Biology subjects

Kulbachinskiy, A.

Publications and source records attributed to Kulbachinskiy, A..

6 recordsLinked to original sources

bioRxiv2026

Tetramerization and RNA-guided filament assembly control Schlafen-Argonaute antiphage defense

Two deeply conserved protein families, Argonaute (Ago) and Schlafen (SLFN), play defense roles in diverse prokaryotes and eukaryotes, including humans. Here, we identify a monophyletic group of proteins broadly distributed across bacteria and archaea that fuse a SLFN domain with an Ago core and a GHKL-family ATPase. Using SLFN-pAgo from Runella zeae as a model, we show that these proteins protect bacterial cells against bacteriophages by employing the Ago core as a guide-dependent sensor and the SLFN domain as a nuclease effector to induce abortive infection via tRNAs cleavage. Structural and biochemical analyses reveal that ATP binding by the GHKL domain drives tetramerization of SLFN-pAgo, reconstituting the canonical SLFN nuclease architecture found in mammalian proteins. Prior to target detection, non-canonical guide RNA binding induces the formation of long helical filaments, locking the SLFN domains in an inactive configuration. Guide-dependent recognition of complementary target DNA triggers massive structural rearrangements leading to filament disassembly and the induction of SLFN nuclease activity. Together, our findings uncover a new antiphage system that employs reversible guide- and ATP-mediated oligomerization to strictly regulate cooperation between an Ago sensor and a SLFN RNAse effector.

molecular biology↗

bioRxiv2020

Structural basis of ribosomal RNA transcription regulation

Ribosomal RNA (rRNA) is the most highly expressed gene in rapidly growing bacteria and is drastically downregulated under stress conditions by the global transcriptional regulator DksA and the alarmone ppGpp. To reveal the mechanism of highly regulated rRNA transcription, we determined cryo-electron microscopy structures of the Escherichia coli RNA polymerase (RNAP) {sigma}70 holoenzyme at different steps of rRNA promoter recognition with and without DksA/ppGpp. RNAP contacts the UP element of rRNA promoter using the dimerized subunit carboxyl-terminal domain and scrunches the template DNA with the {sigma}finger and {beta}lid to select a transcription start site favorable for rRNA expression. Promoter DNA binding to RNAP induces conformational change of the {sigma} domain 2 that opens a gate for DNA loading and ejects {sigma}1.1 from the RNAP cleft to facilitate open complex formation. DksA/ppGpp binding to RNAP also opens the DNA loading gate, but it is not coupled to {sigma}1.1 ejection and impedes the open complex formation of the rRNA promoter due to its G+C rich discriminator sequence. Mutations in {sigma}1.1 or the {beta}lid stabilize the RNAP and rRNA promoter complex and decrease its sensitivity to DksA/ppGpp. These results provide a molecular basis for exceptionally active rRNA transcription and for its vulnerability to DksA/ppGpp.

molecular biology↗

bioRxiv2020

pAgo-induced DNA interference protects bacteria from invader DNA

Members of the conserved Argonaute protein family use small RNA guides to find their mRNA targets to regulate gene expression and suppress mobile genetic elements in eukaryotes. Argonautes are also present in many bacterial and archaeal species 1-3. Unlike eukaryotic proteins, several studied prokaryotic Argonautes use small DNA guides to cleave DNA, a process dubbed DNA interference 4-8. However, the natural functions and targets of DNA interference are poorly understood and the mechanisms of DNA guide generation and target discrimination remain unknown. Here, we studied the in vivo activities of a bacterial Argonaute nuclease CbAgo and demonstrated that it induces cleavage of multicopy genetic elements, including plasmids, transposons and repetitive chromosomal loci. Generation of small DNA guides employed by CbAgo requires cooperation between its intrinsic endonuclease activity and the cellular double-strand break repair machinery. The mechanism of guide generation ensures that small DNA guides are enriched in sequences that target foreign DNA and endows CbAgo with capacity to eliminate plasmids and fight phage infection. Similar principles may underlie the specificity of self-nonself discrimination by diverse defense systems in prokaryotes.

molecular biology↗

bioRxiv2019

Programmable DNA cleavage by Ago nucleases from mesophilic bacteria Clostridium butyricum and Limnothrix rosea

Argonaute (Ago) proteins are the key players in RNA interference in eukaryotes, where they function as RNA-guided RNA endonucleases. Prokaryotic Argonautes (pAgos) are much more diverse than their eukaryotic counterparts but their cellular functions and mechanisms of action remain largely unknown. Some pAgos were shown to use small DNA guides for endonucleolytic cleave of complementary DNA in vitro. However, previously studied pAgos from thermophilic prokaryotes function at elevated temperatures which limits their potential use as a tool in genomic applications. Here, we describe two pAgos from mesophilic bacteria, Clostridium butyricum (CbAgo) and Limnothrix rosea (LrAgo), that act as DNA-guided DNA nucleases at physiological temperatures. In contrast to previously studied pAgos, CbAgo and LrAgo can use not only 5-phosphorylated but also 5-hydroxyl DNA guides, with diminished precision of target cleavage. Both LrAgo and CbAgo can tolerate guide/target mismatches in the seed region, but are sensitive to mismatches in the 3-guide region. CbAgo is highly active under a wide range of conditions and can be used for programmable endonucleolytic cleavage of both single-stranded and double-stranded DNA substrates at moderate temperatures. The biochemical characterization of mesophilic pAgo proteins paths the way for their use for DNA manipulations both in vitro and in vivo.

molecular biology↗

bioRxiv2018

The expanded universe of prokaryotic Argonaute proteins

The members of the ancient family of Argonaute (Ago) proteins are present in all domains of life. The common feature of Ago proteins is the ability to bind small nucleic acid guides and use them for sequence-specific recognition - and sometimes cleavage - of complementary targets. While eukaryotic Ago (eAgo) proteins are key players in RNA interference and related pathways, the properties and functions of these proteins in archaeal and bacterial species have just started to emerge. We undertook comprehensive exploration of prokaryotic Ago (pAgo) proteins in sequenced genomes and almost tripled the number of previously analyzed genes of this family. In comparison with eAgos, pAgos are highly diverse and have likely spread by horizontal gene transfer. Many pAgos contain divergent variants of the conserved domains involved in interactions with nucleic acids and in target cleavage, while having extra domains that are absent in eAgos, suggesting that they might have unusual specificities in the nucleic acid recognition and processing. Many pAgos, including catalytically inactive variants, are associated with putative nucleases, helicases and DNA binding proteins in the same gene or operon, suggesting that they are involved in DNA processing. The great diversity of pAgos revealed by our analysis opens new ways for exploration of their functions in host cells and their use as potential tools in genome editing.

genomics↗

bioRxiv2017

Pausing controls branching between productive and non-productive pathways during initial transcription

Transcription in bacteria is controlled by multiple molecular mechanisms that precisely regulate gene expression. Recently, initial RNA synthesis by the bacterial RNA polymerase (RNAP) has been shown to be interrupted by pauses; however, the pausing determinants and the relationship of pausing with productive and abortive RNA synthesis remain poorly understood. Here, we employed single-molecule FRET and biochemical analysis to disentangle the pausing-related pathways of bacterial initial transcription. We present further evidence that region {sigma}3.2 constitutes a barrier after the initial transcribing complex synthesizes a 6-nt RNA (ITC6), halting transcription. We also show that the paused ITC6 state acts as a checkpoint that directs RNAP, in an NTP-dependent manner, to one of three competing pathways: productive transcription, abortive RNA release, or a new unscrunching/scrunching pathway that blocks transcription initiation. Our results show that abortive RNA release and DNA unscrunching are not as tightly coupled as previously thought.

biophysics↗