DNA recognition by an RNA-guided bacterial Argonaute.

Kevin W Doxzen,Jennifer A Doudna,Emanuele Paci

doi:10.1371/journal.pone.0177097

Abstract

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.

Highlights

Argonaute (Ago) proteins exist in all three domains of life [1,2]
We present a structural analysis of a divergent bacterial Argonaute, M. piezophila Argonaute (MpAgo), bound to an RNA-DNA helix, revealing unique domain orientations and a distinct conformation of the helical substrate
These features extend the diversity of target recognition mechanisms observed for Argonaute proteins

Summary

Introduction

Argonaute (Ago) proteins exist in all three domains of life [1,2]. In eukaryotes, Argonautes are the core component of the RNA interference (RNAi) effector complex. Unlike other Argonautes, the Ago protein from the eubacterium Marinitoga piezophila (MpAgo) has been shown to preferentially bind 50-hydroxylated guide RNAs to target ssDNA [12]. This unique preference adds to the question of how prokaryotic Argonaute guide sequences are generated and what structural features dictate guide and target binding specificity. The crystal structure of RsAgo bound to an RNA-DNA heteroduplex revealed how Argonaute proteins discriminate between nucleic acid type through the duplex structure of the seed sequence, and how the N-terminal domain assists in hybrid duplex stabilization [31]. We present a crystal structure of MpAgo bound to an RNA guide sequence and a complementary DNA target strand, providing insight into preferential targeting of ssDNA. Our structural and biochemical findings provide insight into the diversity of mechanisms of target recognition by Argonaute proteins

Results

A MpAgo L2

Discussion

Materials and methods