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 Alternative Conformations and Motions Adopted by 30S Ribosomal Subunits Visualized by Cryo-Electron Microscopy

2020 ◽  
Author(s):  
Dushyant Jahagirdar ◽  
Vikash Jha ◽  
Kaustuv Basu ◽  
Josue Gomez-Blanco ◽  
Javier Vargas ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
16S Rrna ◽  
Ribosomal Proteins ◽  
Structural Changes ◽  
Ribosomal Subunit ◽  
Conformational Space ◽  
Inactive Conformation ◽  
Cryo Electron Microscopy ◽  
30S Subunit

ABSTRACTIt is only after recent advances in cryo-electron microscopy that is now possible to describe at high resolution structures of large macromolecules that do not crystalize. Purified 30S subunits interconvert between the “active” and “inactive” conformations. The active conformation was described by crystallography in the early 2000s, but the structure of the inactive form at high resolution remains unsolved. Here we used cryo-electron microscopy to obtain the structure of the inactive conformation of the 30S subunit to 3.6Å resolution and study its motions. In the inactive conformation, three nucleotides at the 3’ end of the 16S rRNA cause the region of helix 44 forming the decoding center to adopt an unlatched conformation and the 3’ end of the 16S rRNA positions similarly to the mRNA during translation. Incubation of inactive 30S subunits at 42 °C reverts these structural changes. The position adopted by helix 44 dictates the most prominent motions of the 30S subunit. We found that extended exposures to low magnesium concentrations induces unfolding of large rRNA structural domains. The air-water interface to which ribosome subuints are exposed during sample preparation also peel off some ribosomal proteins. Overall this study provides new insights about the conformational space explored by the 30S ribosomal subunit when the ribosomal particles are free in solution.

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

Acta Naturae ◽  
2012 ◽  
Vol 4 (4) ◽  
pp. 65-72 ◽  
Author(s):  
A. V. Golovin ◽  
G. A. Khayrullina ◽  
B. Kraal ◽  
А. М. Kopylov
Keyword(s):  
16S Rrna ◽  
Ribosomal Protein ◽  
Self Assembly ◽  
Ribosomal Proteins ◽  
Ribosome Biogenesis ◽  
Ribosomal Subunit ◽  
Regulatory Function ◽  
Binary Complex ◽  
30S Subunit ◽  
Cross Links

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 RNAprotein interactions within binary complexes is obligatory for understanding ribosome biogenesis. The non-conventional RNAprotein contact within the binary complex of recombinant ribosomal protein S7 and its 16S rRNA binding site (236 nucleotides) was identified. UVinduced RNAprotein 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 crosslink 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 RNAprotein 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.

Structural Role of rRNAs on the Ribosomal Subunits from E. Coli by Scanning Transmission Electron Microscopy

1981 ◽  
Vol 39 ◽  
pp. 424-425
Author(s):  
M. Boublik ◽  
N. Robakis ◽  
J.S. Wall
Keyword(s):  
Three Dimensional ◽  
Uranyl Acetate ◽  
Dimensional Structure ◽  
Electron Microscopic ◽  
Ribosomal Subunits ◽  
E Coli ◽  
Freeze Dried ◽  
16S Rna ◽  
Scanning Transmission ◽  
And Function

The three-dimensional structure and function of biological supramolecular complexes are, in general, determined and stabilized by conformation and interactions of their macromolecular components. In the case of ribosomes, it has been suggested that one of the functions of ribosomal RNAs is to act as a scaffold maintaining the shape of the ribosomal subunits. In order to investigate this question, we have conducted a comparative TEM and STEM study of the structure of the small 30S subunit of E. coli and its 16S RNA.The conventional electron microscopic imaging of nucleic acids is performed by spreading them in the presence of protein or detergent; the particles are contrasted by electron dense solution (uranyl acetate) or by shadowing with metal (tungsten). By using the STEM on freeze-dried specimens we have avoided the shearing forces of the spreading, and minimized both the collapse of rRNA due to air drying and the loss of resolution due to staining or shadowing. Figure 1, is a conventional (TEM) electron micrograph of 30S E. coli subunits contrasted with uranyl acetate.

Imaging of ribosomal RNA-protein interactions by dark-field STEM

1989 ◽  
Vol 47 ◽  
pp. 250-251
Author(s):  
M. Boublik ◽  
V. Mandiyan ◽  
S. Tumminia ◽  
J.F. Hainfeld ◽  
J.S. Wall
Keyword(s):  
Nucleic Acid ◽  
16S Rrna ◽  
Dark Field ◽  
Radius Of Gyration ◽  
Central Domain ◽  
The Core ◽  
16S Rna ◽  
30S Subunit ◽  
Core Particles

Success in protein-free deposition of native nucleic acid molecules from solutions of selected ionic conditions prompted attempts for high resolution imaging of nucleic acid interactions with proteins, not attainable by conventional EM. Since the nucleic acid molecules can be visualized in the dark-field STEM mode without contrasting by heavy atoms, the established linearity between scattering cross-section and molecular weight can be applied to the determination of their molecular mass (M) linear density (M/L), mass distribution and radius of gyration (RG). Determination of these parameters promotes electron microscopic imaging of biological macromolecules by STEM to a quantitative analytical level. This technique is applied to study the mechanism of 16S rRNA folding during the assembly process of the 30S ribosomal subunit of E. coli. The sequential addition of protein S4 which binds to the 5'end of the 16S rRNA and S8 and S15 which bind to the central domain of the molecule leads to a corresponding increase of mass and increased coiling of the 16S rRNA in the core particles. This increased compactness is evident from the decrease in RG values from 114Å to 91Å (in “ribosomal” buffer consisting of 10 mM Hepes pH 7.6, 60 mM KCl, 2 m Mg(OAc)2, 1 mM DTT). The binding of S20, S17 and S7 which interact with the 5'domain, the central domain and the 3'domain, respectively, continues the trend of mass increase. However, the RG values of the core particles exhibit a reverse trend, an increase to 108Å. In addition, the binding of S7 leads to the formation of a globular mass cluster with a diameter of about 115Å and a mass of ∽300 kDa. The rest of the mass, about 330 kDa, remains loosely coiled giving the particle a “medusa-like” appearance. These results provide direct evidence that 16S RNA undergoes significant structural reorganization during the 30S subunit assembly and show that its interactions with the six primary binding proteins are not sufficient for 16S rRNA coiling into particles resembling the native 30S subunit, contrary to what has been reported in the literature.

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.

Structure and function of ribosomal RNA

1995 ◽  
Vol 73 (11-12) ◽  
pp. 997-1009 ◽  
Author(s):  
Harry F. Noller ◽  
Rachel Green ◽  
Gabriele Heilek ◽  
Vernita Hoffarth ◽  
Alexander Hüttenhofer ◽  
...  
Keyword(s):  
16S Rrna ◽  
Hydroxyl Radical ◽  
Ribosomal Proteins ◽  
23S Rrna ◽  
Active Population ◽  
30S Subunit ◽  
Radical Cleavage ◽  
The Individual ◽  
And Function

A refined model has been developed for the folding of 16S rRNA in the 30S subunit, based on additional constraints obtained from new experimental approaches. One set of constraints comes from hydroxyl radical footprinting of each of the individual 30S ribosomal proteins, using free Fe2+–EDTA complex. A second approach uses localized hydroxyl radical cleavage from a single Fe2+tethered to unique positions on the surface of single proteins in the 30S subunit. This has been carried out for one position on the surface of protein S4, two on S17, and three on S5. Nucleotides in 16S rRNA that are essential for P-site tRNA binding were identified by a modification interference strategy. Ribosomal subunits were partially inactivated by chemical modification at a low level. Active, partially modified subunits were separated from inactive ones by binding 3′-biotin-derivatized tRNA to the 30S subunits and captured with streptavidin beads. Essential bases are those that are unmodified in the active population but modified in the total population. The four essential bases, G926, 2mG966, G1338, and G1401 are a subset of those that are protected from modification by P-site tRNA. They are all located in the cleft of our 30S subunit model. The rRNA neighborhood of the acceptor end of tRNA was probed by hydroxyl radical probing from Fe2+tethered to the 5′ end of tRNA via an EDTA linker. Cleavage was detected in domains IV, V, and VI of 23S rRNA, but not in 5S or 16S rRNA. The sites were all found to be near bases that were protected from modification by the CCA end of tRNA in earlier experiments, except for a set of E-site cleavages in domain IV and a set of A-site cleavages in the α-sarcin loop of domain VI. In vitro genetics was used to demonstrate a base-pairing interaction between tRNA and 23S rRNA. Mutations were introduced at positions C74 and C75 of tRNA and positions 2252 and 2253 of 23S rRNA. Interaction of the CCA end of tRNA with mutant ribosomes was tested using chemical probing in conjunction with allele-specific primer extension. The interaction occurred only when there was a Watson–Crick pairing relationship between positions 74 of tRNA and 2252 of 23S rRNA. Using a novel chimeric in vitro reconstitution method, it was shown that the peptidyl transferase reaction depends on this same Watson–Crick base pair.Key words: rRNA, ribosome, tRNA, hydroxyl radical, ribosomal protein.

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.

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