Biology subjects

Yakunina, M.

Publications and source records attributed to Yakunina, M..

4 recordsLinked to original sources

bioRxiv2024

Mechanisms of RecA filament nucleation on ssDNA by the DprA protein

The DprA (a.k.a. Smf) protein has emerged as a RecA mediator during natural chromosomal transformation, but its ubiquity suggests a possible role in DNA metabolism beyond natural transformation. We show that Bacillus subtilis dprA increases the frequency of Escherichia coli Hfr conjugation. RecA{middle dot}ATP binds and cooperatively polymerises in a 50-nucleotide (nt) poly deoxy T (dT)50 ssDNA to form a dynamic filament with SSB competing for binding, but B. subtilis DprA (DprABsu) counters the inhibitory effects of SSB on RecA{middle dot}ATP filaments. RecA bound to (dT)21 is poorly active as dATPase, with DprABsu significantly improving RecA dATP hydrolysis. RecABsu{middle dot}dATP-(dT)20 complexes were readily formed, while DprABsu exerts an allosteric effect on RecABsu-(dT)15 complexes competent for dATP hydrolysis. Combining experimental data with a full-atomic model of the RecA-DprA-ssDNA complexs spatial structure, we proposed a molecular mechanism for DprA-mediated loading of RecA onto short ssDNA stretches. Our results suggest that steric constraints allow for the participation of only one DNA binding site of the DprA dimer in RecA-mediated dATP hydrolysis.

molecular biology↗

bioRxiv2023

The Dynamics of Synthesis and Localization of Jumbo Phage RNA Polymerases inside Infected Cells

A nucleus-like structure composed of phage-encoded proteins and containing replicating viral DNA is formed in Pseudomonas aeruginosa cells infected by jumbo bacteriophage phiKZ. The PhiKZ genes are transcribed independently from host RNA polymerase (RNAP) by two RNAPs encoded by the phage. The virion RNAP (vRNAP) transcribes early viral genes and must be injected into the cell with phage DNA. The non-virion RNAP (nvRNAP) is composed of early genes products and transcribes late viral genes. In this work, the dynamics of phage RNAPs localization during phage phiKZ infection was studied. We provide direct evidence of PhiKZ vRNAP injection in infected cells and show that it is excluded from the phage nucleus. The nvRNAP is synthesized shortly after the onset of infection and localizes in the nucleus. We propose that spatial separation of two phage RNAPs allows coordinated expression of phage genes belonging to different temporal classes.

molecular biology↗

bioRxiv2023

Single-molecule studies reveal the off-pathway elemental pause state as a target of streptolydigin inhibition of RNA polymerase and its dramatic enhancement by Gre factors

Antibiotic streptolydigin (Stl) inhibits bacterial transcription by blocking the trigger loop folding in the active center of RNA polymerase (RNAP), which is essential for catalysis. We use acoustic force spectroscopy to characterize the dynamics of transcription elongation in ternary elongation complexes of RNAP (ECs) in the presence of Stl at a single-molecule level. We found that Stl induces long-lived stochastic pauses while the instantaneous velocity of transcription between the pauses is unaffected. Stl enhances the short-lived pauses associated with an off-pathway elemental paused state of the RNAP nucleotide addition cycle. Unexpectedly, we found that transcript cleavage factors GreA and GreB, which were thought to be Stl competitors, do not alleviate the streptolydigin-induced pausing; instead, they synergistically increase transcription inhibition by Stl. This is the first known instance of a transcriptional factor enhancing antibiotic activity. We propose a structural model of the EC-Gre-Stl complex that explains the observed Stl activities and provides insight into possible cooperative action of secondary channel factors and other antibiotics binding at the Stl-pocket. These results offer a new strategy for high-throughput screening for prospective antibacterial agents.

biophysics↗

bioRxiv2021

Structure of the Bacteriophage PhiKZ non-Virion RNA Polymerase

Bacteriophage {Phi}KZ is the founding member of a family of massive bacterial viruses. It is considered to have therapeutic potential as its host, Pseudomonas aeruginosa, is an opportunistic, intrinsically antibiotic resistant, pathogen that kills tens of thousands worldwide each year. {Phi}KZ is an incredibly interesting virus, expressing many systems the host already possesses. On infection, it forms a "nucleus", erecting a barrier around its massive genome to exclude host restriction endonucleases and CRISPR-Cas systems. {Phi}KZ infection is independent of the host transcriptional apparatus. It expresses two different multi-subunit RNA polymerases (RNAPs): the virion RNAP (vRNAP) is injected with the viral DNA during infection to transcribe early genes, including those encoding the non-virion RNAP (nvRNAP), which transcribes all further genes. {Phi}KZ nvRNAP is formed by four polypeptides thought to represent homologues of the eubacterial {beta}/{beta}' subunits, and a fifth with unclear homology, but essential for transcription. We have resolved the structure of {Phi}KZ nvRNAP to 3.3 [A], shedding light on its assembly, homology, and the biological role of the fifth subunit: it is an embedded, integral member of the complex, with structural homology and a biochemical role implying that it has evolved from an ancestral homologue to {sigma}-factor.

biochemistry↗