Probing the rRNA environment of ribosomal protein S5 across the subunit interface and inside the 30 S subunit using tethered Fe(II) 1 1Edited by D. E. Draper

Culver, Gloria M.; Heilek, Gabriele; Noller, Harry F.

doi:10.1006/jmbi.1998.2483

Cited by 21 publications

(3 citation statements)

References 30 publications

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“…S5 is a tertiary protein that binds at a helical junction (Culver et al, 1999), formed by h1, h2, h28, h36 and h44 (Fig. 5A), including rRNA segments from all four domains of the 16S rRNA.…”

Section: Discussionmentioning

confidence: 99%

Structural insights into the assembly of the 30S ribosomal subunit in vivo: functional role of S5 and location of the 17S rRNA precursor sequence

Yang¹,

Guo²,

Goto³

et al. 2014

Protein Cell

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The in vivo assembly of ribosomal subunits is a highly complex process, with a tight coordination between protein assembly and rRNA maturation events, such as folding and processing of rRNA precursors, as well as modifications of selected bases. In the cell, a large number of factors are required to ensure the efficiency and fidelity of subunit production. Here we characterize the immature 30S subunits accumulated in a factor-null Escherichia coli strain (∆rsgA∆rbfA). The immature 30S subunits isolated with varying salt concentrations in the buffer system show interesting differences on both protein composition and structure. Specifically, intermediates derived under the two contrasting salt conditions (high and low) likely reflect two distinctive assembly stages, the relatively early and late stages of the 3′ domain assembly, respectively. Detailed structural analysis demonstrates a mechanistic coupling between the maturation of the 5′ end of the 17S rRNA and the assembly of the 30S head domain, and attributes a unique role of S5 in coordinating these two events. Furthermore, our structural results likely reveal the location of the unprocessed terminal sequences of the 17S rRNA, and suggest that the maturation events of the 17S rRNA could be employed as quality control mechanisms on subunit production and protein translation.Electronic supplementary materialThe online version of this article (doi:10.1007/s13238-014-0044-1) contains supplementary material, which is available to authorized users.

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Section: Discussionmentioning

confidence: 99%

Structural insights into the assembly of the 30S ribosomal subunit in vivo: functional role of S5 and location of the 17S rRNA precursor sequence

Yang¹,

Guo²,

Goto³

et al. 2014

Protein Cell

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“…Conjugation of Fe(II)-BABE to IF2 was performed essentially as described in Culver et al (1999). A total of 2 µL 1 M DTT were added to 1 mL of IF2 solution (1 µg/µL in storage buffer).…”

Section: Preparation Of Cu(ii):phenanthroline and Fe(ii)-babe-derivatmentioning

confidence: 99%

Ribosomal localization of translation initiation factor IF2

Marzi¹,

Knight²,

Brandi³

et al. 2003

RNA

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Bacterial translation initiation factor IF2 is a GTP-binding protein that catalyzes binding of initiator fMet-tRNA in the ribosomal P site. The topographical localization of IF2 on the ribosomal subunits, a prerequisite for understanding the mechanism of initiation complex formation, has remained elusive. Here, we present a model for the positioning of IF2 in the 70S initiation complex as determined by cleavage of rRNA by the chemical nucleases Cu(II):1,10-orthophenanthroline and Fe(II):EDTA tethered to cysteine residues introduced into IF2. Two specific amino acids in the GII domain of IF2 are in proximity to helices H3, H4, H17, and H18 of 16S rRNA. Furthermore, the junction of the C-1 and C-2 domains is in proximity to H89 and the thiostrepton region of 23S rRNA. The docking is further constrained by the requisite proximity of the C-2 domain with P-site-bound tRNA and by the conserved GI domain of the IF2 with the large subunit's factor-binding center. Comparison of our present findings with previous data further suggests that the IF2 orientation on the 30S subunit changes during the transition from the 30S to 70S initiation complex.

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“…These effects were exaggerated in a complex of S5 and S12 with 16S rRNA. Moreover, Fe(II)-S5 mediates cleavage of the 59 terminus in 30S subunits (Culver et al 1999). However, a minimal particle of Fe(II)-C73-S8/ S5/16S rRNA failed to recapitulate cleavages observed in Fe(II)-S8/30S subunit (data not shown).…”

Section: Discussionmentioning

confidence: 99%

Interdependencies govern multidomain architecture in ribosomal small subunit assembly

Calidas

Culver²

2010

RNA

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The 30S subunit is composed of four structural domains, the body, platform, head, and penultimate/ultimate stems. The functional integrity of the 30S subunit is dependent upon appropriate assembly and precise orientation of all four domains. We examined 16S rRNA conformational changes during in vitro assembly using directed hydroxyl radical probing mediated by Fe(II)-derivatized ribosomal protein (r-protein) S8. R-protein S8 binds the central domain of 16S rRNA directly and independently and its iron derivatized substituents have been shown to mediate cleavage in three domains of 16S rRNA, thus making it an ideal probe to monitor multidomain orientation during assembly. Cleavages in minimal ribonucleoprotein (RNP) particles formed with Fe(II)-S8 and 16S rRNA alone were compared with that in the context of the fully assembled subunit. The minimal binding site of S8 at helix 21 exists in a structure similar to that observed in the mature subunit, in the absence of other r-proteins. However, the binding site of S8 at the junction of helices 25-26a, which is transcribed after helix 21, is cleaved with differing intensities in the presence and absence of other r-proteins. Also, assembly of the body helps establish an architecture approximating, but perhaps not identical, to the 30S subunit at helix 12 and the 59 terminus. Moreover, the assembly or orientation of the neck is dependent upon assembly of both the head and the body. Thus, a complex interrelationship is observed between assembly events of independent domains and the incorporation of primary binding proteins during 30S subunit formation.

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Probing the rRNA environment of ribosomal protein S5 across the subunit interface and inside the 30 S subunit using tethered Fe(II) 1 1Edited by D. E. Draper

Cited by 21 publications

References 30 publications

Structural insights into the assembly of the 30S ribosomal subunit in vivo: functional role of S5 and location of the 17S rRNA precursor sequence

Structural insights into the assembly of the 30S ribosomal subunit in vivo: functional role of S5 and location of the 17S rRNA precursor sequence

Ribosomal localization of translation initiation factor IF2

Interdependencies govern multidomain architecture in ribosomal small subunit assembly

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