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4Y27

E.coli 23S Sarcin-Ricil Loop, modified with a 2-Me on G2661 and a methylphosphonate on A2662

Summary for 4Y27
Entry DOI10.2210/pdb4y27/pdb
Related3DVZ
Descriptor27-mer 23S Sarcin-Ricil Loop (2 entities in total)
Functional Keywordsrna, modification, ribosome
Biological sourceEscherichia coli
Total number of polymer chains1
Total formula weight8756.31
Authors
Ennifar, E.,Micura, R.,Fluer, S. (deposition date: 2015-02-09, release date: 2015-11-18, Last modification date: 2024-01-10)
Primary citationKoch, M.,Flur, S.,Kreutz, C.,Ennifar, E.,Micura, R.,Polacek, N.
Role of a ribosomal RNA phosphate oxygen during the EF-G-triggered GTP hydrolysis.
Proc.Natl.Acad.Sci.USA, 112:E2561-E2568, 2015
Cited by
PubMed Abstract: Elongation factor-catalyzed GTP hydrolysis is a key reaction during the ribosomal elongation cycle. Recent crystal structures of G proteins, such as elongation factor G (EF-G) bound to the ribosome, as well as many biochemical studies, provide evidence that the direct interaction of translational GTPases (trGTPases) with the sarcin-ricin loop (SRL) of ribosomal RNA (rRNA) is pivotal for hydrolysis. However, the precise mechanism remains elusive and is intensively debated. Based on the close proximity of the phosphate oxygen of A2662 of the SRL to the supposedly catalytic histidine of EF-G (His87), we probed this interaction by an atomic mutagenesis approach. We individually replaced either of the two nonbridging phosphate oxygens at A2662 with a methyl group by the introduction of a methylphosphonate instead of the natural phosphate in fully functional, reconstituted bacterial ribosomes. Our major finding was that only one of the two resulting diastereomers, the SP methylphosphonate, was compatible with efficient GTPase activation on EF-G. The same trend was observed for a second trGTPase, namely EF4 (LepA). In addition, we provide evidence that the negative charge of the A2662 phosphate group must be retained for uncompromised activity in GTP hydrolysis. In summary, our data strongly corroborate that the nonbridging proSP phosphate oxygen at the A2662 of the SRL is critically involved in the activation of GTP hydrolysis. A mechanistic scenario is supported in which positioning of the catalytically active, protonated His87 through electrostatic interactions with the A2662 phosphate group and H-bond networks are key features of ribosome-triggered activation of trGTPases.
PubMed: 25941362
DOI: 10.1073/pnas.1505231112
PDB entries with the same primary citation
Experimental method
X-RAY DIFFRACTION (0.998 Å)
Structure validation

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