Ailong Ke

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 SMK box (SAM-III) translational riboswitch has been identified in the SAM synthetase gene in members of the Lactobacillales. Here we report the 2.2-Å crystal structure of the Enterococcus faecalis SMK 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 SMK 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.

Architecture of the yeast Rrp44-exosome complex suggests routes of RNA recruitment for 3 end processing

The eukaryotic core exosome (CE) is a conserved nine-subunit protein complex important for 3′ end trimming and degradation of RNA. In yeast, the Rrp44 protein constitutively associates with the CE and provides the sole source of processive 3′-to-5′ exoribonuclease activity. Here we present EM reconstructions of the core and Rrp44-bound exosome complexes. The two-lobed Rrp44 protein binds to the RNase PH domain side of the exosome and buttresses the bottom of the exosome-processing chamber. The Rrp44 C-terminal body part containing an RNase II-type active site is anchored to the exosome through a conserved set of interactions mainly to the Rrp45 and Rrp43 subunit, whereas the Rrp44 N-terminal head part is anchored to the Rrp41 subunit and may function as a roadblock to restrict access of RNA to the active site in the body region. The Rrp44–exosome (RE) architecture suggests an active site sequestration mechanism for strict control of 3′ exoribonuclease activity in the RE complex.

Envelope stress is a trigger of CRISPR RNA‐mediated DNA silencing in Escherichia coli

A widespread feature in the genomes of most bacteria and archaea is an array of clustered, regularly interspaced short palindromic repeats (CRISPRs) that, together with a group of CRISPR‐associated (Cas) proteins, mediate immunity against invasive nucleic acids such as plasmids and viruses. Here, the CRISPR‐Cas system was activated in cells expressing a plasmid‐encoded protein that was targeted to the twin‐arginine translocation (Tat) pathway. Expression of this Tat substrate resulted in upregulation of the Cas enzymes and subsequent silencing of the encoding plasmid in a manner that required the BaeSR two‐component regulatory system, which is known to respond to extracytoplasmic stress. Furthermore, we confirm that the CasCDE enzymes form a stable ternary complex and appear to function as the catalytic core of the Cas system to process CRISPR RNA into its mature form. Taken together, our results indicate that the CRISPR‐Cas system targets DNA directly as part of a defence mechanism in bacteria that is overlapping with but not limited to phage infection.

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.

Structural basis for promiscuous PAM recognition in type I–E Cascade from E. coli

Clustered regularly interspaced short palindromic repeats (CRISPRs) and the cas (CRISPR-associated) operon form an RNA-based adaptive immune system against foreign genetic elements in prokaryotes. Type I accounts for 95% of CRISPR systems, and has been used to control gene expression and cell fate. During CRISPR RNA (crRNA)-guided interference, Cascade (CRISPR-associated complex for antiviral defence) facilitates the crRNA-guided invasion of double-stranded DNA for complementary base-pairing with the target DNA strand while displacing the non-target strand, forming an R-loop. Cas3, which has nuclease and helicase activities, is subsequently recruited to degrade two DNA strands. A protospacer adjacent motif (PAM) sequence flanking target DNA is crucial for self versus foreign discrimination. Here we present the 2.45 Å crystal structure of Escherichia coli Cascade bound to a foreign double-stranded DNA target. The 5′-ATG PAM is recognized in duplex form, from the minor groove side, by three structural features in the Cascade Cse1 subunit. The promiscuity inherent to minor groove DNA recognition rationalizes the observation that a single Cascade complex can respond to several distinct PAM sequences. Optimal PAM recognition coincides with wedge insertion, initiating directional target DNA strand unwinding to allow segmented base-pairing with crRNA. The non-target strand is guided along a parallel path 25 Å apart, and the R-loop structure is further stabilized by locking this strand behind the Cse2 dimer. These observations provide the structural basis for understanding the PAM-dependent directional R-loop formation process.

T box RNA decodes both the information content and geometry of tRNA to affect gene expression

The T box leader sequence is an RNA element that controls gene expression by binding directly to a specific tRNA and sensing its aminoacylation state. This interaction controls expression of amino acid-related genes in a negative feedback loop. The T box RNA structure is highly conserved, but its tRNA binding mechanism is only partially understood. Known sequence elements are the specifier sequence, which recognizes the tRNA anticodon, and the antiterminator bulge, which base pairs with the tRNA acceptor end. Here, we reveal the crucial function of the highly conserved stem I distal region in tRNA recognition and report its 2.65-Å crystal structure. The apex of this region contains an intricately woven loop–loop interaction between two conserved motifs, the Adenine-guanine (AG) bulge and the distal loop. This loop–loop structure presents a base triple on its surface that is optimally positioned for base-stacking interactions. Mutagenesis, cross-linking, and small-angle X-ray scattering data demonstrate that the apical base triple serves as a binding platform to dock the tRNA D- and T-loops. Strikingly, the binding platform strongly resembles the D- and T-loop binding elements from RNase P and the ribosome exit site, suggesting that this loop–loop structure may represent a widespread tRNA recognition platform. We propose a two-checkpoint molecular ruler model for tRNA decoding in which the information content of tRNA is first examined through specifier sequence–anticodon interaction, and the length of the tRNA anticodon arm is then measured by the distal loop–loop platform. When both conditions are met, tRNA is secured, and its aminoacylation state is sensed.

Double-stranded endonuclease activity in Bacillus halodurans clustered regularly interspaced short palindromic repeats (CRISPR)-associated Cas2 protein.

Background: Cas2 is universally conserved and essential for new CRISPR spacer acquisition. Results: Bha_Cas2 uses a single metal ion to cleave dsDNA and is likely activated by a pH-dependent conformational change. A method to classify Cas2 into ssRNase and dsDNase is proposed. Conclusion: B. halodurans and T. thermophilus Cas2 are metal-dependent endonucleases. Significance: dsDNase activity is consistent with the direct involvement of Cas2 in new spacer acquisition. The CRISPR (clustered regularly interspaced short palindromic repeats) system is a prokaryotic RNA-based adaptive immune system against extrachromosomal genetic elements. Cas2 is a universally conserved core CRISPR-associated protein required for the acquisition of new spacers for CRISPR adaptation. It was previously characterized as an endoribonuclease with preference for single-stranded (ss)RNA. Here, we show using crystallography, mutagenesis, and isothermal titration calorimetry that the Bacillus halodurans Cas2 (Bha_Cas2) from the subtype I-C/Dvulg CRISPR instead possesses metal-dependent endonuclease activity against double-stranded (ds)DNA. This activity is consistent with its putative function in producing new spacers for insertion into the 5′-end of the CRISPR locus. Mutagenesis and isothermal titration calorimetry studies revealed that a single divalent metal ion (Mg2+ or Mn2+), coordinated by a symmetric Asp pair in the Bha_Cas2 dimer, is involved in the catalysis. We envision that a pH-dependent conformational change switches Cas2 into a metal-binding competent conformation for catalysis. We further propose that the distinct substrate preferences among Cas2 proteins may be determined by the sequence and structure in the β1–α1 loop.

Structure and assembly of the essential RNA ring component of a viral DNA packaging motor

Prohead RNA (pRNA) is an essential component in the assembly and operation of the powerful bacteriophage ϕ29 DNA packaging motor. The pRNA forms a multimeric ring via intermolecular base-pairing interactions between protomers that serves to guide the assembly of the ring ATPase that drives DNA packaging. Here we report the quaternary structure of this rare multimeric RNA at 3.5 Å resolution, crystallized as tetrameric rings. Strong quaternary interactions and the inherent flexibility helped rationalize how free pRNA is able to adopt multiple oligomerization states in solution. These characteristics also allowed excellent fitting of the crystallographic pRNA protomers into previous prohead/pRNA cryo-EM reconstructions, supporting the presence of a pentameric, but not hexameric, pRNA ring in the context of the DNA packaging motor. The pentameric pRNA ring anchors itself directly to the phage prohead by interacting specifically with the fivefold symmetric capsid structures that surround the head-tail connector portal. From these contacts, five RNA superhelices project from the pRNA ring, where they serve as scaffolds for binding and assembly of the ring ATPase, and possibly mediate communication between motor components. Construction of structure-based designer pRNAs with little sequence similarity to the wild-type pRNA were shown to fully support the packaging of ϕ29 DNA.

Visualization of distinct substrate-recruitment pathways in the yeast exosome by EM

The eukaryotic exosome is a multisubunit complex typically composed of a catalytically inactive core and the Rrp44 protein, which contains 3′-to-5′ exo- and endo-RNase activities. RNA substrates have been shown to be recruited through the core to reach Rrp44s exo-RNase (EXO) site. Using single-particle EM and biochemical analysis, we provide visual evidence that two distinct substrate-recruitment pathways exist. In the through-core route, channeling of the single-stranded substrates from the core to Rrp44 induces a characteristic conformational change in Rrp44. In the alternative direct-access route, this conformational change does not take place, and the RNA substrate is visualized to avoid the core and enter Rrp44s EXO site directly. Our results provide mechanistic explanations for several RNA processing scenarios by the eukaryotic exosome and indicate substrate-specific modes of degradation by this complex.

Crystal structure of the S. solfataricus archaeal exosome reveals conformational flexibility in the RNA-binding ring.

Background The exosome complex is an essential RNA 3′-end processing and degradation machinery. In archaeal organisms, the exosome consists of a catalytic ring and an RNA-binding ring, both of which were previously reported to assume three-fold symmetry. Methodology/Principal Findings Here we report an asymmetric 2.9 Å Sulfolobus solfataricus archaeal exosome structure in which the three-fold symmetry is broken due to combined rigid body and thermal motions mainly within the RNA-binding ring. Since increased conformational flexibility was also observed in the RNA-binding ring of the related bacterial PNPase, we speculate that this may reflect an evolutionarily conserved mechanism to accommodate diverse RNA substrates for degradation. Conclusion/Significance This study clearly shows the dynamic structures within the RNA-binding domains, which provides additional insights on mechanism of asymmetric RNA binding and processing.