Biology subjects

Arseniev, A.

Publications and source records attributed to Arseniev, A..

6 recordsLinked to original sources

bioRxiv2023

Catalytically inactive dKbCas12d guided by sgRNA and new insights into its binding through a single-molecule approach

CRISPR-Cas12d is a distinct V-D type system discovered in the metagenomes of Candidate Phyla Radiation bacteria. It stands out from most closely related systems due to its 17-19 nucleotide short spacer region and specialized stabilizing scoutRNAs. We made significant improvements to this system by modifying its scoutRNA to create sgRNA, which greatly simplifies its use. We found mutations in the RuvC domain of the effector protein KbCas12d that resulted in loss of nuclease activity. We obtained two catalytically inactive dKbCas12d variants: D827A and E913A. Using the optical tweezers technique, we demonstrated the high specificity of dKbCas12d in binding targets on individual DNA molecules. Engineered sgRNA and catalytically inactive dKbCas12d variants have promising applications in biotechnology for the precise regulation of gene expression and molecular diagnostics.

biophysics↗

bioRxiv2023

Characterization of Streptococcus uberis Cas9 (SuCas9) - a Type II-A Ortholog Functional in Human Cells

Type II CRISPR-Cas9 RNA-guided nucleases are commonly used for genome engineering. To date, all characterized Cas9-based genome editors, including the widely used SpCas9, have limitations such as their relatively large size and restriction of targets flanked by a specific PAM sequence. Here, we biochemically characterized more compact SpCas9 ortholog, SuCas9, from Streptococcus uberis, a bacterium inhabiting the mammary glands of dairy cattle. SuCas9 recognizes a novel 5'-NNAAA-3' PAM, efficiently cleaves DNA in vitro, and is active in human cells. The study of SuCas9 has the potential to expand the range of applications of CRISPR-Cas9 enzymes in medicine and biotechnology.

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↗

bioRxiv2019

Purification of native CCL7 and its functional interaction with selected chemokine receptors

Chemokine receptors form a major sub-family of G protein-coupled receptors (GPCRs) and they are involved in a number of cellular and physiological processes related to our immune response and regulation. A better structural understanding of ligand-binding, activation, signaling and regulation of chemokine receptors is very important to design potentially therapeutic interventions for human disorders arising from aberrant chemokine signaling. One of the key limitations in probing the structural details of chemokine receptors is the availability of large amounts of purified, homogenous and fully functional chemokine ligands, and the commercially available products, are not affordable for in-depth structural studies. Moreover, production of uniformly isotope-labeled chemokines, for example, suitable for NMR-based structural investigation, also remains challenging. Here, we have designed a streamlined approach to express and purify the human chemokine CCL7 as well as its 15N-, 15N/13C-, 2H/15N/13C-isotope-labeled derivatives, at milligram levels using E. coli expression system. Purified CCL7 not only maintains a well-folded three-dimensional structure as analyzed using circular dichroism and 1H/15N NMR but it also induces coupling of heterotrimeric G-proteins and {beta}-arrestins for selected chemokine receptors in cellular system. Our strategy presented here may be applicable to other chemokines and therefore, provide a potentially generic and cost-effective approach to produce chemokines in large amounts for functional and structural studies.

biochemistry↗

bioRxiv2019

Structural Basis and Mechanism of TrkA Activation by NGF through Ligand-Induced Rotation of Transmembrane Domain Dimers.

Trk receptors are essential for the nervous system development. The molecular mechanism of TrkA activation by its ligand NGF is still unsolved. Recent data indicates that at endogenous levels most of TrkA is in an equilibrium monomer-dimer and the binding of NGF induces an increase of the dimer and oligomer forms of the receptor. An unsolved issue is the role of the transmembrane domain (TMD) in the dimerization of TrkA and the structural details of the TMD in the active dimer receptor. We found that TrkA-TMD can form dimers, identified the structural determinants of the dimer interface in the active receptor and validated this interface using site-directed mutagenesis together with functional and cell differentiation studies. Using in vivo crosslinking we identified a reordering of the extracellular juxtamembrane (JTM) region after ligand binding. Replacement of some residues in the JTM region with cysteine form ligand-independent active dimers and reveal a preferred dimer interface. In addition to that, insertion of leucine residues into the TMD helix induces a ligand-independent TrkA activation suggesting that a rotation of the TMD dimers could be behind TrkA activation by NGF. Altogether our data indicates that the transmembrane and juxtamembrane regions of the receptor play a key role in the dimerization and activation of TrkA by NGF.

biochemistry↗