Evidence that the G2661 region of 23S rRNA is located at the ribosomal binding sites of both elongation factors

Hausner, Thomas-Peter; Atmadja, Johannes; Nierhaus, Knud H.

doi:10.1016/0300-9084(87)90225-2

Cited by 190 publications

(137 citation statements)

References 35 publications

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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.

339

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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Section: Efs and Ribosomal Activationmentioning

confidence: 99%

Why nature really chose phosphate

Kamerlin

Sharma

Prasad

et al. 2013

Quart. Rev. Biophys.

339

448

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“…There are threedimensional structures of EF-G alone (3) and in a complex with GDP (4) from x-ray crystallography; however, little is known of the chemistry of the binding of the factor to ribosomes. There is evidence for the interaction of EF-G with two separate sites in Escherichia coli 23S rRNA: with the thiostrepton region (5)(6)(7)(8)(9)(10)(11) and with the sarcin͞ricin (S͞R) domain (11,12).…”

mentioning

confidence: 99%

“…The conclusion derives from a series of compelling observations: that in eukaryotes these are the partial reactions of protein synthesis that are most adversely affected by the toxins (19) (peptidyltransferase activity, for example, is not affected); that the S͞R domain is the binding site for elongation factors (12); and that EF-Tu and EF-G protect four nucleotides in the S͞R domain from chemical modification (11).…”

mentioning

confidence: 99%

The ribosome-in-pieces: Binding of elongation factor EF-G to oligoribonucleotides that mimic the sarcin/ricin and thiostrepton domains of 23S ribosomal RNA

Munishkin

Wool

1997

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

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An oligoribonucleotide (a 27-mer) that mimics the sarcin͞ricin (S͞R) domain of Escherichia coli 23S rRNA binds elongation factor EF-G; the K d is 6.9 M, whereas for binding to ribosomes it is 0.7 M. Binding saturates when EF-G and the S͞R RNA are equimolar; at saturation 70% of the input RNA is in complexes with EF-G. Binding of EF-G to S͞R RNA does not require GTP but is inhibited by GDP; the inhibition by GDP is overcome by GTP. The effects of mutations of the S͞R domain nucleotides G2655, A2660, and G2661 suggest that EF-G recognizes the conformation of the RNA rather than the identity of the nucleotides. EF-G also binds to an oligoribonucleotide (an 84-mer) that has the thiostrepton region of 23S rRNA; however, EF-G binds independently to S͞R and thiostrepton oligoribonucleotides.

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“…This base is located in the so called cr-sarcin loop in domain six of the 23s rRNA [2]. Since the cleavage blocks the binding of both EF-Tu .…”

mentioning

confidence: 99%

Base 2661 in Escherichia coli 23S rRNA influences the binding of elongation factor Tu during protein synthesis in vivo

Tapio

Isaksson

1991

European Journal of Biochemistry

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The binding of the EF-Tu . GTP . aminoacyl-tRNA ternary complex (EF, elongation factor) to the ribosome is known to be strengthened by a 2661G-to-C mutation in 23s ribosomal RNA, whereas the binding to normal ribosomes is weakened if the factor is in an appropriate mutant form (Aa). In this report we describe the mutual effects by the 2661C alteration in 23s rRNA and EF-Tu(Aa) on bacterial viability and translation efficiency in strains with normal or mutationally altered ribosomes. The rrnB(2661C) allele on a multicopy plasmid was introduced by transformation into Escherichia coli K-12 strains, harbouring either the wild-type or the mutant gene (tufA) for EF-Tu as well as norinal or mutant ribosomal protein S12 (rpsL). Together with wild-type EF-Tu, the 2661 C mutant ribosomes decreased the translation elongation rate in a rpsL+ strain or a non-restrictive rpsL224 strain. This reduction was not seen in strains which harbored EF-Tu(Aa) instead of EF-Tu(As) (As, wildtype form). Nonsense codon suppression by tyrT(Su3) suppressor tRNA was reduced by 2662C in a rpsL224 strain in the presence of EF-Tu(As) but not in the presence of EF-Tu(Aa). The lethal effect obtained by the combination of 2661 C and a restrictive ribosomal protein S12 mutation (rpsL282) disappeared if EF-Tu(As) was replaced by EF-Tu(Aa) in the strain. In such a viable strain, 2662C had no effect on either the translation elongation rate or nonsense codon suppression. Our data suggest that the G base at position 2661 in 23s rRNA is important for binding of EF-Tu during protein synthesis in vivo. The interaction between this base and EF-Tu is strongly influenced by the structure of ribosomal protein S12.The small basic protein cr-sarcin has been shown to inhibit protein synthesis in Escherichia coli 111 by specifically cleaving the phosphodiester bond on the 3'-side of the G2661 residue. This base is located in the so called cr-sarcin loop in domain six of the 23s rRNA [2]. Since the cleavage blocks the binding of both EF-Tu . GTP . aminoacyl-tRNA and EF-G . GTP to the ribosome, the G2661 residue is presumably located at the overlapping region of the binding sites of both elongation factors 121.Recently, a single base substitution mutation was constructed in the cloned rrnB operon of the multi-copy plasmid pKK3535, altering the base at position 2661 in 23s rRNA from G to C [3]. The translational elongation rate of the mutant 2661 C ribosomes, measured in a poly(U)-primed in vitro system, was lowered if the ribosomes also were altered in ribosomal protein S12. This effect was suggested to be the result of stronger binding of EF-Tu . GTP . aminoacyl-tRNA ternary complexes to mutant ribosomes [3].Many streptomycin-resistant mutations in the rpsL gene, encoding ribosomal protein S12, are known to increase the accuracy of translation [4, 51. One such example, giving a hyper-accurate (restrictive) phenotype, is the rpsL282 mutation. In contrast, another mutation in this gene, rpsL224, leads to streptomycin resistance although it still shows an essentially ...

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Evidence that the G2661 region of 23S rRNA is located at the ribosomal binding sites of both elongation factors

Cited by 190 publications

References 35 publications

Why nature really chose phosphate

Why nature really chose phosphate

The ribosome-in-pieces: Binding of elongation factor EF-G to oligoribonucleotides that mimic the sarcin/ricin and thiostrepton domains of 23S ribosomal RNA

Base 2661 in Escherichia coli 23S rRNA influences the binding of elongation factor Tu during protein synthesis in vivo

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