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Exploration of the structural and functional diversity in the metamorphic RfaH subfamily
Cyndi Tabilo-Agurto1,2, Bastián González-Bustos1,2, Javiera Reyes1,2
1Institute for Biological and Medical Engineering, Schools of Engineering, Medicine and Biological Sciences, Pontificia Universidad Católica de Chile, Santiago 7820436, Chile.
The bacterial protein RfaH, crucial for virulence gene expression, can exist in active or autoinhibited states. Researchers identified constitutively active RfaH forms, supporting an evolutionary pathway involving structural changes.
Area of Science:
- Molecular Biology
- Evolutionary Biology
- Structural Biology
Background:
- RfaH, a conserved protein, links RNA polymerase and ribosomes to activate transcription and translation of bacterial virulence genes.
- Unlike its paralog NusG, RfaH exhibits a unique fold-switching mechanism between autoinhibited and active states.
Purpose of the Study:
- To investigate the structural diversity and evolutionary origins of RfaH homologs.
- To identify RfaH variants that function constitutively, bypassing autoinhibition.
Main Methods:
- Computational structure prediction using AlphaFold2 for thousands of RfaH homologs.
- In vivo functional assays to confirm the activity of predicted RfaH structures.
- Phylogenetic and genomic analyses to understand the evolutionary context of RfaH homologs.
Main Results:
- A subset of RfaH homologs (approximately 14%) were predicted to predominantly exist in the active state.
- These monomorphic homologs demonstrated constitutive activity, similar to known RfaH mutants.
- Phylogenetic analysis revealed a distinct clade of monomorphic RfaH proteins associated with virulence operons.
Conclusions:
- The findings support a model of RfaH evolution involving stepwise structural transformations.
- The identification of constitutively active RfaH forms provides insights into the regulation of virulence gene expression.
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