Biology subjects

Said, N.

Publications and source records attributed to Said, N..

7 recordsLinked to original sources

bioRxiv2026

Molecular mechanism of albicidin-dependent transcription activation by the antibiotic resistance factor AlbA

The escalating spread of antimicrobial resistance demands antibiotics with novel mechanisms of action. Albicidin is a potent DNA gyrase inhibitor produced by Xanthomonas species, and bacteria counter its activity through resistance mechanisms including the albicidin-sequestering protein AlbA from Klebsiella oxytoca. AlbA is a MerR family transcription activator that binds albicidin and stimulates expression of the albA gene, but how ligand recognition is coupled to transcription activation has remained unclear. Here, we use cryogenic electron microscopy and complementary biochemical assays to define the mechanism of AlbA-dependent transcription activation. Structures of transcription complexes reveal that AlbA remodels the promoter, repositioning the -35 element for productive sigma70 recognition and RNA polymerase engagement. Albicidin does not measurably alter AlbAs DNA affinity or the architecture of the mature open complex. Instead, transcription kinetics and experiments bypassing promoter opening indicate that albicidin selectively facilitates a promoter opening-dependent transition. AlbA is released upon synthesis of approximately five nucleotides, concomitant with downstream DNA melting, bubble translocation and upstream DNA rewinding rather than DNA scrunching. Together, these findings establish a sequential mechanism in which AlbA remodels promoter architecture, while albicidin tunes the kinetic transition to promoter opening, linking antibiotic recognition to induction of its sequestration machinery.

biochemistry↗

bioRxiv2024

A pervasive large conjugative plasmid mediates multispecies biofilm formation in the intestinal microbiota increasing resilience to perturbations

Although horizontal gene transfer is pervasive in the intestinal microbiota, we understand only superficially the roles of most exchanged genes and how the mobile repertoire affects community dynamics. Similarly, little is known about the mechanisms underlying the ability of a community to recover after a perturbation. Here, we identified and functionally characterized a large conjugative plasmid that is one of the most frequently transferred elements among Bacteroidales species and is ubiquitous in diverse human populations. This plasmid encodes both an extracellular polysaccharide and fimbriae, which promote the formation of multispecies biofilms in the mammalian gut. We use a hybridization-based approach to visualize biofilms in clarified whole colon tissue with unprecedented 3D spatial resolution. These biofilms increase bacterial survival to common stressors encountered in the gut, increasing strain resiliency, and providing a rationale for the plasmids recent spread and high worldwide prevalence.

microbiology↗

bioRxiv2023

Sm-like protein Rof inhibits transcription termination factor Rho by binding site obstruction and conformational insulation

Transcription termination factor {rho} is a hexameric, RNA-dependent NTPase that can adopt active closed-ring and inactive open-ring conformations. The Sm-like protein Rof, a homolog of the RNA chaperone Hfq, inhibits {rho}-dependent termination in vivo but recapitulation of this activity in vitro has proven difficult and the precise mode of Rof action is presently unknown. Our electron microscopic structures of {rho}-Rof and {rho}-RNA complexes show that Rof undergoes pronounced conformational changes to bind {rho} at the protomer interfaces, undercutting {rho} conformational dynamics associated with ring closure and occluding extended primary RNA-binding sites that are also part of interfaces between {rho} and RNA polymerase. Consistently, Rof impedes {rho} ring closure, {rho}-RNA interactions, and {rho} association with transcription elongation complexes. Structure-guided mutagenesis coupled with functional assays confirmed that the observed {rho}-Rof interface is required for Rof-mediated inhibition of cell growth and {rho}-termination in vitro. Bioinformatic analyses revealed that Rof is restricted to Pseudomonadota and that the {rho}-Rof interface is conserved. Genomic contexts of rof differ between Enterobacteriaceae and Vibrionaceae, suggesting distinct modes of Rof regulation. We hypothesize that Rof and other cellular anti-terminators silence {rho} under diverse, but yet to be identified, stress conditions when unrestrained transcription termination by {rho} would be lethal.

biochemistry↗

bioRxiv2023

Transcription termination factor {rho} polymerizes under stress

Bacterial RNA helicase {rho} is a genome sentinel that terminates synthesis of damaged and junk RNAs that are not translated by the ribosome. Co-transcriptional RNA surveillance by {rho} is essential for quality control of the transcriptome during optimal growth. However, it is unclear how bacteria protect their RNAs from overzealous {rho} during dormancy or stress, conditions common in natural habitats. Here we used cryogenic electron microscopy, biochemical, and genetic approaches to show that residue substitutions, ADP, or ppGpp promote hyper-oligomerization of Escherichia coli {rho}. Our results demonstrate that nucleotides bound at subunit interfaces control {rho} switching from active hexamers to inactive higher-order oligomers and extended filaments. Polymers formed upon exposure to antibiotics or ppGpp disassemble when stress is relieved, thereby directly linking termination activity to cellular physiology. Inactivation of {rho} through hyper-oligomerization is a regulatory strategy shared by RNA polymerases, ribosomes, and metabolic enzymes across all life.

microbiology↗

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↗

bioRxiv2023

Widespread gene regulator Psu inhibits transcription termination factor rho by forced hyper-oligomerization

Many bacteriophages modulate the host transcription machinery for efficient expression of their own genomes. Phage P4 polarity suppression protein, Psu, is a building block of the viral capsid and inhibits the hexameric transcription termination factor, {rho}, by presently unknown mechanisms. We elucidated cryogenic electron microscopy structures of {rho}-Psu complexes, showing that Psu dimers laterally clamp two inactive, open {rho} rings and promote their expansion to higher-oligomeric states. Systematic ATPase, nucleotide binding and nucleic acid binding studies revealed that Psu hinders {rho} ring closure and traps nucleotides in their binding pockets on {rho}. Structure-guided mutagenesis in combination with growth, pull-down and termination assays further delineated the functional {rho}-Psu interfaces. Bioinformatic analyses suggested that, in addition to guarding its own genome against {rho}, Psu enables expression of diverse phage-defense systems commonly found in P4-like mobile genetic elements across bacteria. Thus, Psu is a widespread gene regulator that inhibits {rho} via forced hyper-oligomerization.

molecular biology↗

bioRxiv2020

SPARC promotes insulin secretion through down-regulation of RGS4 protein in pancreatic beta-cells

SPARC-deficient mice have been shown to exhibit impaired glucose tolerance and insulin secretion, but the underlying mechanism remains unknown. Here, we show that SPARC enhanced the promoting effect of Muscarinic receptor agonist oxotremorine-M on insulin secretion in cultured mouse islets. Overexpression of SPARC down-regulated RGS4, a negative regulator of {beta}-cell M3 muscarinic receptors. Conversely, knockdown of SPARC up-regulated RGS4 in Min6 cells. RGS4 was up-regulated in islets from sparc -/- mice, which correlated with decreased glucose-stimulated insulin secretion (GSIS). Furthermore, inhibition of RGS4 restored GSIS in sparc -/- mice, and knockdown of RGS4 partially decreased the promoting effect of SPARC on oxotremorine-M stimulated insulin secretion. Phosphoinositide 3-kinase (PI3K) inhibitor LY-294002 abolished SPARC-induced down-regulation of RGS4. Taken together, our data revealed that SPARC promoted GSIS by inhibiting RGS4 in pancreatic {beta} cells.

cell biology↗