Structural basis for target site selection in RNA-guided DNA transposition systems.

CRISPR-associated transposition systems allow guide RNA-directed integration of a single DNA cargo in one orientation at a fixed distance from a programmable target sequence. We used cryo-electron microscopy (cryo-EM) to define the mechanism that underlies this process by characterizing the transposition regulator, TnsC, from a type V-K CRISPR-transposase system. In this scenario, polymerization of adenosine triphosphate-bound TnsC helical filaments could explain how polarity information is passed to the transposase. TniQ caps the TnsC filament, representing a universal mechanism for target information transfer in Tn7/Tn7-like elements. Transposase-driven disassembly establishes delivery of the element only to unused protospacers. Finally, TnsC transitions to define the fixed point of insertion, as revealed by structures with the transition state mimic ADP•AlF3 These mechanistic findings provide the underpinnings for engineering CRISPR-associated transposition systems for research and therapeutic applications.

Local-to-global signal transduction at the core of a Mn2+ sensing riboswitch.

The widespread Mn2+-sensing yybP-ykoY riboswitch controls the expression of bacterial Mn2+ homeostasis genes. Here, we first determine the crystal structure of the ligand-bound yybP-ykoY riboswitch aptamer from Xanthomonas oryzae at 2.96 Å resolution, revealing two conformations with docked four-way junction (4WJ) and incompletely coordinated metal ions. In >100 µs of MD simulations, we observe that loss of divalents from the core triggers local structural perturbations in the adjacent docking interface, laying the foundation for signal transduction to the regulatory switch helix. Using single-molecule FRET, we unveil a previously unobserved extended 4WJ conformation that samples transient docked states in the presence of Mg2+. Only upon adding sub-millimolar Mn2+, however, can the 4WJ dock stably, a feature lost upon mutation of an adenosine contacting Mn2+ in the core. These observations illuminate how subtly differing ligand preferences of competing metal ions become amplified by the coupling of local with global RNA dynamics.

ATP/ADP modulates gp16-pRNA conformational change in the Phi29 DNA packaging motor.

Packaging of phage phi29 genome requires the ATPase gp16 and prohead RNA (pRNA). The highly conserved pRNA forms the interface between the connector complex and gp16. Understanding how pRNA interacts with gp16 under packaging conditions can shed light on the molecular mechanism of the packaging motor. Here, we present 3D models of the pRNA-gp16 complex and its conformation change in response to ATP or ADP binding. Using a combination of crystallography, small angle X-ray scattering and chemical probing, we find that the pRNA and gp16 forms a 'Z'-shaped complex, with gp16 specifically binds to pRNA domain II. The whole complex closes in the presence of ATP, and pRNA domain II rotates open as ATP hydrolyzes, before resetting after ADP is released. Our results suggest that pRNA domain II actively participates in the packaging process.

Sortase-mediated fluorescent labeling of CRISPR complexes.

Fluorescent labeling of proteins is a critical requirement for single-molecule imaging studies. Many protein labeling strategies require harsh conditions or large epitopes that can inactivate the target protein, either by decreasing the protein's enzymatic activity or by blocking protein-protein interactions. Here, we provide a detailed protocol to efficiently label CRISPR-Cas complexes with a small fluorescent peptide via sortase-mediated transpeptidation. The sortase tag consists of just a few amino acids that are specifically recognized at either the N- or the C-terminus, making this strategy advantageous when the protein is part of a larger complex. Sortase is active at high ionic strength, 4°C, and with a broad range of organic fluorophores. We discuss the design, optimization, and single-molecule fluorescent imaging of CRISPR-Cas complexes on DNA curtains. Sortase-mediated transpeptidation is a versatile addition to the protein labeling toolkit.

Structure basis for RNA-guided DNA degradation by Cascade and Cas3.

Type I CRISPR-Cas system features a sequential target-searching and degradation process on double-stranded DNA by the RNA-guided Cascade (CRISPR associated complex for antiviral defense) complex and the nuclease-helicase fusion enzyme Cas3, respectively. Here, we present a 3.7-angstrom-resolution cryo-electron microscopy (cryo-EM) structure of the Type I-E Cascade/R-loop/Cas3 complex, poised to initiate DNA degradation. Cas3 distinguishes Cascade conformations and only captures the R-loop-forming Cascade, to avoid cleaving partially complementary targets. Its nuclease domain recruits the nontarget strand (NTS) DNA at a bulged region for the nicking of single-stranded DNA. An additional 4.7-angstrom-resolution cryo-EM structure captures the postnicking state, in which the severed NTS retracts to the helicase entrance, to be threaded for adenosine 5'-triphosphate-dependent processive degradation. These snapshots form the basis for understanding RNA-guided DNA degradation in Type I-E CRISPR-Cas systems.

Structures of Large RNAs and RNA-Protein Complexes: Toward Structure Determination of Riboswitches.

Riboswitches are widespread and important regulatory elements. They are typically present in the mRNA of the gene under their regulation, where they form complex three-dimensional structures that can bind an effector and regulate either transcription or translation of the mRNA. Structural biology has been essential to our understanding of their ligand recognition and conformational switching mechanisms, but riboswitch determination presents several important complications. Overcoming these challenges requires a synergistic approach using rational design of the constructs and supporting methods to biochemically validate the designs and resulting structures.

Mn(2+)-sensing mechanisms of yybP-ykoY orphan riboswitches.

Gene regulation in cis by riboswitches is prevalent in bacteria. The yybP-ykoY riboswitch family is quite widespread, yet its ligand and function remained unknown. Here, we characterize the Lactococcus lactis yybP-ykoY orphan riboswitch as a Mn(2+)-dependent transcription-ON riboswitch, with a ∼30-40 µM affinity for Mn(2+). We further determined its crystal structure at 2.7 Å to elucidate the metal sensing mechanism. The riboswitch resembles a hairpin, with two coaxially stacked helices tethered by a four-way junction and a tertiary docking interface. The Mn(2+)-sensing region, strategically located at the highly conserved docking interface, has two metal binding sites. Whereas one site tolerates the binding of either Mg(2+) or Mn(2+), the other site strongly prefers Mn(2+) due to a direct contact from the N7 of an invariable adenosine. Mutagenesis and a Mn(2+)-free E. coli yybP-ykoY structure further reveal that Mn(2+) binding is coupled with stabilization of the Mn(2+)-sensing region and the aptamer domain.

Structures of CRISPR Cas3 offer mechanistic insights into Cascade-activated DNA unwinding and degradation.

CRISPR drives prokaryotic adaptation to invasive nucleic acids such as phages and plasmids, using an RNA-mediated interference mechanism. Interference in type I CRISPR-Cas systems requires a targeting Cascade complex and a degradation machine, Cas3, which contains both nuclease and helicase activities. Here we report the crystal structures of Thermobifida fusca Cas3 bound to single-stranded (ss) DNA substrate and show that it is an obligate 3'-to-5' ssDNase that preferentially accepts substrate directly from the helicase moiety. Conserved residues in the HD-type nuclease coordinate two irons for ssDNA cleavage. We demonstrate ATP coordination and conformational flexibility of the SF2-type helicase domain. Cas3 is specifically guided toward Cascade-bound target DNA by a PAM sequence, through physical interactions with both the nontarget substrate strand and the CasA protein. The sequence of recognition events ensures well-controlled DNA targeting and degradation of foreign DNA by Cascade and Cas3.

Common themes and differences in SAM recognition among SAM riboswitches.

The recent discovery of short cis-acting RNA elements termed riboswitches has caused a paradigm shift in our understanding of genetic regulatory mechanisms. The three distinct superfamilies of S-adenosyl-l-methionine (SAM) riboswitches are the most commonly found riboswitch classes in nature. These RNAs represent three independent evolutionary solutions to achieve specific SAM recognition. This review summarizes research on 1) modes of gene regulatory mechanisms, 2) common themes and differences in ligand recognition, and 3) ligand-induced conformational dynamics among SAM riboswitch families. The body of work on the SAM riboswitch families constitutes a useful primer to the topic of gene regulatory RNAs as a whole. This article is part of a Special Issue entitled: Riboswitches.

Elastic coupling between RNA degradation and unwinding by an exoribonuclease.

Rrp44 (Dis3) is a key catalytic subunit of the yeast exosome complex and can processively digest structured RNA one nucleotide at a time in the 3' to 5' direction. Its motor function is powered by the energy released from the hydrolytic nuclease reaction instead of adenosine triphosphate hydrolysis as in conventional helicases. Single-molecule fluorescence analysis revealed that instead of unwinding RNA in single base pair steps, Rrp44 accumulates the energy released by multiple single nucleotide step hydrolysis reactions until about four base pairs are unwound in a burst. Kinetic analyses showed that RNA unwinding, not cleavage or strand release, determines the overall RNA degradation rate and that the unwinding step size is determined by the nonlinear elasticity of the Rrp44/RNA complex, but not by duplex stability.

Variable sequences outside the SAM-binding core critically influence the conformational dynamics of the SAM-III/SMK box riboswitch.

The S(MK) box (SAM-III) translational riboswitches were identified in S-adenosyl-l-methionine (SAM) synthetase metK genes in members of Lactobacillales. This riboswitch switches between two alternative conformations in response to intracellular SAM concentration and controls metK expression at the level of translation initiation. We previously reported the crystal structure of the SAM-bound S(MK) box riboswitch. In this study, we combined selective 2'-hydroxyl acylation analyzed by primer extension chemical probing with mutagenesis to probe the ligand-induced conformational switching mechanism. We revealed that while the majority of the apo S(MK) box RNA molecules exist in an alternatively base-paired (ON) conformation, a subset of them pre-organize into a SAM-bound-like (READY) conformation, which, upon SAM exposure, is selectively stabilized into the SAM-bound (OFF) conformation through an induced-fit mechanism. Mutagenesis showed that the ON state is only slightly more stable than the READY state, as several single-nucleotide substitutions in a hypervariable region outside the SAM-binding core can alter the folding landscape to favor the READY state. Such S(MK) variants display a "constitutively OFF" behavior both in vitro and in vivo. Time-resolved and temperature-dependent selective 2'-hydroxyl acylation analyzed by primer extension analyses revealed adaptation of the S(MK) box RNA to its mesothermal working environment. The latter analysis revealed that the SAM-bound S(MK) box RNA follows a two-step folding/unfolding process.

SAM recognition and conformational switching mechanism in the Bacillus subtilis yitJ S box/SAM-I riboswitch.

S-box (SAM-I) riboswitches are a widespread class of riboswitches involved in the regulation of sulfur metabolism in Gram-positive bacteria. We report here the 3.0-Å crystal structure of the aptamer domain of the Bacillus subtilis yitJ S-box (SAM-I) riboswitch bound to S-adenosyl-L-methionine (SAM). The RNA folds into two sets of helical stacks spatially arranged by tertiary interactions including a K-turn and a pseudoknot at a four-way junction. The tertiary structure is further stabilized by metal coordination, extensive ribose zipper interactions, and SAM-mediated tertiary interactions. Despite structural differences in the peripheral regions, the SAM-binding core of the B. subtilis yitJ riboswitch is virtually superimposable with the previously determined Thermoanaerobacter tengcongensis yitJ riboswitch structure, suggesting that a highly conserved ligand-recognition mechanism is utilized by all S-box riboswitches. SHAPE (selective 2'-hydroxyl acylation analyzed by primer extension) chemical probing analysis further revealed that the alternative base-pairing element in the expression platform controls the conformational switching process. In the absence of SAM, the apo yitJ aptamer domain folds predominantly into a pre-binding conformation that resembles, but is not identical with, the SAM-bound state. We propose that SAM enters the ligand-binding site through the "J1/2-J3/4" gate and "locks" down the SAM-bound conformation through an induced-fit mechanism. Temperature-dependent SHAPE revealed that the tertiary interaction-stabilized SAM-binding core is extremely stable, likely due to the cooperative RNA folding behavior. Mutational studies revealed that certain modifications in the SAM-binding region result in loss of SAM binding and constitutive termination, which suggests that these mutations lock the RNA into a form that resembles the SAM-bound form in the absence of SAM.

Crystal structures of the SAM-III/S(MK) riboswitch reveal the SAM-dependent translation inhibition mechanism.

Three distinct classes of S-adenosyl-L-methionine (SAM)-responsive riboswitches have been identified that regulate bacterial gene expression at the levels of transcription attenuation or translation inhibition. The S(MK) box (SAM-III) translational riboswitch has been identified in the SAM synthetase gene in members of the Lactobacillales. Here we report the 2.2-A crystal structure of the Enterococcus faecalis S(MK) box riboswitch. The Y-shaped riboswitch organizes its conserved nucleotides around a three-way junction for SAM recognition. The Shine-Dalgarno sequence, which is sequestered by base-pairing with the anti-Shine-Dalgarno sequence in response to SAM binding, also directly participates in SAM recognition. The riboswitch makes extensive interactions with the adenosine and sulfonium moieties of SAM but does not appear to recognize the tail of the methionine moiety. We captured a structural snapshot of the S(MK) box riboswitch sampling the near-cognate ligand S-adenosyl-L-homocysteine (SAH) in which SAH was found to adopt an alternative conformation and fails to make several key interactions.

RNA-mediated interaction between the peptide-binding and GTPase domains of the signal recognition particle.

The signal recognition particle (SRP) targets nascent proteins to cellular membranes for insertion or secretion by recognizing polypeptides containing an N-terminal signal sequence as they emerge from the ribosome. GTP-dependent binding of SRP to its receptor protein leads to controlled release of the nascent chain into a membrane-spanning translocon pore. Here we show that the association of the SRP with its receptor triggers a marked conformational change in the complex, localizing the SRP RNA and the adjacent signal peptide-binding site at the SRP-receptor heterodimer interface. The orientation of the RNA suggests how peptide binding and GTP hydrolysis can be coupled through direct structural contact during cycles of SRP-directed protein translocation.