Biology subjects

Knauer, S. H.

Publications and source records attributed to Knauer, S. H..

5 recordsLinked to original sources

bioRxiv2023

Concerted transformation of a hyper-paused transcription complex and its reinforcing protein

RfaH, a paralog of the universally conserved NusG, binds to RNA polymerases (RNAP) and ribosomes to activate expression of virulence genes. In free, autoinhibited RfaH, an -helical KOW domain sequesters the RNAP-binding site. Upon recruitment to RNAP paused at an ops site, KOW is released and refolds into a {beta}-barrel, which binds the ribosome. Our structures of ops-paused transcription elongation complexes alone and bound to the autoinhibited and activated RfaH reveal swiveled, pre-translocated pause states stabilized by an ops hairpin in the non-template DNA. Autoinhibited RfaH binds and twists the ops hairpin, expanding the RNA:DNA hybrid to 11 base pairs and triggering the KOW release. Once activated, RfaH hyper-stabilizes the pause, which thus requires anti-backtracking factors for escape. Our results suggest that the entire RfaH cycle is solely determined by the ops and RfaH sequences and provide insights into mechanisms of recruitment and metamorphosis of NusG homologs across all life. HIGHLIGHTS- The nontemplate DNA strand of an ops-paused transcription complex forms a hairpin - Autoinhibited RfaH binds and twists the ops hairpin to expand the RNA:DNA hybrid - RfaH-hairpin contacts are solely responsible for triggering RfaH activation - Upon recruitment, RfaH hyper-stabilizes the pause and promotes RNAP backtracking

biochemistry↗

bioRxiv2022

Structural and thermodynamic analyses of the β-to-α transformation in RfaH reveal principles of fold-switching proteins

The two-domain protein RfaH, a paralog of the universally conserved NusG/Spt5 transcription factors, is regulated by autoinhibition coupled to the reversible conformational switch of its 60-residue C-terminal KOW domain between an -hairpin and a {beta}-barrel. In contrast, NusG/Spt5-KOW domains only occur in the {beta}-barrel state. To understand the principles underlying the drastic fold switch in RfaH, we elucidated the thermodynamic stability and the structural dynamics of two RfaH- and four NusG/Spt5-KOW domains by combining biophysical and structural biology methods. We find that the RfaH-KOW {beta}-barrel is thermodynamically less stable than that of most NusG/Spt5-KOWs and we show that it is in equilibrium with a globally unfolded species, which, strikingly, contains two helical regions that prime the transition towards the -hairpin. Our results suggest that transiently structured elements in the unfolded conformation might drive the global folding transition in metamorphic proteins in general.

biophysics↗

bioRxiv2019

Escherichia coli NusG links the lead ribosome with the transcription elongation complex

It has been known for more than 50 years that transcription and translation are physically coupled in bacteria, but whether or not this coupling may be mediated by the two-domain protein N-utilization substance (Nus) G in Escherichia coli is still heavily debated. Here, we combine integrative structural biology and functional analyses to provide conclusive evidence that NusG can physically link transcription with translation by contacting both RNA polymerase and the ribosome. We present a cryo-electron microscopy structure of a NusG:70S ribosome complex and nuclear magnetic resonance spectroscopy data revealing simultaneous binding of NusG to RNAP and the intact 70S ribosome, providing the first direct structural evidence for NusG-mediated coupling. Furthermore, in vivo reporter assays show that recruitment of NusG occurs late in transcription and strongly depends on translation. Thus, our data suggest that coupling occurs initially via direct RNAP:ribosome contacts and is then mediated by NusG.

molecular biology↗

bioRxiv2019

Differential local stability governs the metamorphic fold-switch of bacterial virulence factor RfaH

A regulatory factor RfaH, present in many Gram-negative bacterial pathogens, is required for transcription and translation of long operons encoding virulence determinants. Escherichia coli RfaH action is controlled by a unique large-scale structural rearrangement triggered by recruitment to transcription elongation complexes through a specific DNA sequence within these operons. Upon recruitment, the C-terminal domain of this two-domain protein refolds from an -hairpin, which is bound to the RNA polymerase binding site within the N-terminal domain of RfaH, into an unbound {beta}-barrel that interacts with the ribosome to enable translation. Although structures of the autoinhibited (-hairpin) and active ({beta}-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), while the rest of this domain is highly flexible (>40% deuteron incorporation) and its flexibility only decreases in the {beta}-folded state. Computationally-predicted {Delta}Gs agree with these results by displaying similar anisotropic stability within the tip of the -hairpin and on neighboring N-terminal domain residues. Remarkably, the {beta}-folded state shows comparable stability to non-metamorphic 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 diseases.\n\nSignificanceInfections caused by Gram-negative bacteria are a worldwide health threat due to rapid acquisition of antibiotic resistance. RfaH, a protein essential for virulence in several Gram-negative pathogens, undergoes a large-scale structural rearrangement in which one RfaH domain completely refolds. Refolding transforms RfaH from an inactive state that restricts RfaH recruitment to a few target genes into an active state that binds to, and couples, transcription and translation machineries to elicit dramatic activation of gene expression. However, the molecular basis of this unique conformational change is poorly understood. Here, we combine molecular dynamics and structural biology to unveil the hotspots that differentially stabilize both states of RfaH. Our findings provide novel insights that will guide design of inhibitors blocking RfaH action.

biophysics↗

bioRxiv2018

The universally-conserved transcription factor RfaH is recruited to a hairpin structure of the non-template DNA strand

RfaH, a transcription regulator of the universally conserved NusG/Spt5 family, utilizes a unique mode of recruitment to elongating RNA polymerase to activate virulence genes. RfaH function depends critically on an ops sequence, an exemplar of a consensus pause, in the non-template DNA strand of the transcription bubble. We used structural and functional analyses to elucidate the role of ops in RfaH recruitment. Our results demonstrate that ops induces pausing to facilitate RfaH binding and establishes direct contacts with RfaH. Strikingly, the non-template DNA forms a hairpin in the RfaH: ops complex structure, flipping out a conserved T residue that is specifically recognized by RfaH. Molecular modeling and genetic evidence support the notion that ops hairpin is required for RfaH recruitment. We argue that both the sequence and the structure of the non-template strand are read out by transcription factors, expanding the repertoire of transcriptional regulators in all domains of life.

biochemistry↗