DNA recognition by an RNA-guided bacterial Argonaute

• Published in: PloS one Vol. 12; no. 5; p. e0177097
• Main Authors: Doxzen, Kevin W, Doudna, Jennifer A
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 17.05.2017; Public Library of Science (PLoS)
• Subjects: Alanine; Analysis; Argonaute 2 protein; Argonaute Proteins - chemistry; Argonaute Proteins - metabolism; Bacteria - genetics; Bacteria - metabolism; Base pairs; BASIC BIOLOGICAL SCIENCES; Biochemistry; Biodegradation; Biology; Biology and life sciences; Biophysics; Cleavage; Comparative analysis; Crystal structure; Crystallization; Crystallography; Defensive behavior; Degradation; Degradation products; Deoxyribonucleic acid; DNA; DNA structure; DNA, Bacterial - chemistry; DNA, Bacterial - metabolism; E coli; Engineering; Enzymes; Gene expression; Gene mapping; Gene regulation; Genetic aspects; Gram-positive bacteria; Immunological tolerance; Kinetics; Microbiology; MicroRNAs; Mode of action; Models, Biological; Molecular biology; Molecular Conformation; Nucleotide sequence; Phosphates; Physical Sciences; Prokaryotes; Protein Binding; Protein Interaction Domains and Motifs; Protein structure; Proteins; Residues; RNA; RNA, Bacterial - chemistry; RNA, Bacterial - metabolism; Science & Technology - Other Topics; Substrates; Target recognition; Thermodynamics; Transcription; Translation; Unwinding; United States > US; California
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0177097

Argonaute (Ago) proteins are widespread in prokaryotes and eukaryotes and share a four-domain architecture capable of RNA- or DNA-guided nucleic acid recognition. Previous studies identified a prokaryotic Argonaute protein from the eubacterium Marinitoga piezophila (MpAgo), which binds preferentially to 5'-hydroxylated guide RNAs and cleaves single-stranded RNA (ssRNA) and DNA (ssDNA) targets. Here we present a 3.2 Å resolution crystal structure of MpAgo bound to a 21-nucleotide RNA guide and a complementary 21-nucleotide ssDNA substrate. Comparison of this ternary complex to other target-bound Argonaute structures reveals a unique orientation of the N-terminal domain, resulting in a straight helical axis of the entire RNA-DNA heteroduplex through the central cleft of the protein. Additionally, mismatches introduced into the heteroduplex reduce MpAgo cleavage efficiency with a symmetric profile centered around the middle of the helix. This pattern differs from the canonical mismatch tolerance of other Argonautes, which display decreased cleavage efficiency for substrates bearing sequence mismatches to the 5' region of the guide strand. This structural analysis of MpAgo bound to a hybrid helix advances our understanding of the diversity of target recognition mechanisms by Argonaute proteins.