Poster abstracts
Poster number 47 submitted by Cyndi Tabilo Agurto
Tracing the metamorphic behavior of RfaH through evolution
Cyndi Tabilo-Agurto (Institute for Biological and Medical Engineering, Schools of Engineering, Medicine and Biological Sciences, Pontificia Universidad Catlica de Chile, Santiago 7820436, Chile; ANID, Millennium Science), Irina Artsimovitch (Department of Microbiology and Center for RNA Biology, The Ohio State University, Columbus, OH 43210, USA), Cesar A. Ramirez-Sarmiento (Institute for Biological and Medical Engineering, Schools of Engineering, Medicine and Biological Sciences, Pontificia Universidad Catlica de Chile, Santiago 7820436, Chile; ANID, Millennium Science)
Abstract:
NusG factors are transcription elongation regulators found in all domains of life, modulating gene expression by binding to RNA polymerase (RNAP). In Escherichia coli, NusG acts as a general regulator [1], while its specialized paralog, RfaH, functions as a virulence factor [2]. RfaH is activated upon binding to its ops DNA target when its C-terminal (CTD) KOW domain fold-switches from an α-helical hairpin to a β-barrel [3]. It is estimated that ~20% of specialized NusG proteins can switch folds [4], but the evolutionary origins of this metamorphic behavior remain unknown.
In this work, we address the evolutionary emergence of RfaH’s metamorphic behavior through ancestral sequence reconstruction (ASR). A phylogenetic tree was built using homologs from GenBank and UniProt. Structural predictions using AlphaFold2 revealed that UniProt ancestors predominantly had RfaH-like CTDs, whereas GenBank ancestors displayed NusG-like structures. Ancestral proteins that were overexpressed in a soluble form were analyzed using in vitro transcription assays. Results indicate that most ancestors facilitated RNA chain elongation, albeit less efficiently than extant RfaH, and bound ops similarly to RfaH; however, some exhibited unique behaviors, delaying RNAP elongation upstream of the ops site. These findings suggest the existence of an ancestral recognition mechanism that preceded the evolution of ops-specific recruitment.
FUNDING: ANID FONDECYT 1240205, ANID PFCHA 21231023, Millennium Science Initiative Program ICN17_022, NIH R01 GM067153, EMBO Global Investigator Network
References:
[1] Sullivan, S. L., & Gottesman, M. E. (1992). Requirement for E. coli NusG protein in factor-dependent transcription termination. Cell, 68(5), 989-994.
[2] Leeds, J. A., & Welch, R. A. (1996). RfaH enhances elongation of Escherichia coli hlyCABD mRNA. Journal of bacteriology, 178(7), 1850-1857
[3] Burmann, B.M., Knauer, S.H., Sevostyanova, A., Schweimer, K., Mooney, R.A., Landick, R., Artsimovitch, I., and Rosch, P. (2012). An alpha helix to beta barrel domain switch transforms the transcription factor RfaH into a translation factor. Cell 150, 291-303. 10.1016/j.cell.2012.05.042
[4] Porter, L. L., Kim, A. K., Rimal, S., Looger, L. L., Majumdar, A., Mensh, B. D., ... & Strub, M. P. (2022). Many dissimilar NusG protein domains switch between α-helix and β-sheet folds. Nature Communications, 13(1), 3802.
Keywords: Metamorphic protein RfaH, Ancestral sequence reconstruction, Transcription