Approaching the Golden Fleece a Molecule at a Time: Biophysical Insights into Argonaute-Instructed Nucleic Acid Interactions

Abstract

Argonaute proteins act at the core of nucleic acid-guided interference pathways that regulate gene expression and defend organisms against foreign genetic elements in all domains of life. Here, we review recent biophysical studies on how Argonaute proteins instruct oligonucleotides in the process of target finding, binding, cleavage, and release, as measured at high spatiotemporal resolution by single-molecule approaches. In the context of previous structural, biochemical, and computational studies, a model emerges for how Argonaute proteins manipulate the thermodynamic rules for nucleic acid hybridization to convey efficiency and specificity to RNA- and DNA-guided regulatory processes.

Specific structural elements of the T-box riboswitch drive the two-step binding of the tRNA ligand

“…Two-step binding mechanisms have been observed in a variety of protein or nucleic acid mediated biological process, including T-cell receptor (TCR) recognition of the major histocompatibility complex (MHC) presenting peptides, where TCRs scan the MHC scaffold first, followed by sensing of specific MHC-presenting peptides ( Wu et al, 2002 ); interaction of the signal recognition particle (SRP) receptor with the membrane, where SRP receptors interact with the membrane in a dynamic mode followed by an SRP-induced conformational transition into a stable binding mode ( Hwang Fu et al, 2017 ), DNA interrogation by CRISPR Cas9-crRNA, where Cas9-crRNA recognizes the protospacer adjacent motif (PAM) on the target DNA followed by sensing of the spacer sequence and triggering R-loop formation ( Sternberg et al, 2014 ); and RNA-induced silencing complexes (RISCs) binding to their mRNA targets, where dynamic sampling of the ‘sub-seed’ region occurs before targeting stably across the full seed region ( Chandradoss et al, 2015 ; Herzog and Ameres, 2015 ; Salomon et al, 2015 ; Salomon et al, 2016 ). In all of these cases, a two-step binding mechanism provides a good balance between sensitivity and specificity.…”

FUS Regulates Activity of MicroRNA-Mediated Gene Silencing

“…The miRNA–mRNA pairing is mediated by a 6–8 nucleotide short seed region and miRISC utilizes the guide strand to target mRNAs by Watson-Crick base pairing to partial complementary sequences within their 3′ untranslated regions (UTR) or coding regions. It is considered a major challenge for miRISC to locate the target mRNAs with high efficiency and specificity (Herzog and Ameres, 2015), and it remains unknown if there are other players that facilitate AGOs in miRNA target recognition. The regulation of gene expression by miRNAs can be altered positively or negatively by RNA-binding proteins associated with the target mRNAs (Bhattacharyya et al, 2006; Kedde et al, 2007; Vasudevan et al, 2007).…”

Structural requirements for protein-catalyzed annealing of U4 and U6 RNAs during di-snRNP assembly

“…Proteins that stimulate the annealing of RNA to target nucleic acids are ubiquitously important in biology, with examples including the Argonaute family proteins ( 1 , 2 ), the CRISPR-Cas systems ( 3 ) and Hfq ( 4 , 5 ). Additionally, RNA remodeling proteins are a broad class of proteins that chaperone the proper folding of RNAs, which have a high propensity to misfold into stable alternative structures ( 6 ).…”