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Members of the conserved Argonaute (Ago) protein family provide defence against invading nucleic acids in eukaryotes in the process of RNA interference. Many prokaryotes also contain Ago proteins that are predicted to be active nucleases; however, their functional activities in host cells remain poorly understood. Here, we characterize the in vitro and in vivo properties of the SeAgo protein from the mesophilic cyanobacterium Synechococcus elongatus. We show that SeAgo is a DNA-guided nuclease preferentially acting on single-stranded DNA targets, with non-specific guide-independent activity observed for double-stranded substrates. The SeAgo gene is steadily expressed in S. elongatus; however, its deletion or overexpression does not change the kinetics of cell growth. When purified from its host cells or from heterologous E. coli, SeAgo is loaded with small guide DNAs whose formation depends on the endonuclease activity of the argonaute protein. SeAgo co-purifies with SSB proteins suggesting that they may also be involved in DNA processing. The SeAgo-associated small DNAs are derived from diverse genomic locations, with certain enrichment for the proposed sites of chromosomal replication initiation and termination, but show no preference for an endogenous plasmid. Therefore, promiscuous genome sampling by SeAgo does not have great effects on cell physiology and plasmid maintenance.

Members of the conserved Argonaute protein family use small RNA guides to locate their mRNA targets and regulate gene expression and suppress mobile genetic elements in eukaryotes.sup.1,2. Argonautes are also present in many bacterial and archaeal species.sup.3-5. Unlike eukaryotic proteins, several prokaryotic Argonaute proteins use small DNA guides to cleave DNA, a process known as DNA interference.sup.6-10. 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 analyse the activity of a bacterial Argonaute nuclease from Clostridium butyricum (CbAgo) in vivo. We show that CbAgo targets multicopy genetic elements and suppresses the propagation of plasmids and infection by phages. CbAgo induces DNA interference between homologous sequences and triggers DNA degradation at double-strand breaks in the target DNA. The loading of CbAgo with locus-specific small DNA guides depends on both its intrinsic endonuclease activity and the cellular double-strand break repair machinery. A similar interaction was reported for the acquisition of new spacers during CRISPR adaptation, and prokaryotic genomes that encode Ago nucleases are enriched in CRISPR-Cas systems. These results identify molecular mechanisms that generate guides for DNA interference and suggest that the recognition of foreign nucleic acids by prokaryotic defence systems involves common principles. Argonaute protein from the bacterium C. butyricum targets multicopy genetic elements and functions in the suppression of plasmid and phage propagation, and there appears to be a DNA-mediated immunity pathway in prokaryotes., Author(s): Anton Kuzmenko [sup.1] [sup.2] , Anastasiya Oguienko [sup.1] , Daria Esyunina [sup.1] , Denis Yudin [sup.1] [sup.4] , Mayya Petrova [sup.1] , Alina Kudinova [sup.1] , Olga Maslova [sup.1] [...]

RNA‐dependent RNA polymerase (RdRp) plays a key role in the replication of RNA viruses, including SARS‐CoV‐2. Processive RNA synthesis by RdRp is crucial for successful genome replication and expression, especially in the case of very long coronaviral genomes. Here, we analysed the activity of SARS‐CoV‐2 RdRp (the nsp12–nsp7–nsp8 complex) on synthetic primer–templates of various structures, including substrates with mismatched primers or template RNA modifications. It has been shown that RdRp cannot efficiently extend RNA primers containing mismatches and has no intrinsic RNA cleavage activity to remove the primer 3′‐end, thus necessitating the action of exoribonuclease for proofreading. Similar to DNA‐dependent RNA polymerases, RdRp can perform processive pyrophosphorolysis of the nascent RNA product but this reaction is also blocked in the presence of mismatches. Furthermore, we have demonstrated that several natural post‐transcriptional modifications in the RNA template, which do not prevent complementary interactions (N6‐methyladenosine, 5‐methylcytosine, inosine and pseudouridine), do not change RdRp processivity. At the same time, certain modifications of RNA bases and ribose residues strongly block RNA synthesis, either prior to nucleotide incorporation (3‐methyluridine and 1‐methylguanosine) or immediately after it (2'‐O‐methylation). The results demonstrate that the activity of SARS‐CoV‐2 RdRp can be strongly inhibited by common modifications of the RNA template suggesting a way to design novel antiviral compounds. [ABSTRACT FROM AUTHOR]

Bacterial RNA polymerase (RNAP) serves as a primase during replication of single‐stranded plasmids and filamentous phages. Primer RNA (prRNA) synthesis from the origin regions of these replicons depends on the σ factor that normally participates in promoter recognition. However, it was proposed that σ may not be required for origin recognition but is rather involved in RNA extension by RNAP. Here, by analyzing the natural replication origin of bacteriophage M13 and synthetic ssDNA templates, we show that interactions of σ with promoter‐like motifs stabilize priming complexes and can control prRNA synthesis by trapping RNAP on the template. Thus, the σ factor is involved in both DNA recognition and RNA priming, unifying its functions in transcription initiation from double‐ and single‐stranded templates. [ABSTRACT FROM AUTHOR]