The sarcin–ricin loop of 23S rRNA is essential for assembly of the functional core of the 50S ribosomal subunit

Lancaster, Laura; Lambert, Nicole; Maklan, Eric J.; Horan, Lucas H.; Noller, Harry F.

doi:10.1261/rna.1202108

Cited by 53 publications

(58 citation statements)

References 72 publications

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“…Here, it is essential to consider the effect of the sarcin-ricin loop (SRL) of the 23S rRNA, and in particular residue A2662 (see Fig. S2), whose critical role has been pointed out before (14)(15)(16). Significantly, our simulations of the EF-Tu′/ribosome complex found that a proper H84 positioning, without any additional artificial force constraints, required the presence of the SRL loop with its A2662 residue.…”

Section: Figmentioning

confidence: 91%

“…Breakthrough in the elucidation of the ribosome structure (2-13) and careful biochemical studies (1)(2)(3)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25) allow one to begin thinking about the nature of the mechanism of the elongation process. In particular, the elucidation of structure of the EF-Tu/ribosome complex (our study refers to the structure of EF-Tu in the complex as EF-Tu′) (9) opens the way for the exploration of the activation process at the molecular level.…”

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confidence: 99%

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Converting structural information into an allosteric-energy-based picture for elongation factor Tu activation by the ribosome

Adamczyk

Warshel

2011

Proc. Natl. Acad. Sci. U.S.A.

130

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The crucial process of aminoacyl-tRNA delivery to the ribosome is energized by the GTPase reaction of the elongation factor Tu (EF-Tu). Advances in the elucidation of the structure of the EF-Tu/ribosome complex provide the rare opportunity of gaining a detailed understanding of the activation process of this system. Here, we use quantitative simulation approaches and reproduce the energetics of the GTPase reaction of EF-Tu with and without the ribosome and with several key mutants. Our study provides a novel insight into the activation process. It is found that the critical H84 residue is not likely to behave as a general base but rather contributes to an allosteric effect, which includes a major transition state stabilization by the electrostatic effect of the P loop and other regions of the protein. Our findings have general relevance to GTPase activation, including the processes that control signal transduction.enzymatic catalysis | preorganization | allostery T he elongation cycle of protein synthesis uses the elongation factor Tu (EF-Tu) with GTP to deliver aminoacyl-tRNA (aa-tRNA) to the mRNA-programmed ribosome. More specifically, the GTP-bound state of EF-Tu forms a high-affinity ternary complex with the aa-tRNA. Upon binding of the ternary complex to the ribosome, the aa-tRNA occupies the A site and, when the codon-anticodon interaction is cognate, the GTPase activity of the EF-Tu is increased significantly. The conformational change following GTP hydrolysis to GDP and a leaving phosphate group (P i ) leads to dissociation of EF-Tu from the ribosome and accommodation of the aa-tRNA on the A site for peptidyl transfer (see Fig. 1 for a schematic description; for additional details, see refs. 1-3).Breakthrough in the elucidation of the ribosome structure (2-13) and careful biochemical studies (1-3, 14-25) allow one to begin thinking about the nature of the mechanism of the elongation process. In particular, the elucidation of structure of the EF-Tu/ribosome complex (our study refers to the structure of EF-Tu in the complex as EF-Tu′) (9) opens the way for the exploration of the activation process at the molecular level. However, despite major biochemical and structural breakthroughs, a detailed explanation of how the codon recognition in the 30S subunit leads to GTP hydrolysis remains elusive. That is, although it is known that the precise positioning of H84 is critical for efficient catalysis (1,9,(11)(12)(13)(23)(24)(25)(26), the energetics of this positioning and its ultimate role in stabilizing the transition state (TS) are unclear. More precisely, it is frequently assumed that the conserved H84 is moved to a catalytic configuration and then serves as a general base, but this assumption is problematic (see ref. 1 and Reproducing the Overall Catalytic and Mutational Effects below) and has similar pitfalls as in the highly related case of the Ras-RasGAP (RasGAP) system. That is, where it was originally assumed that Q61 in the RasGAP system serves as a general base, but this assumption has been shown to ...

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Section: Figmentioning

confidence: 91%

mentioning

confidence: 99%

Converting structural information into an allosteric-energy-based picture for elongation factor Tu activation by the ribosome

Adamczyk

Warshel

2011

Proc. Natl. Acad. Sci. U.S.A.

130

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“…Here, it is important to consider the effect of the SRL of the 23S rRNA subunit, and, in particular the role of residue A2662 (see Fig. 33 for relative positions), whose critical role has been already pointed out elsewhere (Hausner et al 1987 ;Lancaster et al 2008 ;Moazed et al 1988). Significantly, our simulations of the EF-Tu'/ribosome complex found that the presence of the SRL loop (with its A2662 residue) was essential for the proper positioning of H84 (without any additional artificial force constraints).…”

Section: Efs and Ribosomal Activationmentioning

confidence: 99%

Why nature really chose phosphate

Kamerlin

Sharma

Prasad

et al. 2013

Quart. Rev. Biophys.

333

448

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Phosphoryl transfer plays key roles in signaling, energy transduction, protein synthesis, and maintaining the integrity of the genetic material. On the surface, it would appear to be a simple nucleophile displacement reaction. However, this simplicity is deceptive, as, even in aqueous solution, the low-lying d-orbitals on the phosphorus atom allow for eight distinct mechanistic possibilities, before even introducing the complexities of the enzyme catalyzed reactions. To further complicate matters, while powerful, traditional experimental techniques such as the use of linear free-energy relationships (LFER) or measuring isotope effects cannot make unique distinctions between different potential mechanisms. A quarter of a century has passed since Westheimer wrote his seminal review, ‘Why Nature Chose Phosphate’ (Science 235 (1987), 1173), and a lot has changed in the field since then. The present review revisits this biologically crucial issue, exploring both relevant enzymatic systems as well as the corresponding chemistry in aqueous solution, and demonstrating that the only way key questions in this field are likely to be resolved is through careful theoretical studies (which of course should be able to reproduce all relevant experimental data). Finally, we demonstrate that the reason that naturereallychose phosphate is due to interplay between two counteracting effects: on the one hand, phosphates are negatively charged and the resulting charge-charge repulsion with the attacking nucleophile contributes to the very high barrier for hydrolysis, making phosphate esters among the most inert compounds known. However, biology is not only about reducing the barrier to unfavorable chemical reactions. That is, the same charge-charge repulsion that makes phosphate ester hydrolysis so unfavorable also makes it possible to regulate, by exploiting the electrostatics. This means that phosphate ester hydrolysis can not only be turned on, but also be turned off, by fine tuning the electrostatic environment and the present review demonstrates numerous examples where this is the case. Without this capacity for regulation, it would be impossible to have for instance a signaling or metabolic cascade, where the action of each participant is determined by the fine-tuned activity of the previous piece in the production line. This makes phosphate esters the ideal compounds to facilitate life as we know it.

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“…In contrast, mutations to the loop E motif are extremely detrimental to the function of the inhibitor (Townsend et al 2008b). Loop E motifs are known to interact with proteins and to be involved in RNA-RNA interactions (Hausner et al 1987;Moazed et al 1988;Leontis and Westhof 1998;Lancaster et al 2008). Both types of interactions may play a role in the ciRNA's function.…”

Section: Discussionmentioning

confidence: 99%

Structural architecture of an RNA that competitively inhibits RNase L

Keel¹,

Jha²,

Kieft³

2011

RNA

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Activation of RNase L endonuclease activity is part of the mammalian innate immune response to viral infection. The poliovirus RNA genome contains a sequence in its protein-coding region that can act as a competitive inhibitor of RNase L. Mutation, sequence, and functional analysis of this competitive inhibitor RNA (ciRNA) revealed that its activity depends on specific sequences, showed that a loop-loop hairpin interaction forms in the ciRNA, and suggested the presence of a loop E motif. These features lead to the hypothesis that the ciRNA's function is conferred in part by a specific three-dimensional folded RNA architecture. By using a combination of biophysical, mutational, and functional studies, we have mapped features of the threedimensional architecture of the ciRNA in its unbound form. We show that the loop-loop interaction forms in the free ciRNA and affects the overall structure, perhaps forming long-range tertiary interactions with the loop E motif. Local tight RNA-RNA backbone packing occurs in parts of the structure, but the fold appears to be less stable than many other tightly packed RNAs. This feature may allow the ciRNA to accommodate the translocation of ribosomes and polymerase across this multifunctional region of the viral RNA but also to function as an RNase L inhibitor.

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The sarcin–ricin loop of 23S rRNA is essential for assembly of the functional core of the 50S ribosomal subunit

Cited by 53 publications

References 72 publications

Converting structural information into an allosteric-energy-based picture for elongation factor Tu activation by the ribosome

Converting structural information into an allosteric-energy-based picture for elongation factor Tu activation by the ribosome

Why nature really chose phosphate

Structural architecture of an RNA that competitively inhibits RNase L

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