In bacteria, RNA polymerase (RNAP) initiates transcription by synthesizing short transcripts that are either released or extended to allow RNAP escape from the promoter. The mechanism of initial is unclear due presence transient intermediates and molecular heterogeneity. Here, we studied on a lac promoter using single-molecule fluorescence observations DNA scrunching immobilized complexes. Our work revealed long pause ("initiation pause," ∼20 s) after synthesis 6-mer RNA; such pauses can...

Abstract Osteoporosis, which results from excessive bone resorption by osteoclasts, is the major cause of morbidity for elder people. Identification clinically relevant regulators needed to develop novel therapeutic strategies. Rho GTPases have essential functions in osteoclasts regulating actin dynamics. This particular importance because cytoskeleton generate sealing zone, an osteoclast-specific structure ultimately mediating resorption. Here we report that atypical Rac1 exchange factor...

Transcription in bacteria is controlled by multiple molecular mechanisms that precisely regulate gene expression. It has been recently shown initial RNA synthesis the bacterial polymerase (RNAP) interrupted pauses; however, pausing determinants and relationship of with productive abortive remain poorly understood. Using single-molecule FRET biochemical analysis, here we show pause encountered RNAP after a 6-nt (ITC6) renders promoter escape strongly dependent on NTP concentration....

In addition to genomic RNA, HIV-1 particles package cellular and spliced viral RNAs. order determine the encapsidation mechanisms of these RNAs, we determined packaging efficiencies specificities singly fully HIV mRNAs different host RNAs species: 7SL U6 snRNA GAPDH mRNA using RT-QPCR. Except mRNA, all are selectively encapsidated. Singly harboring Rev-responsible element, which do not contain this motif, enriched in virions similar levels, even though they exported from nucleus by routes....

RbpA is an RNA polymerase (RNAP)-binding protein whose presence increases the tolerance levels of Mycobacteria to first-line anti-tuberculosis drug rifampicin by unknown mechanism. Here, we show that role Mycobacterium tuberculosis in resistance indirect because it does not affect sensitivity RNAP while stimulates transcription controlled housekeeping σ A -factor. The regulated stress-related F was affected RbpA. binding site maps β subunit Sandwich-Barrel Hybrid Motif, which has previously...

RbpA, a transcriptional activator that is essential for Mycobacterium tuberculosis replication and survival during antibiotic treatment, binds to RNA polymerase (RNAP) in the absence of promoter DNA. It has been hypothesized RbpA stimulates housekeeping gene expression by promoting assembly σA subunit with core RNAP. Here, using purified vitro transcription system M. tuberculosis, we show functions promoter-dependent manner as companion RNAP DNA unwinding formation catalytically active open...

Self-assembly of macromolecules into higher-order symmetric structures is fundamental for the regulation biological processes. Higher-order structure self-assembly by gene expression machinery, such as bacterial DNA-dependent RNA polymerase (RNAP), has never been reported before. Here, we show that stress-response σB factor from human pathogen, Mycobacterium tuberculosis, induces RNAP holoenzyme oligomerization a supramolecular complex composed eight units. Cryo-electron microscopy revealed...

Retroviruses replicate by converting their positive sense genomic RNA into double-stranded DNA that is subsequently integrated the host genome. This conversion catalyzed reverse transcriptase (RT) early after virus entry target cell and chaperoned nucleocapsid protein (NC). In HIV-1, NC composed of small basic domains flanking two highly conserved CCHC zinc fingers specifically interact with RT. Through specific interactions RT, possibly cellular factors, were found to play critical roles in...

Abstract Initiation of transcription requires the formation “open” promoter complex (RPo). For this, σ subunit bacterial RNA polymerase (RNAP) binds to nontemplate strand −10 element sequence promoters and nucleates DNA unwinding. This is accompanied by a cascade conformational changes on RNAP, exact mechanics which remains elusive. Here, using single-molecule Förster resonance energy transfer cryo-electron microscopy, we explored landscape RNAP from human pathogen Mycobacterium tuberculosis...

Transcription initiation is a major step in gene regulation for all organisms. In bacteria, the promoter DNA first recognized by RNA polymerase (RNAP) to yield an initial closed complex. This complex subsequently undergoes conformational changes resulting strand separation form transcription bubble and RNAP-promoter open complex; however, series sequence of changes, factors that influence them are unclear. To address landscape transitions initiation, we applied single-molecule Förster...

Reverse transcription of the genomic RNA by reverse transcriptase occurs soon after HIV-1 infection target cells. The viral nucleocapsid (NC) protein chaperones this process via its nucleic acid annealing activities and interactions with enzyme. To function, NC needs two conserved zinc fingers flanking basic residues. We recently reported a new role for NC, whereby it negatively controls in course virus formation. Indeed, deleting causes activation producer investigate we used viruses subtle...

Mycobacterial activator RbpA stimulates transcription by stabilizing open conformation of the RNA polymerase σ subunit.

The transcriptional activator RbpA associates with Mycobacterium tuberculosis RNA polymerase (MtbRNAP) during transcription initiation, and stimulates formation of the MtbRNAP-promoter open complex (RPo). Here, we explored influence promoter motifs on RbpA-mediated activation MtbRNAP containing stress-response σB subunit. We show that both 'extended −10' motif (T-17G-16T-15G-14) stabilized RPo allowed opening at suboptimal temperatures. Furthermore, in presence T-17G-16T-15G-14 motif, was...

Abstract Background Murine Leukemia Virus (MLV) assembly has been long thought to occur exclusively at the plasma membrane. Current models of retroviral particle describe recruitment host vacuolar protein sorting machinery cell surface induce budding new particles. Previous fluorescence microscopy study reported vesicular traffic MLV components (Gag, Env and RNA). Here, electron (EM) associated with immunolabeling approaches were used go deeply into "prototypic" in chronically infected...

Abstract We have shown previously that HIV actively and selectively packages the spliced RNAs into progeny virions. In present study, by using a RT-QPCR QPCR strategies, we show viral are in infectious particles consequently participate, along with unspliced genomic RNA, to some of early steps infection such as reverse transcription step. This work provides first quantitative data on fully RNAs, also called transcripts, target cells but inside The latter results were obtained measuring...

Technological breakthroughs in electron microscopy (EM) have made it possible to solve structures of biological macromolecular complexes and raise novel challenges, specifically related sample preparation heterogeneous assemblies such as DNA-protein, protein-protein, membrane protein assemblies. Here, we built a V-shaped DNA origami scaffolding molecular system template proteins at user-defined positions space. This various sizes, juxtaposes combinations biomolecules into complex...

All cellular genetic information is transcribed into RNA by multisubunit polymerases (RNAPs). The basal transcription initiation factors of RNAPs stimulate the initial synthesis via poorly understood mechanisms. Here, we explored mechanism employed bacterial factor σ in promoter-independent transcription. We found that RNAP holoenzyme lacking promoter-binding domain σ4 ineffective de novo and displays high propensity to pausing upon extension RNAs 3 7 nucleotides length. nucleotide at 3' end...

Abstract Transcription in bacteria is controlled by multiple molecular mechanisms that precisely regulate gene expression. Recently, initial RNA synthesis the bacterial polymerase (RNAP) has been shown to be interrupted pauses; however, pausing determinants and relationship of with productive abortive remain poorly understood. Here, we employed single-molecule FRET biochemical analysis disentangle pausing-related pathways transcription. We present further evidence region σ 3.2 constitutes a...

ABSTRACT Initiation of transcription requires the formation “open” promoter complex (RPo). For this, σ subunit bacterial RNA polymerase (RNAP) binds to non-template strand -10 element sequence promoters and nucleates DNA unwinding. This is accompanied by a cascade conformational changes on RNAP mechanics which remains elusive. Here, using single-molecule Förster resonance energy transfer cryo-electron microscopy, we explored landscape from human pathogen Mycobacterium tuberculosis upon...

Address: 1CNRS UMR 5236-UMI/UMII, CPBS Equipe «Assemblage et Replication des Retrovirus», Institut de Biologie, Montpellier, France, 2Department of Pathogen Biology, Tongji Medical College Huazhong University Science and Technology, Wuhan, PR China, 3Molecular Virology Section, Laboratory Molecular Microbiology National Institute Allergy Infectious Diseases, Institutes Health, Bethesda, Maryland 20892, USA 4LaboRetro, Unite Virologie humaine INSERM U758, IFR128, ENS, 46 allee d'Italie, Lyon,...

Background HIV particles include two-copies of full-length genomic RNA (gRNA) that is selectively incorporated into the viral as a non-covalent dimer. packaging virus dependent upon specific interaction between gRNA and nucleocapsid protein (NC) domain Gag precursor. Selection HIV-1 involves so-called Psi region located immediately upstream gag start codon folded three stem-loops important for genome (SL1 to SL3). In particular, SL1 mediates dimerization, presumably prerequisite packaging....

ABSTRACT The basal transcription factors of cellular RNA polymerases (RNAPs) stimulate the initial synthesis via poorly understood mechanisms. Here, we explored mechanism employed by bacterial factor σ in promoter-independent transcription. We found that RNAP holoenzyme lacking promoter-binding domain σ4 is ineffective de novo initiation and displays high propensity to pausing upon extension RNAs 3 7 nucleotides length. stabilizes short RNA:DNA hybrids suppresses stimulating active-center...