Tabilo-Agurto, C. | Search bioRxiv

bioRxiv2026

Exploration of the structural and functional diversity in the metamorphic RfaH subfamily

RfaH, a member of the universally conserved NusG/Spt5 family, activates transcription and translation of virulence genes by bridging the transcribing RNA polymerase to the translating ribosome. Unlike its monomorphic, constitutively active paralog NusG, RfaH fold-switches from an autoinhibited state, wherein its C-terminal domain forms an -helical hairpin bound to the N-terminal domain, into an active NusG-like state through C-terminal domain dissociation and refolding into a {beta}-barrel. RfaH has been proposed to evolve from NusG via gene duplication by first activating genes adjacent to its production site, before developing autoinhibition to selectively control distant genes. Using AlphaFold2, we predicted the structures of thousands of RfaH homologs and identified a subset ([~]14%) that appear predominantly folded in the active state. In vivo assays confirmed that these putative monomorphic homologs exhibit constitutive activity comparable to known RfaH mutants. Phylogenetic and genomic analyses revealed that these monomorphic proteins form a distinct clade and are preferentially located within or next to long virulence-associated operons. Together, these results further support a stepwise evolutionary model of RfaH specialization through structural transformation.

biochemistry↗

bioRxiv2022

Developing a temperature-inducible transcriptional rheostat in Neurospora crassa

Heat shock protein (hsp) encoding genes, part of the highly conserved Heat Shock Response (HSR), are known to be induced by thermal stress in several organisms. In Neurospora crassa, three hsp genes, hsp30, hsp70, and hsp80, have been characterized; however, the role of defined cis-elements in their response to discrete changes in temperature remains largely unexplored. To fill this gap, while also aiming to obtain a reliable fungal heat-shock inducible system, we analyzed different sections of each hsp promoter, by assessing the expression of real-time transcriptional reporters. Whereas all three promoters, and their resected versions, were acutely induced by high temperatures, only hsp30 displayed a broad range of expression and high tunability amply exciding other inducible promoter systems existing in Neurospora, such as Quinic acid- or light-inducible ones. As proof of concept, we employed one of these promoters to control the expression of clr-2, which encodes for the master regulator of Neurospora cellulolytic capabilities. The resulting strain fails to grow on cellulose at 25{degrees}C, whereas it robustly grows if heat shock pulses are delivered daily. Additionally, we designed two hsp30 synthetic promoters and characterized these, as well as the native promoters, to a gradient of high temperatures, yielding a wide range of responses to thermal stimuli. Thus, Neurospora hsp30-based promoters represent a new set of modular elements that can be used as a transcriptional rheostat to adjust the expression of a gene of interest or for the implementation of regulated circuitries for synthetic biology and biotechnological strategies. ImportanceTimely and dynamic response to strong temperature rises is paramount for organismal biology. At the same time, inducible promoters are a powerful tool for fungal biotechnological and synthetic biology endeavors. In this work, we analyzed the activity of several N. crassa heat shock protein (hsp) promoters upon a wide range of temperatures, observing that hsp30 exhibits remarkable sensitivity and dynamic range of expression as we chartered the response of this promoter to subtle increases in temperature, while also building synthetic promoters based on hsp30 cis-elements. As proof of concept, we analyzed the ability of hsp30 to provide tight control of a central process such as cellulose degradation. While this study provides an unprecedented description of the regulation of the N. crassa hsp genes it also contributes with a noteworthy addition to the molecular toolset of transcriptional controllers in filamentous fungi.

molecular biology↗