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

2021 ◽  
Vol 17 (9) ◽  
pp. e1008882
Author(s):  
Pablo Galaz-Davison ◽  
Ernesto A. Román ◽  
César A. Ramírez-Sarmiento
Keyword(s):  
Molecular Dynamics ◽  
Virulence Factors ◽  
Elongation Factor ◽  
Active Role ◽  
Structural Rearrangement ◽  
Umbrella Sampling ◽  
Protein Fold ◽  
Terminal Domain ◽  
Rnap Binding

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.

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

2021 ◽  
Author(s):  
Pablo Galaz-Davison ◽  
Ernesto A Román ◽  
Cesar A. Ramirez-Sarmiento
Keyword(s):  
Molecular Dynamics ◽  
Virulence Factors ◽  
Elongation Factor ◽  
Active Role ◽  
Structural Rearrangement ◽  
Umbrella Sampling ◽  
Protein Fold ◽  
Native State ◽  
Terminal Domain ◽  
Rnap Binding

The bacterial elongation factor RfaH promotes the expression of virulence factors by specifically binding to RNA polymerases (RNAP) stalled at a DNA signal known as ops. 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 ops 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, most studies employ tertiary biases towards each native state, hampering the analysis of sequence-encoded interactions on fold-switching. 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 also by favoring β-barrel rupture into an intermediate from which fold-switching proceeds.

The Role of the N-Terminal Domain in the Regulation of the “Constitutively Active” Conformation of Protein Kinase CK2α: Insight from a Molecular Dynamics Investigation

ChemMedChem ◽  
2011 ◽  
Vol 6 (7) ◽  
pp. 1207-1216 ◽  
Author(s):  
Andrea Cristiani ◽  
Giorgio Costa ◽  
Giorgio Cozza ◽  
Flavio Meggio ◽  
Leonardo Scapozza ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Protein Kinase ◽  
Active Conformation ◽  
Terminal Domain ◽  
Constitutively Active

scholarly journals Active role of elongation factor G in maintaining the mRNA reading frame during translation

2019 ◽  
Vol 5 (12) ◽  
pp. eaax8030 ◽  
Author(s):  
Bee-Zen Peng ◽  
Lars V. Bock ◽  
Riccardo Belardinelli ◽  
Frank Peske ◽  
Helmut Grubmüller ◽  
...  
Keyword(s):  
Elongation Factor ◽  
Active Role ◽  
Transfer Rna ◽  
Specific Interactions ◽  
Elongation Factor G ◽  
Reading Frame ◽  
A Site ◽  
Factor G ◽  
Domain 4

During translation, the ribosome moves along the mRNA one codon at a time with the help of elongation factor G (EF-G). Spontaneous changes in the translational reading frame are extremely rare, yet how the precise triplet-wise step is maintained is not clear. Here, we show that the ribosome is prone to spontaneous frameshifting on mRNA slippery sequences, whereas EF-G restricts frameshifting. EF-G helps to maintain the mRNA reading frame by guiding the A-site transfer RNA during translocation due to specific interactions with the tip of EF-G domain 4. Furthermore, EF-G accelerates ribosome rearrangements that restore the ribosome’s control over the codon-anticodon interaction at the end of the movement. Our data explain how the mRNA reading frame is maintained during translation.

scholarly journals Interaction between P-TEFb and the C-Terminal Domain of RNA Polymerase II Activates Transcriptional Elongation from Sites Upstream or Downstream of Target Genes

2002 ◽  
Vol 22 (1) ◽  
pp. 321-331 ◽  
Author(s):  
Ran Taube ◽  
Xin Lin ◽  
Dan Irwin ◽  
Koh Fujinaga ◽  
B. Matija Peterlin
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Target Genes ◽  
Elongation Factor ◽  
Transcriptional Elongation ◽  
Cyclin T1 ◽  
Positive Transcription Elongation Factor ◽  
Terminal Domain ◽  
Activation Of Transcription

ABSTRACT Transcriptional elongation by RNA polymerase II (RNAPII) is regulated by the positive transcription elongation factor b (P-TEFb). P-TEFb is composed of Cdk9 and C-type cyclin T1 (CycT1), CycT2a, CycT2b, or CycK. The role of the C-terminal region of CycT1 and CycT2 remains unknown. In this report, we demonstrate that these sequences are essential for the activation of transcription by P-TEFb via DNA, i.e., when CycT1 is tethered upstream or downstream of promoters and coding sequences. A histidine-rich stretch, which is conserved between CycT1 and CycT2 in this region, bound the C-terminal domain of RNAPII. This binding was required for the subsequent expression of full-length transcripts from target genes. Thus, P-TEFb could mediate effects of enhancers on the elongation of transcription.

Exploring the role of elongation Factor-Like 1 (EFL1) in Shwachman-Diamond syndrome through molecular dynamics

2019 ◽  
Vol 38 (17) ◽  
pp. 5219-5229
Author(s):  
Pietro Delre ◽  
Domenico Alberga ◽  
Abril Gijsbers ◽  
Nuria Sánchez-Puig ◽  
Orazio Nicolotti ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Elongation Factor ◽  
Shwachman Diamond Syndrome

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

2019 ◽  
Author(s):  
P. Galaz-Davison ◽  
J.A. Molina ◽  
S. Silletti ◽  
E.A. Komives ◽  
S.H. Knauer ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Local Stability ◽  
Large Scale ◽  
Target Genes ◽  
Structural Rearrangement ◽  
Virulence Determinants ◽  
Gram Negative ◽  
Rapid Acquisition ◽  
Terminal Domain ◽  
Polymerase Binding

AbstractA 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 α-hairpin, which is bound to the RNA polymerase binding site within the N-terminal domain of RfaH, into an unbound β-barrel that interacts with the ribosome to enable translation. Although structures of the autoinhibited (α-hairpin) and active (β-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 β-folded state. Computationally-predicted ΔGs agree with these results by displaying similar anisotropic stability within the tip of the α-hairpin and on neighboring N-terminal domain residues. Remarkably, the β-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.SignificanceInfections 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.

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