Nonbridging phosphate oxygens in 16S rRNA important for 30S subunit assembly and association with the 50S ribosomal 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°.

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Characterization of 16S rRNA Processing with Pre-30S Subunit Assembly Intermediates from E. coli

Journal of Molecular Biology ◽

10.1016/j.jmb.2018.04.009 ◽

2018 ◽

Vol 430 (12) ◽

pp. 1745-1759 ◽

Cited By ~ 16

Author(s):

Brian A. Smith ◽

Neha Gupta ◽

Kevin Denny ◽

Gloria M. Culver

Keyword(s):

16S Rrna ◽

Rrna Processing ◽

Subunit Assembly ◽

E Coli ◽

30S Subunit

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Analysis of Conformational Changes in 16S rRNA During the Course of 30S Subunit Assembly

Journal of Molecular Biology ◽

10.1016/j.jmb.2005.09.056 ◽

2005 ◽

Vol 354 (2) ◽

pp. 340-357 ◽

Cited By ~ 30

Author(s):

Kristi L. Holmes ◽

Gloria M. Culver

Keyword(s):

16S Rrna ◽

Conformational Changes ◽

Subunit Assembly ◽

30S Subunit

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Abstract P-27: The 30S Ribosomal Subunit Assembly Factor Rbfa Plays a Key Role in the Formation of the Central Pseudoknot and in the Correct Docking of Helix 44 of the Decoding Center

International Journal of Biomedicine ◽

10.21103/ijbm.11.suppl_1.p27 ◽

2021 ◽

Vol 11 (Suppl_1) ◽

pp. S23-S24

Author(s):

Elena Maksimova ◽

Alexey Korepanov ◽

Olesya Kravchenko ◽

Timur Baymukhametov ◽

Alexander Myasnikov ◽

...

Keyword(s):

Ribosome Biogenesis ◽

Ribosomal Subunit ◽

Coli Strain ◽

Assembly Process ◽

Subunit Assembly ◽

Head Domain ◽

Multi Stage ◽

30S Subunit ◽

Assembly Factor

Background: Ribosome biogenesis is a complicated multi-stage process. In the cell, 30S ribosomal subunit assembly is fast and efficient, proceeding with the help of numerous assembly protein factors. The exact role of most assembly factors and mechanistic details of their operation remain unclear. The combination of genetic modification with cryo-EM analysis is widely used to identify the role of protein factors in assisting specific steps of the ribosome assembly process. The strain with knockout of a single assembly factor gene accumulates immature ribosomal particles which structural characterization reveals the information about the reactions catalyzed by the corresponding factor. Methods: We isolated the immature 30S subunits (pre-30S subunits) from the Escherichia coli strain lacking the rbfA gene (ΔrbfA) and characterized them by cryo-electron microscopy (cryo-EM). Results: Deletion of the assembly factor RbfA caused a substantial distortion of the structure of an important central pseudoknot which connects three major domains of 30S subunit and is necessary for ribosome stability. It was shown that the relative order of the assembly of the 3′ head domain and the docking of the functionally important helix 44 depends on the presence of RbfA. The formation of the central pseudoknot may promote stabilization of the head domain, likely through the RbfA-dependent maturation of the neck helix 28. The cryo-EM maps for pre-30S subunits were divided into the classes corresponding to consecutive assembly intermediates: from the particles with completely unresolved head domain and unfolded central pseudoknot to almost mature 30S subunits with well-resolved body, platform, and head domains and with partially distorted helix 44. Cryo-EM analysis of ΔrbfA 30S particles revealing the accumulation of two predominant classes of early and late intermediates (obtained at 2.7 Å resolutions) allowed us to suggest that RbfA participate in two stages of the 30S subunit assembly and is deeper involved in the maturation process than previously thought. Conclusion: In summary, RbfA acts at two distinctive 30S assembly stages: early formation of the central pseudoknot including the folding of the head, and positioning of helix 44 in the decoding center at a later stage. An update to the model of factor-dependent 30S maturation was proposed, suggesting that RfbA is involved in most of the subunit assembly process.

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Site-Directed Mutants of 16S rRNA Reveal Important RNA Domains for KsgA Function and 30S Subunit Assembly

Biochemistry ◽

10.1021/bi101005r ◽

2011 ◽

Vol 50 (5) ◽

pp. 854-863 ◽

Cited By ~ 11

Author(s):

Pooja M. Desai ◽

Gloria M. Culver ◽

Jason P. Rife

Keyword(s):

16S Rrna ◽

Subunit Assembly ◽

30S Subunit ◽

Rna Domains

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

Acta Naturae ◽

10.32607/20758251-2012-4-4-65-72 ◽

2012 ◽

Vol 4 (4) ◽

pp. 65-72 ◽

Cited By ~ 1

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.

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

10.1101/2020.03.21.001677 ◽

2020 ◽

Cited By ~ 2

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.

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

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100153221 ◽

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.

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