Insertional Inactivation ◽  
Pathway Regulation ◽  
Reading Frames

ABSTRACT The antibiotic hygromycin A (HA) binds to the 50S ribosomal subunit and inhibits protein synthesis in gram-positive and gram-negative bacteria. The HA biosynthetic gene cluster in Streptomyces hygroscopicus NRRL 2388 contains 29 open reading frames, which have been assigned putative roles in biosynthesis, pathway regulation, and self-resistance. The hyg21 gene encodes an O-phosphotransferase with a proposed role in self-resistance. We observed that insertional inactivation of hyg21 in S. hygroscopicus leads to a greater than 90% decrease in HA production. The wild type and the hyg21 mutant were comparably resistant to HA. Using Escherichia coli as a heterologous host, we expressed and purified Hyg21. Kinetic analyses revealed that the recombinant protein catalyzes phosphorylation of HA (Km = 30 ± 4 μM) at the C-2‴ position of the fucofuranose ring in the presence of ATP (Km = 200 ± 20 μM) or GTP (Km = 350 ± 60 μM) with a k cat of 2.2 ± 0.1 min−1. The phosphorylated HA is inactive against HA-sensitive ΔtolC E. coli and Streptomyces lividans. Hyg21 also phosphorylates methoxyhygromycin A and desmethylenehygromycin A with k cat and Km values similar to those observed with HA. Phosphorylation of the naturally occurring isomers of 5‴-dihydrohygromycin A and 5‴-dihydromethoxyhygromycin A was about 12 times slower than for the corresponding non-natural isomers. These studies demonstrate that Hyg21 is an O-phosphotransferase with broad substrate specificity, tolerating changes in the aminocyclitol moiety more than in the fucofuranose moiety, and that phosphorylation by Hyg21 is one of several possible mechanisms of self-resistance in S. hygroscopicus NRRL 2388.

scholarly journals Regulation of Ribosomal Protein OperonsrplM-rpsI,rpmB-rpmG, andrplU-rpmAat the Transcriptional and Translational Levels

2016 ◽  
Vol 198 (18) ◽  
pp. 2494-2502 ◽  
Author(s):  
Leonid V. Aseev ◽  
Ludmila S. Koledinskaya ◽  
Irina V. Boni
Keyword(s):  
Ribosomal Proteins ◽  
Ribosome Biogenesis ◽  
Ribosomal Subunit ◽  
General Rule ◽  
Single Copy ◽  
Translation Initiation Region ◽  
Content Type ◽  
Regulatory Pathways ◽  
E Coli

ABSTRACTIt is widely assumed that in the best-characterized model bacteriumEscherichia coli, transcription units encoding ribosomal proteins (r-proteins) and regulation of their expression have been already well defined. However, transcription start sites for severalE. colir-protein operons have been established only very recently, so that information concerning the regulation of these operons at the transcriptional or posttranscriptional level is still missing. This paper describes for the first time thein vivoregulation of three r-protein operons,rplM-rpsI,rpmB-rpmG, andrplU-rpmA. The results demonstrate that transcription of all three operons is subject to ppGpp/DksA-dependent negative stringent control under amino acid starvation, in parallel with the rRNA operons. By using single-copy translational fusions with the chromosomallacZgene, we show here that at the translation level only one of these operons,rplM-rpsI, is regulated by the mechanism of autogenous repression involving the 5′ untranslated region (UTR) of the operon mRNA, whilerpmB-rpmGandrplU-rpmAare not subject to this type of regulation. This may imply that translational feedback control is not a general rule for modulating the expression ofE. colir-protein operons. Finally, we report that L13, a primary protein in 50S ribosomal subunit assembly, serves as a repressor ofrplM-rpsIexpressionin vivo, acting at a target within therplMtranslation initiation region. Thus, L13 represents a novel example of regulatory r-proteins in bacteria.IMPORTANCEIt is important to obtain a deeper understanding of the regulatory mechanisms responsible for coordinated and balanced synthesis of ribosomal components. In this paper, we highlight the major role of a stringent response in regulating transcription of three previously unexplored r-protein operons, and we show that only one of them is subject to feedback regulation at the translational level. Improved knowledge of the regulatory pathways controlling ribosome biogenesis may promote the development of novel antibacterial agents.

scholarly journals Improving the Methanol Tolerance of an Escherichia coli Methylotroph via Adaptive Laboratory Evolution Enhances Synthetic Methanol Utilization

2021 ◽  
Vol 12 ◽  
Author(s):  
R. Kyle Bennett ◽  
Gwendolyn J. Gregory ◽  
Jacqueline E. Gonzalez ◽  
Jie Ren Gerald Har ◽  
Maciek R. Antoniewicz ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Ribosomal Subunit ◽  
Biological Properties ◽  
Methanol Tolerance ◽  
Adaptive Laboratory Evolution ◽  
Laboratory Evolution ◽  
E Coli ◽  
Translational Accuracy ◽  
Methanol Utilization ◽  
Carbon Compounds

There is great interest in developing synthetic methylotrophs that harbor methane and methanol utilization pathways in heterologous hosts such as Escherichia coli for industrial bioconversion of one-carbon compounds. While there are recent reports that describe the successful engineering of synthetic methylotrophs, additional efforts are required to achieve the robust methylotrophic phenotypes required for industrial realization. Here, we address an important issue of synthetic methylotrophy in E. coli: methanol toxicity. Both methanol, and its oxidation product, formaldehyde, are cytotoxic to cells. Methanol alters the fluidity and biological properties of cellular membranes while formaldehyde reacts readily with proteins and nucleic acids. Thus, efforts to enhance the methanol tolerance of synthetic methylotrophs are important. Here, adaptive laboratory evolution was performed to improve the methanol tolerance of several E. coli strains, both methylotrophic and non-methylotrophic. Serial batch passaging in rich medium containing toxic methanol concentrations yielded clones exhibiting improved methanol tolerance. In several cases, these evolved clones exhibited a > 50% improvement in growth rate and biomass yield in the presence of high methanol concentrations compared to the respective parental strains. Importantly, one evolved clone exhibited a two to threefold improvement in the methanol utilization phenotype, as determined via 13C-labeling, at non-toxic, industrially relevant methanol concentrations compared to the respective parental strain. Whole genome sequencing was performed to identify causative mutations contributing to methanol tolerance. Common mutations were identified in 30S ribosomal subunit proteins, which increased translational accuracy and provided insight into a novel methanol tolerance mechanism. This study addresses an important issue of synthetic methylotrophy in E. coli and provides insight as to how methanol toxicity can be alleviated via enhancing methanol tolerance. Coupled improvement of methanol tolerance and synthetic methanol utilization is an important advancement for the field of synthetic methylotrophy.

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