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The ribosome modulates the structural dynamics of the conserved GTPase HflX and triggers tight nucleotide binding.
Fischer, Jeffrey J; Coatham, Mackenzie L; Bear, Shey Eagle; Brandon, Harland E; De Laurentiis, Evelina I; Shields, Michael J; Wieden, Hans-Joachim.
Afiliação
  • Fischer JJ; Department of Chemistry and Biochemistry, University of Lethbridge, Lethbridge, Alberta T1K 3M4, Canada.
Biochimie ; 94(8): 1647-59, 2012 Aug.
Article em En | MEDLINE | ID: mdl-22554723
The universally conserved GTPase HflX is a putative translation factor whose GTPase activity is stimulated by the 70S ribosome as well as the 50S but not the 30S ribosomal subunit. However, the details and mechanisms governing this interaction are only poorly understood. In an effort to further elucidate the functional mechanism of HflX, we examined its interaction with the 70S ribosome, the two ribosomal subunits (50S and 30S), as well as its ability to interact with guanine nucleotides in the respective ribosomal complexes using a highly purified in vitro system. Binding studies reported here demonstrate that HflX not only interacts with 50S and 70S particles, but also with the 30S subunit, independent of the nucleotide-bound state. A detailed pre-steady-state kinetic analysis of HflX interacting with a non-hydrolyzable analog of mant-GTP, coupled with an enzymatic probing assay utilizing limited trypsinolysis, reveal that HflX·GTP exists in a structurally distinct 50S- and 70S-bound form that stabilizes GTP binding up to 70 000-fold and that may represent the "GTPase-activated" state. This activation is likely required for efficient GTP-hydrolysis, and may be similar to that observed in elongation factor G. Results reported here address the surprising low affinity of free HflX for GTP and suggest that cellular HflX will mainly exist in the HflX·GTP·ribosome-bound form. A minimal model for the functional cycle of HflX is proposed.
Assuntos

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Proteínas de Ligação ao GTP / Proteínas de Escherichia coli / Subunidades Ribossômicas Maiores de Bactérias / Subunidades Ribossômicas Menores de Bactérias / Nucleotídeos de Guanina Tipo de estudo: Prognostic_studies Idioma: En Ano de publicação: 2012 Tipo de documento: Article

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Proteínas de Ligação ao GTP / Proteínas de Escherichia coli / Subunidades Ribossômicas Maiores de Bactérias / Subunidades Ribossômicas Menores de Bactérias / Nucleotídeos de Guanina Tipo de estudo: Prognostic_studies Idioma: En Ano de publicação: 2012 Tipo de documento: Article