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A Transient, Excited Species of the Autoinhibited α-State of the Bacterial Transcription Factor RfaH May Represent an Early Intermediate on the Fold-Switching Pathway.
Cai, Mengli; Agarwal, Nipanshu; Garrett, Daniel S; Baber, James; Clore, G Marius.
Afiliación
  • Cai M; Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-0520, United States.
  • Agarwal N; Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-0520, United States.
  • Garrett DS; Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-0520, United States.
  • Baber J; Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-0520, United States.
  • Clore GM; Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-0520, United States.
Biochemistry ; 63(16): 2030-2039, 2024 Aug 20.
Article en En | MEDLINE | ID: mdl-39088556
ABSTRACT
RfaH is a two-domain transcription factor in which the C-terminal domain switches fold from an α-helical hairpin to a ß-roll upon binding the ops-paused RNA polymerase. To ascertain the presence of a sparsely populated excited state that may prime the autoinhibited resting state of RfaH for binding ops-paused RNA polymerase, we carried out a series of NMR-based exchange experiments to probe for conformational exchange on the millisecond time scale. Quantitative analysis of these data reveals exchange between major ground (∼95%) and sparsely populated excited (∼5%) states with an exchange lifetime of ∼3 ms involving residues at the interface between the N-terminal and C-terminal domains formed by the ß3/ß4 hairpin and helix α3 of the N-terminal domain and helices α4 and α5 of the C-terminal domain. The largest 15N backbone chemical shift differences are associated with the ß3/ß4 hairpin, leading us to suggest that the excited state may involve a rigid body lateral displacement/rotation away from the C-terminal domain to adopt a position similar to that seen in the active RNA polymerase-bound state. Such a rigid body reorientation would result in a reduction in the interface between the N- and C-terminal domains with the possible introduction of a cavity or cavities. This hypothesis is supported by the observation that the population of the excited species and the exchange rate of interconversion between ground and excited states are reduced at a high (2.5 kbar) pressure. Mechanistic implications for fold switching of the C-terminal domain in the context of RNA polymerase binding are discussed.
Asunto(s)

Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Pliegue de Proteína / Proteínas de Escherichia coli Idioma: En Revista: Biochemistry Año: 2024 Tipo del documento: Article País de afiliación: Estados Unidos Pais de publicación: Estados Unidos

Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Pliegue de Proteína / Proteínas de Escherichia coli Idioma: En Revista: Biochemistry Año: 2024 Tipo del documento: Article País de afiliación: Estados Unidos Pais de publicación: Estados Unidos