Dataset Information

Differential Local Stability Governs the Metamorphic Fold Switch of Bacterial Virulence Factor RfaH.

ABSTRACT: RfaH, a two-domain protein from a universally conserved NusG/Spt5 family of regulators, is required for the transcription and translation of long virulence and conjugation operons in many Gram-negative bacterial pathogens. Escherichia coli RfaH action is controlled by a unique large-scale structural rearrangement triggered by recruitment to transcription elongation complexes through a specific DNA element. Upon recruitment, the C-terminal domain of RfaH refolds from an ?-hairpin, which is bound to RNA polymerase binding site within the N-terminal domain, into an unbound ?-barrel that interacts with the ribosome. Although structures of the autoinhibited (?-hairpin) and active (?-barrel) states and plausible refolding pathways have been reported, how this reversible switch is encoded within RfaH sequence and structure is poorly understood. Here, we combined hydrogen-deuterium exchange measurements by mass spectrometry and nuclear magnetic resonance with molecular dynamics to evaluate the differential local stability between both RfaH folds. Deuteron incorporation reveals that the tip of the C-terminal hairpin (residues 125-145) is stably folded in the autoinhibited state (?20% deuteron incorporation), whereas the rest of this domain is highly flexible (>40% deuteron incorporation), and its flexibility only decreases in the ?-folded state. Computationally predicted ?G agree with these results by displaying similar anisotropic stability within the tip of the ?-hairpin and on neighboring N-terminal domain residues. Remarkably, the ?-folded state shows comparable structural flexibility than nonmetamorphic homologs. Our findings provide information critical for understanding the metamorphic behavior of RfaH and other chameleon proteins and for devising targeted strategies to combat bacterial infections.

SUBMITTER: Galaz-Davison P

PROVIDER: S-EPMC6950767 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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Differential Local Stability Governs the Metamorphic Fold Switch of Bacterial Virulence Factor RfaH.

Galaz-Davison Pablo P Molina José Alejandro JA Silletti Steve S Komives Elizabeth A EA Knauer Stefan H SH Artsimovitch Irina I Ramírez-Sarmiento César A CA

Biophysical journal 20191121 1

RfaH, a two-domain protein from a universally conserved NusG/Spt5 family of regulators, is required for the transcription and translation of long virulence and conjugation operons in many Gram-negative bacterial pathogens. Escherichia coli RfaH action is controlled by a unique large-scale structural rearrangement triggered by recruitment to transcription elongation complexes through a specific DNA element. Upon recruitment, the C-terminal domain of RfaH refolds from an α-hairpin, which is bound ...[more]

PMID: 31810657

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Project description:The molecular machine necessary for protein synthesis, the ribosome, is generally considered constitutively functioning and lacking any inherent regulatory capacity. Yet ribosomes are commonly heterogeneous in composition and the impact of ribosome heterogeneity on translation is not well understood. Here, we determined that changes in ribosome protein composition govern gene expression in the intracellular bacterial pathogen Francisella tularensis. F. tularensis encodes three distinct homologs for bS21, a ribosomal protein involved in translation initiation, and analysis of purified F. tularensis ribosomes revealed they are heterogeneous with respect to bS21. The loss of one homolog, bS21-2, resulted in significant changes to the cellular proteome unlinked to changes in the transcriptome. Among the reduced proteins were components of the type VI secretion system (T6SS), an essential virulence factor encoded by the Francisella Pathogenicity Island. Furthermore, loss of bS21-2 led to an intramacrophage growth defect. Although multiple bS21 homologs complemented the loss of bS21-2 with respect to T6SS protein abundance, bS21-2 was uniquely necessary for robust intramacrophage growth, suggesting bS21-2 modulates additional virulence gene(s) distinct from the T6SS. Our results indicate that ribosome composition in F. tularensis, either directly or indirectly, posttranscriptionally modulates gene expression and virulence. Our findings are consistent with a model in which bS21 homologs function as posttranscriptional regulators, allowing preferential translation of specific subsets of mRNAs, likely at the stage of translation initiation. This work also raises the possibility that bS21 in other organisms may function similarly and that ribosome heterogeneity may permit many bacteria to posttranscriptionally regulate gene expression. IMPORTANCE While bacterial ribosomes are commonly heterogeneous in composition (e.g., incorporating different homologs for a ribosomal protein), how heterogeneity impacts translation is unclear. We found that the intracellular human pathogen Francisella tularensis has heterogeneous ribosomes, incorporating one of three homologs for ribosomal protein bS21. Furthermore, one bS21 homolog posttranscriptionally governs the expression of the F. tularensis type VI secretion system, an essential virulence factor. This bS21 homolog is also uniquely important for robust intracellular growth. Our data support a model in which bS21 heterogeneity leads to modulation of translation, providing another source of posttranscriptional gene regulation. Regulation of translation by bS21, or other sources of ribosomal heterogeneity, may be a conserved mechanism to control gene expression across the bacterial phylogeny.

Dataset Information

Differential Local Stability Governs the Metamorphic Fold Switch of Bacterial Virulence Factor RfaH.

Publications

Differential Local Stability Governs the Metamorphic Fold Switch of Bacterial Virulence Factor RfaH.

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