Impact des pauses transcriptionnelles sur le repliement des riborégulateurs

Pour s’adaptater à l’environnement et répondre aux besoins énergétiques, tous les organismes doivent pouvoir contrôler l’expression de certains gènes. La trancription constitue le premier échelon de cette régulation en déterminant le niveau d’ARNm produit au cours du temps. Chez les procaryotes, une seule ARNp est responsable de la synthèse de l’ensemble des ARN de la cellule et celle-ci est soumise à différents types de régulation. Ce contrôle peut s’effectuer à tous les niveaux par des processus faisant intervenir bon nombre de facteurs externes ou intrinsèques à la transcription comme les pauses de l’ARNp. Les riborégulateurs sont des ARN structurés majoritairement retrouvés dans la région 5’ non traduite des ARNm bactériens qui vont réguler l’expression du gène situé en aval. Suite à la liaison d’un métabolite particulier, appelé ligand, le riborégulateur change de conformation induisant une réponse directe qui déterminera si un gène est exprimé ou non. Au cours de mes travaux j’ai établi le lien qu’il existait entre les pauses de l’ARNp au cours de la transcription et la structure des riborégulateurs. En prenant pour modèles deux riborégulateurs liant le TPP, j’ai identifié une conformation des riborégulateurs qui est réfractaire à la liaison du ligand. Cette structure appelée «Anti-P1» se forme lorsque l’ARNp pause à la fin du riborégulateur, ce qui détermine une fenêtre de liaison cotranscriptionnelle du ligand

Unprecedented tunability of riboswitch structure and regulatory function by sub-millimolar variations in physiological Mg2+

Riboswitches are cis-acting regulatory RNA biosensors that rival the efficiency of those found in proteins. At the heart of their regulatory function is the formation of a highly specific aptamer–ligand complex. Understanding how these RNAs recognize the ligand to regulate gene expression at physiological concentrations of Mg2+ ions and ligand is critical given their broad impact on bacterial gene expression and their potential as antibiotic targets. In this work, we used single-molecule FRET and biochemical techniques to demonstrate that Mg2+ ions act as fine-tuning elements of the amino acid-sensing lysC aptamer's ligand-free structure in the mesophile Bacillus subtilis. Mg2+ interactions with the aptamer produce encounter complexes with strikingly different sensitivities to the ligand in different, yet equally accessible, physiological ionic conditions. Our results demonstrate that the aptamer adapts its structure and folding landscape on a Mg2+-tunable scale to efficiently respond to changes in intracellular lysine of more than two orders of magnitude. The remarkable tunability of the lysC aptamer by sub-millimolar variations in the physiological concentration of Mg2+ ions suggests that some single-aptamer riboswitches have exploited the coupling of cellular levels of ligand and divalent metal ions to tightly control gene expression.Publisher PDFPeer reviewe

Trans regulation in the Ultrabithorax gene of Drosophila: alterations in the promoter enhance transvection

PMCID: PMC556824We report a genetic and molecular study of UbxMX6 and Ubx195rx1, two mutations in the Ultrabithorax (Ubx) locus which appear to have a strong effect on the activity of the homologous Ubx gene. These mutations show the characteristic embryonic and adult phenotypes of Ubx null alleles, and also fail to produce any detectable Ubx product. Yet, genetic and phenotypic analyses involving a large number of trans heterozygous combinations of UbxMX6 and Ubx195rx1 with different classes of Ubx mutations, indicate that they hyperactivate the homologous gene. This effect is induced on wildtype or mutant forms of Ubx, provided that the pairing in the bithorax region is normal, i.e. these mutations have a strong positive effect on transvection. We also show that, unlike all the other known cases of transvection in Ubx, this is not zeste-dependent. Southern analyses indicate that UbxMX6 is a 3.4 kb deletion, and Ubx195rx1 is an approximately 11 kb insertion of foreign DNA, both in the promoter region. We speculate that the region altered in the mutations may have a wildtype function to ensure cis-autonomy of the regulation of Ubx transcription.This work was supported by grants from the DGICYT and the Fundación Ramón Areces.Peer reviewe

Impact des pauses transcriptionnelles sur le repliement des riborégulateurs

Pour s’adaptater à l’environnement et répondre aux besoins énergétiques, tous les organismes doivent pouvoir contrôler l’expression de certains gènes. La trancription constitue le premier échelon de cette régulation en déterminant le niveau d’ARNm produit au cours du temps. Chez les procaryotes, une seule ARNp est responsable de la synthèse de l’ensemble des ARN de la cellule et celle-ci est soumise à différents types de régulation. Ce contrôle peut s’effectuer à tous les niveaux par des processus faisant intervenir bon nombre de facteurs externes ou intrinsèques à la transcription comme les pauses de l’ARNp. Les riborégulateurs sont des ARN structurés majoritairement retrouvés dans la région 5’ non traduite des ARNm bactériens qui vont réguler l’expression du gène situé en aval. Suite à la liaison d’un métabolite particulier, appelé ligand, le riborégulateur change de conformation induisant une réponse directe qui déterminera si un gène est exprimé ou non. Au cours de mes travaux j’ai établi le lien qu’il existait entre les pauses de l’ARNp au cours de la transcription et la structure des riborégulateurs. En prenant pour modèles deux riborégulateurs liant le TPP, j’ai identifié une conformation des riborégulateurs qui est réfractaire à la liaison du ligand. Cette structure appelée «Anti-P1» se forme lorsque l’ARNp pause à la fin du riborégulateur, ce qui détermine une fenêtre de liaison cotranscriptionnelle du ligand

A nascent riboswitch helix orchestrates robust transcriptional regulation through signal integration

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/192779/1/Chauvier 2024 - Nature Communications.zipDescription of Chauvier 2024 - Nature Communications.zip : All single molecule datasets + Matlab codesSEL

Dynamic competition between a ligand and transcription factor NusA governs riboswitch-mediated transcription regulation

Co-transcriptional RNA folding is widely assumed to influence the timely control of gene expression, but our understanding remains limited. In bacteria, the fluoride (F-) sensing riboswitch is a transcriptional control element essential to defend against toxic F- levels. Using this model riboswitch, we find that its ligand F- and essential bacterial transcription factor NusA compete for binding the co-transcriptionally folding RNA, opposing each other’s modulation of downstream pausing and termination by RNA polymerase. Single molecule fluorescence assays probing active transcription elongation complexes discover that NusA unexpectedly binds highly reversibly, frequently interrogating the complex for emerging, co-transcriptionally folding RNA duplexes. NusA thus fine-tunes the transcription rate in dependence of the ligand-responsive higher order structure of the riboswitch. At the high NusA concentrations found intracellularly, this dynamic modulation is expected to lead to adaptive bacterial transcription regulation with fast response times.GM131922, GM062357, GM118524Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/170438/1/EC25-No Fluoride - NusA.ziphttp://deepblue.lib.umich.edu/bitstream/2027.42/170438/2/EC25-With Fluoride - NusA.ziphttp://deepblue.lib.umich.edu/bitstream/2027.42/170438/3/HisP - NusA.ziphttp://deepblue.lib.umich.edu/bitstream/2027.42/170438/4/Matlab scripts.ziphttp://deepblue.lib.umich.edu/bitstream/2027.42/170438/5/PEC48-A10U-No Fluoride - NusA.ziphttp://deepblue.lib.umich.edu/bitstream/2027.42/170438/6/PEC48-A10U-U38A-No Fluoride - NusA.ziphttp://deepblue.lib.umich.edu/bitstream/2027.42/170438/7/PEC48-A10U-U38A-With Fluoride - NusA.ziphttp://deepblue.lib.umich.edu/bitstream/2027.42/170438/8/PEC48-A10U-With Fluoride - NusA.ziphttp://deepblue.lib.umich.edu/bitstream/2027.42/170438/9/PEC48-U38A-No Fluoride - NusA.ziphttp://deepblue.lib.umich.edu/bitstream/2027.42/170438/10/PEC48-U38A-With Fluoride - NusA.ziphttp://deepblue.lib.umich.edu/bitstream/2027.42/170438/11/PEC48-WT-No Fluoride - Competition unlabeled NusA.ziphttp://deepblue.lib.umich.edu/bitstream/2027.42/170438/12/PEC48-WT-No Fluoride - NusA.ziphttp://deepblue.lib.umich.edu/bitstream/2027.42/170438/13/PEC48-WT-With Fluoride - NusA.ziphttp://deepblue.lib.umich.edu/bitstream/2027.42/170438/14/PEC72-No Fluoride - NusA.ziphttp://deepblue.lib.umich.edu/bitstream/2027.42/170438/15/PEC72-With Fluoride - NusA.ziphttp://deepblue.lib.umich.edu/bitstream/2027.42/170438/16/PEC72-No Fluoride - SiMKARTS.ziphttp://deepblue.lib.umich.edu/bitstream/2027.42/170438/17/PEC72-With Fluoride - SiMKARTS.ziphttp://deepblue.lib.umich.edu/bitstream/2027.42/170438/18/RealTime Trx-No Fluoride-25micros rNTPs - 100nM NusA.ziphttp://deepblue.lib.umich.edu/bitstream/2027.42/170438/19/RealTime Trx-No Fluoride-25micros rNTPs - No NusA.ziphttp://deepblue.lib.umich.edu/bitstream/2027.42/170438/20/RealTime Trx-No Fluoride-100micros rNTPs - 100nM NusA.ziphttp://deepblue.lib.umich.edu/bitstream/2027.42/170438/21/RealTime Trx-No Fluoride-100micros rNTPs - No NusA.ziphttp://deepblue.lib.umich.edu/bitstream/2027.42/170438/22/RealTime Trx-No Fluoride-250micros rNTPs - 100nM NusA.ziphttp://deepblue.lib.umich.edu/bitstream/2027.42/170438/23/RealTime Trx-No Fluoride-250micros rNTPs - No NusA.ziphttp://deepblue.lib.umich.edu/bitstream/2027.42/170438/24/RealTime Trx-U38A-No Fluoride-25micros rNTPs - 2nM NusA-Cy5.ziphttp://deepblue.lib.umich.edu/bitstream/2027.42/170438/25/RealTime Trx-U38A-With Fluoride-25micros rNTPs - 2nM NusA-Cy5.ziphttp://deepblue.lib.umich.edu/bitstream/2027.42/170438/26/RealTime Trx-With Fluoride-25micros rNTPs - No NusA.ziphttp://deepblue.lib.umich.edu/bitstream/2027.42/170438/27/RealTime Trx-WT-No Fluoride-25micros rNTPs - 2nM NusA-Cy5.ziphttp://deepblue.lib.umich.edu/bitstream/2027.42/170438/28/RealTime Trx-WT-With Fluoride-25micros rNTPs - 2nM NusA-Cy5.ziphttp://deepblue.lib.umich.edu/bitstream/2027.42/170438/29/RT72-No Fluoride-1mM rNTPs - SiMKARTS.ziphttp://deepblue.lib.umich.edu/bitstream/2027.42/170438/30/RT72-No Fluoride-1mM rNTPs-With NusA - SiMKARTS.ziphttp://deepblue.lib.umich.edu/bitstream/2027.42/170438/31/RT72-No Fluoride-100micros rNTPs - SiMKARTS.ziphttp://deepblue.lib.umich.edu/bitstream/2027.42/170438/32/RT72-With Fluoride CoTrx-1mM rNTPs - SiMKARTS.ziphttp://deepblue.lib.umich.edu/bitstream/2027.42/170438/33/RT72-With Fluoride PostTrx-1mM rNTPs - SiMKARTS.ziphttp://deepblue.lib.umich.edu/bitstream/2027.42/170438/34/RT72-With Fluoride-1mM rNTPs-With NusA - SiMKARTS.ziphttp://deepblue.lib.umich.edu/bitstream/2027.42/170438/35/RT72-With Fluoride-100micros rNTPs - SiMKARTS.ziphttp://deepblue.lib.umich.edu/bitstream/2027.42/170438/36/Terminator Only-1mM rNTPs - SiMKARTS.zipDescription of EC25-No Fluoride - NusA.zip : NusA binding assay - Single molecule traces obtained at EC-25 in the absence of FluorideDescription of EC25-With Fluoride - NusA.zip : NusA binding assay - Single molecule traces obtained at EC-25 in the presence of FluorideDescription of HisP - NusA.zip : NusA binding assay - Single molecule traces obtained at HisPDescription of Matlab scripts.zip : Matlab codes used in this studyDescription of PEC48-A10U-No Fluoride - NusA.zip : NusA binding assay - Single molecule traces obtained at PEC48-A10U mutant in the absence of FluorideDescription of PEC48-A10U-U38A-No Fluoride - NusA.zip : NusA binding assay - Single molecule traces obtained at PEC48-A10U-U38A mutant in the absence of FluorideDescription of PEC48-A10U-U38A-With Fluoride - NusA.zip : NusA binding assay - Single molecule traces obtained at PEC48-A10U-U38A mutant in the presence of FluorideDescription of PEC48-A10U-With Fluoride - NusA.zip : NusA binding assay - Single molecule traces obtained at PEC48-A10U mutant in the presence of FluorideDescription of PEC48-U38A-No Fluoride - NusA.zip : NusA binding assay - Single molecule traces obtained at PEC48-U38A mutant in the absence of FluorideDescription of PEC48-U38A-With Fluoride - NusA.zip : NusA binding assay - Single molecule traces obtained at PEC48-U38A mutant in the presence of FluorideDescription of PEC48-WT-No Fluoride - Competition unlabeled NusA.zip : NusA binding assay - Single molecule traces obtained at PEC48-WT in the presence of unlabeled NusADescription of PEC48-WT-No Fluoride - NusA.zip : NusA binding assay - Single molecule traces obtained at PEC48-WT in the absence of FluorideDescription of PEC48-WT-With Fluoride - NusA.zip : NusA binding assay - Single molecule traces obtained at PEC48-WT in the presence of FluorideDescription of PEC72-No Fluoride - NusA.zip : NusA binding assay - Single molecule traces obtained at PEC72 in the absence of FluorideDescription of PEC72-With Fluoride - NusA.zip : NusA binding assay - Single molecule traces obtained at PEC72 in the presence of FluorideDescription of PEC72-No Fluoride - SiMKARTS.zip : SiM-KARTS - Single molecule traces obtained at PEC72 in the absence of FluorideDescription of PEC72-With Fluoride - SiMKARTS.zip : SiM-KARTS - Single molecule traces obtained at PEC72 in the presence of FluorideDescription of RealTime Trx-No Fluoride-25micros rNTPs - 100nM NusA.zip : Real Time transcription - 25µM rNTPs with 100nM NusADescription of RealTime Trx-No Fluoride-25micros rNTPs - No NusA.zip : Real Time transcription - 25µM rNTPsDescription of RealTime Trx-No Fluoride-100micros rNTPs - 100nM NusA.zip : Real Time transcription - 100µM rNTPs with 100nM NusADescription of RealTime Trx-No Fluoride-100micros rNTPs - No NusA.zip : Real Time transcription - 100µM rNTPsDescription of RealTime Trx-No Fluoride-250micros rNTPs - 100nM NusA.zip : Real Time transcription - 250µM rNTPs with 100nM NusADescription of RealTime Trx-No Fluoride-250micros rNTPs - No NusA.zip : Real Time transcription - 250µM rNTPsDescription of RealTime Trx-U38A-No Fluoride-25micros rNTPs - 2nM NusA-Cy5.zip : Real Time transcription - U38A mutant - 25µM rNTPs with 2nM NusA-Cy5 in the absence of FluorideDescription of RealTime Trx-U38A-With Fluoride-25micros rNTPs - 2nM NusA-Cy5.zip : Real Time transcription - U38A mutant - 25µM rNTPs with 2nM NusA-Cy5 in the presence of FluorideDescription of RealTime Trx-With Fluoride-25micros rNTPs - No NusA.zip : Real Time transcription - 25µM rNTPs in the presence of FluorideDescription of RealTime Trx-WT-No Fluoride-25micros rNTPs - 2nM NusA-Cy5.zip : Real Time transcription - WT - 25µM rNTPs with 2nM NusA-Cy5 in the absence of FluorideDescription of RealTime Trx-WT-With Fluoride-25micros rNTPs - 2nM NusA-Cy5.zip : Real Time transcription - WT - 25µM rNTPs with 2nM NusA-Cy5 in the presence of FluorideDescription of RT72-No Fluoride-1mM rNTPs - SiMKARTS.zip : SiM-KARTS - Single molecule traces obtained at RT72 in the absence of Fluoride - 1mM rNTPsDescription of RT72-No Fluoride-1mM rNTPs-With NusA - SiMKARTS.zip : SiM-KARTS - Single molecule traces obtained at RT72 in the absence of Fluoride - 1mM rNTPs with NusADescription of RT72-No Fluoride-100micros rNTPs - SiMKARTS.zip : SiM-KARTS - Single molecule traces obtained at RT72 in the absence of Fluoride - 100µM rNTPsDescription of RT72-With Fluoride CoTrx-1mM rNTPs - SiMKARTS.zip : SiM-KARTS - Single molecule traces obtained at RT72 in the presence of Fluoride (Co-Trx) - 1mM rNTPsDescription of RT72-With Fluoride PostTrx-1mM rNTPs - SiMKARTS.zip : SiM-KARTS - Single molecule traces obtained at RT72 in the presence of Fluoride (Post-Trx) - 1mM rNTPsDescription of RT72-With Fluoride-1mM rNTPs-With NusA - SiMKARTS.zip : SiM-KARTS - Single molecule traces obtained at RT72 in the presence of Fluoride - 1mM rNTPs with NusADescription of RT72-With Fluoride-100micros rNTPs - SiMKARTS.zip : SiM-KARTS - Single molecule traces obtained at RT72 in the presence of Fluoride - 100µM rNTPsDescription of Terminator Only-1mM rNTPs - SiMKARTS.zip : SiM-KARTS - Single molecule traces obtained at the terminator only construct - 1mM rNTPsSEL