Ribosome Mechanics Informs about Mechanism

Zimmermann, Michael T.; Jia, Kejue; Jernigan, Robert L.

doi:10.1016/j.jmb.2015.12.003

Cited by 20 publications

(21 citation statements)

References 66 publications

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“…Despite its simplicity, the normal mode analysis (NMA) of ENM can be used to predict a few low‐frequency modes of collective motions, which often capture those conformational changes observed between experimentally solved protein structures in different functional states . Numerous studies have established ENM as a useful and efficient means to probe structural dynamics of large biomolecular complexes (including a DNA translocase/helicase and protein‐RNA complexes like ribosome) with virtually no limit in timescale or system size (see reviews).…”

Section: Introductionmentioning

confidence: 99%

Probing the structural dynamics of the CRISPR‐Cas9 RNA‐guided DNA‐cleavage system by coarse‐grained modeling

Zheng

2017

Proteins

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In the adaptive immune systems of many bacteria and archaea, the Cas9 endonuclease forms a complex with specific guide/scaffold RNA to identify and cleave complementary target sequences in foreign DNA. This DNA targeting machinery has been exploited in numerous applications of genome editing and transcription control. However, the molecular mechanism of the Cas9 system is still obscure. Recently, high-resolution structures have been solved for Cas9 in different structural forms (e.g., unbound forms, RNA-bound binary complexes, and RNA-DNA-bound tertiary complexes, corresponding to an inactive state, a pre-target-bound state, and a cleavage-competent or product state), which offered key structural insights to the Cas9 mechanism. To further probe the structural dynamics of Cas9 interacting with RNA and DNA at the amino-acid level of details, we have performed systematic coarse-grained modeling using an elastic network model and related analyses. Our normal mode analysis predicted a few key modes of collective motions that capture the observed conformational changes featuring large domain motions triggered by binding of RNA and DNA. Our flexibility analysis identified specific regions with high or low flexibility that coincide with key functional sites (such as DNA/RNA-binding sites, nuclease cleavage sites, and key hinges). We also identified a small set of hotspot residues that control the energetics of functional motions, which overlap with known functional sites and offer promising targets for future mutagenesis efforts to improve the specificity of Cas9. Finally, we modeled the conformational transitions of Cas9 from the unbound form to the binary complex and then the tertiary complex, and predicted a distinct sequence of domain motions. In sum, our findings have offered rich structural and dynamic details relevant to the Cas9 machinery, and will guide future investigation and engineering of the Cas9 systems. Proteins 2017; 85:342-353. © 2016 Wiley Periodicals, Inc.

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Section: Introductionmentioning

confidence: 99%

Probing the structural dynamics of the CRISPR‐Cas9 RNA‐guided DNA‐cleavage system by coarse‐grained modeling

Zheng

2017

Proteins

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“…On one hand, single particle cryo-EM and X-ray structural studies, while informing on the conformational change amplitudes, do not contain information on forces or time-scales. On the other hand, molecular dynamics (MD) simulations and normal mode calculations are particularly challenging because of the size and complexity of ribosome structures121314151617. Dynamics is also very sensitive to environment, which leads to a well-structured system being inactive under certain solvent conditions18, and it is now fully accepted that further to structure being adapted to function, appropriate molecular motions are also necessary.…”

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confidence: 99%

The fluctuating ribosome: thermal molecular dynamics characterized by neutron scattering

Zaccaı̈

Natali

Peters

et al. 2016

Sci Rep

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Conformational changes associated with ribosome function have been identified by X-ray crystallography and cryo-electron microscopy. These methods, however, inform poorly on timescales. Neutron scattering is well adapted for direct measurements of thermal molecular dynamics, the ‘lubricant’ for the conformational fluctuations required for biological activity. The method was applied to compare water dynamics and conformational fluctuations in the 30 S and 50 S ribosomal subunits from Haloarcula marismortui, under high salt, stable conditions. Similar free and hydration water diffusion parameters are found for both subunits. With respect to the 50 S subunit, the 30 S is characterized by a softer force constant and larger mean square displacements (MSD), which would facilitate conformational adjustments required for messenger and transfer RNA binding. It has been shown previously that systems from mesophiles and extremophiles are adapted to have similar MSD under their respective physiological conditions. This suggests that the results presented are not specific to halophiles in high salt but a general property of ribosome dynamics under corresponding, active conditions. The current study opens new perspectives for neutron scattering characterization of component functional molecular dynamics within the ribosome.

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“…Due to the much higher GC and G content than other groups of rRNA segments (see Table 3 ), ESLs could be expected to have higher potency for interaction with proteins (see Jones et al, 2001 ; Biot et al, 2004 ; Ellis et al, 2007 for the affinities of protein amino acids for nucleobases and the backbone ribose and phosphate). For mRNPs associated with the ER (Cui and Palazzo, 2014 ; Reid and Nicchitta, 2015 ) or with other subcellular networks (Jansen, 1999 ) this may aid a competitive detachment of the mRNP protein component prior to mRNA entrance in ribosome′s translation tunnel (Zimmermann et al, 2016 ). To obtain a rough estimate of the interactive potential with proteins, segments of 28S and 18S rRNAs were examined for frequencies of H bonding and of van der Waals interaction of the nucleobases with protein amino acids (calculated using parameters from Table 5 in Jones et al, 2001 ).…”

Section: Resultsmentioning

confidence: 99%

“…Both helical and single-stranded parts of ES could compete with mRNAs for protein components of mRNPs to facilitate their separation. Interaction of ES parts with mRNAs and/or mRNP proteins might help entrance of mRNA into the mRNA tunnel of the ribosome (Zimmermann et al, 2016 ). The extraction of mRNAs from mRNPs by rRNA ES does not have to discriminate between mRNA sectors; based on both the GC content and density of the matches, the release of 5′utr could occur preferentially.…”

Section: Discussionmentioning

confidence: 99%

The Expansion Segments of 28S Ribosomal RNA Extensively Match Human Messenger RNAs

et al. 2018

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Eukaryote ribosomal RNAs (rRNAs) have expanded in the course of phylogeny by addition of nucleotides in specific insertion areas, the expansion segments. These number about 40 in the larger (25–28S) rRNA (up to 2,400 nucleotides), and about 12 in the smaller (18S) rRNA (<700 nucleotides). Expansion of the larger rRNA shows a clear phylogenetic increase, with a dramatic rise in mammals and especially in hominids. Substantial portions of expansion segments in this RNA are not bound to ribosomal proteins, and may engage extraneous interactants, including messenger RNAs (mRNAs). Studies on the ribosome-mRNA interaction have focused on proteins of the smaller ribosomal subunit, with some examination of 18S rRNA. However, the expansion segments of human 28S rRNA show much higher density and numbers of mRNA matches than those of 18S rRNA, and also a higher density and match numbers than its own core parts. We have studied that with frequent and potentially stable matches containing 7–15 nucleotides. The expansion segments of 28S rRNA average more than 50 matches per mRNA even assuming only 5% of their sequence as available for such interaction. Large expansion segments 7, 15, and 27 of 28S rRNA also have copious long (≥10-nucleotide) matches to most human mRNAs, with frequencies much higher than in other 28S rRNA parts. Expansion segments 7 and 27 and especially segment 15 of 28S rRNA show large size increase in mammals compared to other metazoans, which could reflect a gain of function related to interaction with non-ribosomal partners. The 28S rRNA expansion segment 15 shows very high increments in size, guanosine, and cytidine nucleotide content and mRNA matching in mammals, and especially in hominids. With these segments (but not with other 28S rRNA or any 18S rRNA expansion segments) the density and number of matches are much higher in 5′-terminal than in 3′-terminal untranslated mRNA regions, which may relate to mRNA mobilization via 5′ termini. Matches in the expansion segments 7, 15, and 27 of human 28S rRNA appear as candidates for general interaction with mRNAs, especially those associated with intracellular matrices such as the endoplasmic reticulum.

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Ribosome Mechanics Informs about Mechanism

Cited by 20 publications

References 66 publications

Probing the structural dynamics of the CRISPR‐Cas9 RNA‐guided DNA‐cleavage system by coarse‐grained modeling

Probing the structural dynamics of the CRISPR‐Cas9 RNA‐guided DNA‐cleavage system by coarse‐grained modeling

The fluctuating ribosome: thermal molecular dynamics characterized by neutron scattering

The Expansion Segments of 28S Ribosomal RNA Extensively Match Human Messenger RNAs

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