Prokaryotic Argonaute Protein from Natronobacterium gregoryi Requires RNAs To Activate for DNA Interference In Vivo

• Published in: mBio Vol. 13; no. 2; p. e0365621
• Main Authors: Xing, Jiani, Ma, Lixia, Cheng, Xinzhen, Ma, Jinrong, Wang, Ruyu, Xu, Kun, Mymryk, Joe S, Zhang, Zhiying
• Format: Journal Article
• Language: English
• Published: United States American Society for Microbiology 26-04-2022
• Subjects: activation; Argonaute Proteins - genetics; bacteriophage T7; Bacteriophages - genetics; Biotechnology; DNA - metabolism; DNA interference; Eukaryota - genetics; in vivo; Natronobacterium - genetics; Natronobacterium - metabolism; NgAgo; Prokaryotic Cells - metabolism; Research Article; RNA; NgAgo; in vivo; activation; bacteriophage T7; DNA interference
• ISSN: 2150-7511; 2150-7511
• DOI: 10.1128/mbio.03656-21

The Argonaute proteins are present in all three domains of life, which are archaea, bacteria, and eukarya. Unlike the eukaryotic Argonaute proteins, which use small RNA guides to target mRNAs, some prokaryotic Argonaute proteins (pAgos) use a small DNA guide to interfere with DNA and/or RNA targets. However, the mechanisms of pAgo natural function remain unknown. Here, we investigate the mechanism by which pAgo from Natronobacterium gregoryi (NgAgo) targets plasmid and bacteriophage T7 DNA using a heterologous Escherichia coli-based model system. We show that NgAgo expressed from a plasmid linearizes its expression vector. Cotransformation assays demonstrate that NgAgo requires an RNA in
that is transcribed from the bacteriophage T7 promoter to activate cleavage of a cotransformed plasmid, reminiscent of the
-RNA function in CRISPR/Cas9. We propose a mechanism to explain how NgAgo eliminates invading foreign DNA and bacteriophage. By leveraging this discovery, we show that NgAgo can be programmed to target a plasmid or a chromosome locus.
We revealed the mechanism that explains how the NgAgo eliminates the invading foreign DNA and bacteriophage in bacterial cells at 37°C, and by leveraging this discovery, NgAgo can be programmed to target a plasmid or a chromosome locus.