Temperature-dependent RNP Conformational Rearrangements: Analysis of Binary Complexes of Primary Binding Proteins with 16 S rRNA

Dutcă, Laura-M.; Jagannathan, Indu; Grondek, Joel; Culver, Gloria M.

doi:10.1016/j.jmb.2007.02.064

Cited by 11 publications

(8 citation statements)

References 42 publications

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“…Our data are consistent with a substantial folding and architectural organization of the 5′ domain early in assembly (see Refs. 10,[14][15][16][17] and with the final alignment of helix 44 occurring later in the assembly cascade. 10,12 These results, taken together with our earlier work, suggest that there are different means by which domains or elements of 16S rRNA are organized.…”

Section: Discussionmentioning

confidence: 99%

Assembly of the 5′ and 3′ Minor Domains of 16S Ribosomal RNA as Monitored by Tethered Probing from Ribosomal Protein S20

Dutca

Culver

2008

Journal of Molecular Biology

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The ribosomal protein (r-protein) S20 is a primary binding protein. As such, it interacts directly and independently with the 5' domain as well as the 3' minor domain of 16S ribosomal RNA (rRNA) in minimal particles and the fully assembled 30S subunit. The interactions observed between r-protein S20 and the 5' domain of 16S rRNA are quite extensive, while those between r-protein S20 and the 3' minor domain are significantly more limited. In this study, directed hydroxyl radical probing mediated by Fe(II)-derivatized S20 proteins was used to monitor the folding of 16S rRNA during r-protein association and 30S subunit assembly. An analysis of the cleavage patterns in the minimal complexes [16S rRNA and Fe(II)-S20] and the fully assembled 30S subunit containing the same Fe(II)-derivatized proteins shows intriguing similarities and differences. These results suggest that the two domains, 5' and 3' minor, are organized relative to S20 at different stages of assembly. The 5' domain acquires, in a less complex ribonucleoprotein particle than the 3' minor domain, the same architecture as observed in mature subunits. These results are similar to what would be predicted of subunit assembly by the 5'-to-3' direction assembly model.

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Section: Discussionmentioning

confidence: 99%

Assembly of the 5′ and 3′ Minor Domains of 16S Ribosomal RNA as Monitored by Tethered Probing from Ribosomal Protein S20

Dutca

Culver

2008

Journal of Molecular Biology

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“…A binary complex of S4 and the 16S rRNA in which the two components have been heated separately does not display the same pattern of protection, suggesting that the protein confers the conformational change directly to the RNA, allowing subsequent proteins to bind and ensuring the hierarchy of assembly. Subsequent studies of the remaining five primary binding proteins indicated a range of behavior (15). The other two 5′ domain primary proteins S17 and S20 bound to the 16S rRNA in a temperature-independent manner, while the central domain primary proteins S8 and S15 conferred only small differences to the chemical footprint of the binary complex upon heating.…”

Section: Binding Of Primary Proteinsmentioning

confidence: 99%

A Complex Assembly Landscape for the 30S Ribosomal Subunit

Sykes

Williamson

2009

Annu. Rev. Biophys.

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The ribosome is a complex macromolecular machine responsible for protein synthesis in the cell. It consists of two subunits, each of which contains both RNA and protein components. Ribosome assembly is subject to intricate regulatory control and is aided by a multitude of assembly factors in vivo, but can also be carried out in vitro. The details of the assembly process remain unknown even in the face of atomic structures of the entire ribosome and after more than three decades of research. Some of the earliest research on ribosome assembly produced the Nomura assembly map of the small subunit, revealing a hierarchy of protein binding dependencies for the 20 proteins involved and suggesting the possibility of a single intermediate. Recent work using a combination of RNA footprinting and pulse-chase quantitative mass spectrometry paints a picture of small subunit assembly as a dynamic and varied landscape, with sequential and hierarchical RNA folding and protein binding events finally converging on complete subunits. Proteins generally lock tightly into place in a 5′ to 3′ direction along the ribosomal RNA, stabilizing transient RNA conformations, while RNA folding and the early stages of protein binding are initiated from multiple locations along the length of the RNA.

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“…Reorganization of S4 complexes may be linked to refolding of the N-terminal domain of S4, which is disordered in the free protein 22. By contrast, there is no evidence for temperature-dependent reorganization of the S17 and S20 complexes 23.…”

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Global Stabilization of rRNA Structure by Ribosomal Proteins S4, S17, and S20

Ramaswamy

Woodson

2009

Journal of Molecular Biology

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SummaryRibosomal proteins stabilize the folded structure of the rRNA and enable the recruitment of further proteins to the complex. Quantitative hydroxyl radical footprinting was used to measure the extent to which three different primary assembly proteins, S4, S17 and S20, stabilize the 3D structure of the E. coli 16S 5′ domain. The stability of the complexes was perturbed by varying the concentration of MgCl 2 . Each protein influences the stability of the rRNA tertiary interactions beyond its immediate binding site. S4 and S17 stabilize the entire 5′ domain, while S20 has a more local effect. Multi-stage folding of individual helices within the 5′ domain shows that each protein stabilizes a different ensemble of structural intermediates, that include non-native interactions at low Mg 2+ . We propose that the combined interactions of S4, S17 and S20 with different helical junctions bias the free energy landscape toward a few RNA conformations that are competent to add the secondary assembly protein S16 in the next step of assembly. Keywordsribosome; ribosomal protein; RNA folding; hydroxyl radical footprinting; RNA-protein interactions Ribosome biogenesis is one of the most important synthetic tasks undertaken by the cell. Among bacteria, short generation times require that several hundreds of ribosomes are produced per minute in order to produce the tens of thousands of ribosomes needed by each new cell 1;2 . A critical question is how the RNA and protein components of the ribosome interact to achieve the native subunits and avoid nonfunctional complexes.Studies on the reconstitution of the 30S ribosomal subunit have shown that binding of ribosomal proteins is coupled to folding of the rRNA 3;4 . The addition of the primary assembly proteins, which bind directly to the 16S rRNA, stabilize the helices with which they interact, and enable further assembly of each 30S domain by pre-ordering the binding sites for secondary and tertiary assembly proteins 5;6 . The extent to which individual ribosomal proteins stabilize the structure of the rRNA beyond their immediate binding site is thus directly connected to the hierarchy and cooperativity of assembly 4;7 .

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Temperature-dependent RNP Conformational Rearrangements: Analysis of Binary Complexes of Primary Binding Proteins with 16 S rRNA

Cited by 11 publications

References 42 publications

Assembly of the 5′ and 3′ Minor Domains of 16S Ribosomal RNA as Monitored by Tethered Probing from Ribosomal Protein S20

Assembly of the 5′ and 3′ Minor Domains of 16S Ribosomal RNA as Monitored by Tethered Probing from Ribosomal Protein S20

A Complex Assembly Landscape for the 30S Ribosomal Subunit

Global Stabilization of rRNA Structure by Ribosomal Proteins S4, S17, and S20

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