The Loop 2 Region of Ribosomal Protein uS5 Influences Spectinomycin Sensitivity, Translational Fidelity, and Ribosome Biogenesis

Kamath, Divya; Gregory, Steven T.; O’Connor, Michael B.

doi:10.1128/aac.01186-16

Cited by 8 publications

(3 citation statements)

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“…The restored C-terminus also reduces, but does not eliminate, stop codon readthrough, which remains substantially above wild type in both the Δ5rev4 and Δ5rev5 mutants. This is consistent with our previous studies on miscoding, where only a minority of the uS4, uS5 or uS12 mutants display processing or assembly defects [7, 27, 28]. Thus, while the C-terminus has a major effect on both miscoding and ribosome profiles, ribosome biogenesis appears to be less sensitive than decoding accuracy to the identity of the residues in the C-terminal region of uS4.…”

Section: Resultssupporting

confidence: 91%

The C-terminus of ribosomal protein uS4 contributes to small ribosomal subunit biogenesis and the fidelity of translation

et al. 2017

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Ribosomal protein uS4 is an essential ribosomal component involved in multiple functions, including mRNA decoding. Structural analyses indicate that during decoding, the interface between the C-terminus of uS4 and protein uS5 is disrupted and in agreement with this, C-terminal uS4 truncation mutants are readily isolated on the basis of their increased miscoding phenotypes. The same mutants can also display defects in small subunit assembly and 16S rRNA processing and some are temperature sensitive for growth. Starting with one such temperature sensitive Escherichia coli uS4 mutant, we have isolated temperature insensitive derivatives carrying additional, intragenic mutations that restore the C-terminus and ameliorate the ribosomal defects. At least one of these suppressors has no detectable ribosome biogenesis phenotype, yet still miscodes, suggesting that the C-terminal requirements for ribosome assembly are less rigid than for mRNA decoding. In contrast to the uS4 C-terminal mutants that increase miscoding, two Salmonella enterica uS4 mutants with altered C-termini have been reported as being error-restrictive. Here, reconstruction experiments demonstrate that contrary to the previous reports, these mutants have a distinct error-prone, increased misreading phenotype, consistent with the behavior of the equivalent E. coli mutants and their likely structural effects on uS4-uS5 interactions.

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Section: Resultssupporting

confidence: 91%

The C-terminus of ribosomal protein uS4 contributes to small ribosomal subunit biogenesis and the fidelity of translation

et al. 2017

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“…3d). This agrees well with reports showing that mutations in the loop 2 regions confer resistance to Spt and affect translation fidelity 117,118 . Whether Spt is bound to ribosomes of the other four classes could not be unambiguously determined due to low resolutions.…”

Section: Supplementary Discussionsupporting

confidence: 93%

Visualizing translation dynamics at atomic detail inside a bacterial cell

Xue

Lenz

Zimmermann-Kogadeeva

et al. 2021

Preprint

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Translation is the fundamental process of protein synthesis and is catalysed by the ribosome in all living cells. Here, we use cryo-electron tomography and sub-tomogram analysis to visualize the dynamics of translation inside the prokaryote Mycoplasma pneumoniae. We first obtain an in-cell atomic model for the M. pneumoniae ribosome that reveals distinct extensions of ribosomal proteins. Classification then resolves thirteen ribosome states that differ in conformation and composition and reflect intermediates during translation. Based on these states, we animate translation elongation and demonstrate how antibiotics reshape the translation landscape inside cells. During translation elongation, ribosomes often arrange in a defined manner to form polysomes. By mapping the intracellular three-dimensional organization of translating ribosomes, we show that their association into polysomes exerts a local coordination mechanism that is mediated by the ribosomal protein L9. Our work demonstrates the feasibility of visualizing molecular processes at atomic detail inside cells.

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“…However, it could be partially (residues 17-33) localized in the hibernating state 70S map. uS5 encircles the mRNA entry site and plays an important role in subunit assembly and maintaining translation fidelity [238].…”

Section: Smegmatis Small Ribosomal Subunitmentioning

confidence: 99%

Cryo-EM structure of the Mycobacterium smegmatis ribosome

Mishra¹

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vi 1.8.1 Mechanism of formation of hibernating ribosome dimers (100S ribosome) 1.8.2 Physiological significance of hibernating ribosome 1.9 Trans-translating Ribosome 1.9.1 Structure of tmRNA 1.9.2 The mechanism of trans-translation 1.9.3 Physiological significance of trans-translation 1.10 Mycobacterial Ribosome 1.11 Cryo-EM 1.12 Aim of this dissertation 2 Materials and Methods 2.1 Materials 2.1.1 Buffers, Media and Salts 2.1.2 Microbiology strains 2.1.3 Buffer Composition 2.1.4 Instruments and accessories 2.1.5 Computer tools and softwares 2.2 Methods 2.2.1 Purification of 70S ribosomes from M. smegmatis 2.2.2 Ribosome protein identification by SDS PAGE and Mass Spectrometry 2.2.3 Purification of 70S ribosomes from M. smegmatis growing in different growth phases 2.2.4 Cryo-Electron Microscopy 2.2.5 Cryo-EM Single Particle Analysis (Data Processing) 2.2.6 Current capabilities and limitations of cryo-EM and Single Particle Analysis 2.2.7 Cryo-EM imaging of M. smegmatis ribosome 2.2.8 Model building and refinement 2.2.9 Model validation 2.2.10 Figure generation vii 3 Results Cryo-EM structures of M. smegmatis ribosomes in different functional states 3.1 Purification profile of M. smegmatis ribosomes in log and stationary phase 3.2 Micrograph inspection 3.3 Image Processing 3.3.1 2D classification 3.3.2 3D classification and 3D refinement 3.3.3 Resolution estimation and Resmap calculation for the 70S P/P state maps 3.3.4 Model Validation 3.3.5 Resolution estimation for the Hibernating and Trans-translating maps 3.4 Structure of M. smegmatis 70S ribosome in P/P state 3.4.1 M. smegmatis small ribosomal subunit 3.4.2 M. smegmatis large ribosomal subunit 3.4.3 Species specific alteration in the 23S rRNA of the LSU in the M. smegmatis ribosome 3.4.4 Species specific alteration in r-proteins clustered near the functional sites of M. smegmatis ribosome 3.5 Structure of M. smegmatis 70S ribosome in the Hibernating state 3.5.1 Hibernation Promoting Factor 3.5.2 Structure of mRNA entry and exit sites of M. smegmatis 70S ribosome 3.6 Trans-Translating state of M. smegmatis 70S ribosome 3.6.1 Unique mechanism of tmRNA during trans-translation 4 Discussion 5 Conclusion 6 References 7 Appendix viii 8 Online access of cryo-EM maps and models 162 9 Author's publications related to this thesis during the

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The Loop 2 Region of Ribosomal Protein uS5 Influences Spectinomycin Sensitivity, Translational Fidelity, and Ribosome Biogenesis

Cited by 8 publications

References 44 publications

The C-terminus of ribosomal protein uS4 contributes to small ribosomal subunit biogenesis and the fidelity of translation

The C-terminus of ribosomal protein uS4 contributes to small ribosomal subunit biogenesis and the fidelity of translation

Visualizing translation dynamics at atomic detail inside a bacterial cell

Cryo-EM structure of the Mycobacterium smegmatis ribosome

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