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 α-helical KOW domain sequesters the RNAP-binding site. Upon recruitment to RNAP paused at an ops site, KOW is released and refolds into a β-barrel, which binds the ribosome. Here, we report structures of ops-paused transcription elongation complexes alone and bound to the autoinhibited and activated RfaH, which 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

The energy landscapes of metamorphic proteins

Most proteins fold into a unique three-dimensional structure called the native state. Recently some examples have been found of so-called metamorphic proteins that undergo reversible large-scale structural transformations between different native states. In this thesis, we develop simulation methods and models to study the thermodynamics of these transformations, both at the coarse-grained and all-atom levels. Because our understanding of the physics fold switching is incomplete, our models utilize in part so-called structure-based or Gō-like potentials, which provide energetic bias towards one, or more, native states. We employ these computational methods to two different fold switch systems: the bacterial protein RfaH and the engineered fold switch system GA/GB. Our models are developed and tested on experimental data for these systems. We study both equilibrium properties, such as stability properties and the characteristics of their energy landscapes, and kinetic properties, such as the mechanism that trigger fold switching and molecular details of the fold switch process. We also study, for the GA/GB system, what role macromolecular crowding effects play for controlling which of the native states is most stable

The N-terminal domain of RfaH plays an active role in protein fold-switching

The bacterial elongation factor RfaH promotes the expression of virulence factors by specifically binding to RNA polymerases (RNAP) paused at a DNA signal. This behavior is unlike that of its paralog NusG, the major representative of the protein family to which RfaH belongs. Both proteins have an N-terminal domain (NTD) bearing an RNAP binding site, yet NusG C-terminal domain (CTD) is folded as a β-barrel while RfaH CTD is forming an α-hairpin blocking such site. Upon recognition of the specific DNA exposed by RNAP, RfaH is activated via interdomain dissociation and complete CTD structural rearrangement into a β-barrel structurally identical to NusG CTD. Although RfaH transformation has been extensively characterized computationally, little attention has been given to the role of the NTD in the fold-switching process, as its structure remains unchanged. Here, we used Associative Water-mediated Structure and Energy Model (AWSEM) molecular dynamics to characterize the transformation of RfaH, spotlighting the sequence-dependent effects of NTD on CTD fold stabilization. Umbrella sampling simulations guided by native contacts recapitulate the thermodynamic equilibrium experimentally observed for RfaH and its isolated CTD. Temperature refolding simulations of full-length RfaH show a high success towards α-folded CTD, whereas the NTD interferes with βCTD folding, becoming trapped in a β-barrel intermediate. Meanwhile, NusG CTD refolding is unaffected by the presence of RfaH NTD, showing that these NTD-CTD interactions are encoded in RfaH sequence. Altogether, these results suggest that the NTD of RfaH favors the α-folded RfaH by specifically orienting the αCTD upon interdomain binding and by favoring β-barrel rupture into an intermediate from which fold-switching proceeds.Fil: Galaz Davison, Pablo. Pontificia Universidad Católica de Chile; Chile. Universidad Católica de Chile; ChileFil: Roman, Ernesto Andres. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Ramírez Sarmiento, César A.. Pontificia Universidad Católica de Chile; Chile. Universidad Católica de Chile; Chil

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

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

Premature rho-dependent transcription termination in escherichia coli : link to translation and gene regulation

Transcription termination factor Rho is an essential protein in Escherichia coli and related bacteria. The primary function of Rho is to clear unproductive RNA polymerases from the DNA template to minimize negative effects associated with uncontrolled transcription. Although most of the Rho termination events are constitutive, premature Rho-mediated termination was observed at 3% of all affected transcripts indicating active regulation of Rho activity. In this work, we investigated the regulatory mechanism behind premature Rho-dependent transcription termination in two unrelated genes: suhB and topAI. We show that in both cases transcription is terminated inside the coding gene as a consequence of translational repression under normal growth conditions. The repression of translation at suhB and topAI is different at the mechanistic level. Interestingly, both genes are adapted to sense the translational status of the cell in order to regulate expression. Both genes utilize Rho as a second layer of repression when needed. This emphasizes the importance of Rho in the surveillance of unproductive transcription, and how this feature can be utilized in gene regulation. We also established an in vivo Rho activity assay that is suitable for large-scale high-throughput molecule screen. Future applications of this assay might yield nove

The Protein-protein Interaction Map of the Treponema pallidum Flagellar Apparatus

Diese Studie stellt den ersten umfassenden Versuch dar, die Protein-Protein-Interaktionen (PPIs) eines bakteriellen Flagellenapparats genomweit zu erfassen. Wir haben dafür Treponema pallidum ausgewählt, also Spirochäten, die auch die Syphilis auslösen. T. pallidum hat ein kleines Genom mit nur 1,041 Genen, die alle in klonierter Form vorliegen. Die Flagellen von Spirochäten besitzen ausserdem einige spezielle Eigenschaften wie die periplasmatische Lokalisation der zwei polaren Flagellenbündel, welche sie für vergleichende Analysen besonders interessant machen Im Lauf dieses Projekts habe ich eine Yeast two-hybrid-Bibliothek des Treponema pallidum-Proteoms konstruiert und damit umfassende genom-weite Screens mit allen 75 Flagellen- und Motilitäts-Proteinen durchgeführt. Insgesamt wurden dabei 268 PPIs zwischen 174 verschiedenen Proteinen identifiziert. Davon waren zuvor nur 15 homologe Interaktionen (“Interologe”) aus der Literatur bekannt. Um die funktionelle Bedeutung dieser neuen Interaktionen für die bakterielle Motilität zu untersuchen, habe ich zuerst 23 Orthologe aller Treponema-Interaktoren in E. coli und 30 in B. subtilis vorhergesagt. Die Gene von 23 Orthologen wurden dann in E. coli und/oder B. subtilis (7 Gene) mutiert und deren Phänotyp in Motilitätsassays überprüft. Ich fand dabei 10 neue Proteine in E. coli (amiA, bcsC, rfaH, rpe, rpmJ, ycfH, yciM, yggH, yggW, und yncE) und 5 Proteine in B. subtilis (yaaT , yhbE/YhbF, yllB, yloS und yviF), deren Treponema-Homologe mit Flagellenproteinen interagierten und deren Mutanten einen Motilitätsphänotyp zeigen. Aufgrund ihrer Interaktionen und Phänotypen konnte ich 8 bisher nicht charakterisierten Proteinen eine Rolle als Motilitätsgene zuordnen, und zwar TP0046 (yaaT), TP0048 (yhbE/yhbF), TP0383 (yllB), TP0421 (yncE), TP0561 (ydjH), TP0658 (yviF), TP0712 (HP1034) and TP0979 (tatD). Basierend auf den 268 two-hybrid-Interaktionen konnte ich ausserdem 1455 Interaktionen in 30 anderen Bakterien vorhersagen. Mit 35 Flagellenproteinen von 30 Bakterienarten habe ich ausserdem einen phylogenetischen “Super-Stammbaum” erstellt, der die Flagellenapparate in einen evolutionären Kontext stellt und belegt, dass die Spirochäten einen ungewöhnlichen und abgeleiteten Flagellenapparat aufweisen. Ein Protein unbekannter Funktion, TP0658 (yviF in B. subtilis) wurde näher untersucht. Die Y2H-Interaktionen von TP0658 mit Flagellinen (flaB1-3) wurden biochemisch verifiziert. TP0658 bindet an eine C-terminale Sequenz von FlaB1 zwischen L231 und D247 (dem so genannten C-Loop der Flagelline). Dieses epitop ist in B. subtilis und anderen Bakterien konserviert. Ein hochkonserviertes Asparagin (N237 in FlaB1 bzw. N255 in B. subtilis hag) ist entscheidend für die Bindung. Eine ΔyviF Mutante in B. subtilis hatte einen starken Motilitätsphänotyp und keine nachweisbaren Flagelline. Koexpression von TP0658 und FlaB1 in E. coli stabilisiert FlaB1. Interessanterweise binden sowohl TP0658 als auch FliS (ein bekanntes aber nicht verwandtes Chaperon) an das gleiche Epitop in Flagellin, welches bei dessen Polymerisierung beteiligt ist. Meine Daten sprechen daher dafür dass TP658 und seine Verwandten “Assembly-Chaperone” der bakteriellen Flagelle sind