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Picornaviruses are small RNA viruses that hijack host cell machinery to promote their replication. During infection, these viruses express two proteases, 2A pro and 3C pro , which process viral proteins. They also subvert a number of host functions, including innate immune responses, host protein synthesis, and intracellular transport, by utilizing poorly understood mechanisms for rapidly and specifically targeting critical host proteins. Here, we used proteomic tools to characterize 2A pro interacting partners, functions, and targeting mechanisms. Our data indicate that, initially, 2A pro primarily targets just two cellular proteins: eukaryotic translation initiation factor eIF4G (a critical component of the protein synthesis machinery) and Nup98 (an essential component of the nuclear pore complex, responsible for nucleocytoplasmic transport). The protease appears to employ two different cleavage mechanisms; it likely interacts with eIF3L, utilizing the eIF3 complex to proteolytically access the eIF4G protein but also directly binds and degrades Nup98. This Nup98 cleavage results in only a marginal effect on nuclear import of proteins, while nuclear export of proteins and mRNAs were more strongly affected. Collectively, our data indicate that 2A pro selectively inhibits protein translation, key nuclear export pathways, and cellular mRNA localization early in infection to benefit viral replication at the expense of particular cell functions., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Binding of dsDNA by cyclic GMP-AMP (cGAMP) synthase (cGAS) triggers formation of the metazoan second messenger c[G(2',5')pA(3',5')p], which binds the signaling protein STING with subsequent activation of the interferon (IFN) pathway. We show that human hSTING(H232) adopts a "closed" conformation upon binding c[G(2',5')pA(3',5')p] and its linkage isomer c[G(2',5')pA(2',5')p], as does mouse mSting(R231) on binding c[G(2',5')pA(3',5')p], c[G(3',5')pA(3',5')p] and the antiviral agent DMXAA, leading to similar "closed" conformations. Comparing hSTING to mSting, 2',5'-linkage-containing cGAMP isomers were more specific triggers of the IFN pathway compared to the all-3',5'-linkage isomer. Guided by structural information, we identified a unique point mutation (S162A) placed within the cyclic-dinucleotide-binding site of hSTING that rendered it sensitive to the otherwise mouse-specific drug DMXAA, a conclusion validated by binding studies. Our structural and functional analysis highlights the unexpected versatility of STING in the recognition of natural and synthetic ligands within a small-molecule pocket created by the dimerization of STING., (Copyright © 2013 Elsevier Inc. All rights reserved.)

We have solved the crystal structure of human ARGONAUTE1 (hAGO1) bound to endogenous 5'-phosphorylated guide RNAs. To identify changes that evolutionarily rendered hAGO1 inactive, we compared our structure with guide-RNA-containing and cleavage-active hAGO2. Aside from mutation of a catalytic tetrad residue, proline residues at positions 670 and 675 in hAGO1 introduce a kink in the cS7 loop, forming a convex surface within the hAGO1 nucleic-acid-binding channel near the inactive catalytic site. We predicted that even upon restoration of the catalytic tetrad, hAGO1-cS7 sterically hinders the placement of a fully paired guide-target RNA duplex into the endonuclease active site. Consistent with this hypothesis, reconstitution of the catalytic tetrad with R805H led to low-level hAGO1 cleavage activity, whereas combining R805H with cS7 substitutions P670S and P675Q substantially augmented hAGO1 activity. Evolutionary amino acid changes to hAGO1 were readily reversible, suggesting that loading of guide RNA and pairing of seed-based miRNA and target RNA constrain its sequence drift., (Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.)

Recent studies identified cyclic GMP-AMP (cGAMP) as a metazoan second messenger triggering an interferon response. cGAMP is generated from GTP and ATP by cytoplasmic dsDNA sensor cGAMP synthase (cGAS). We combined structural, chemical, biochemical, and cellular assays to demonstrate that this second messenger contains G(2',5')pA and A(3',5')pG phosphodiester linkages, designated c[G(2',5')pA(3',5')p]. We show that, upon dsDNA binding, cGAS is activated through conformational transitions, resulting in formation of a catalytically competent and accessible nucleotide-binding pocket for generation of c[G(2',5')pA(3',5')p]. We demonstrate that cyclization occurs in a stepwise manner through initial generation of 5'-pppG(2',5')pA prior to cyclization to c[G(2',5')pA(3',5')p], with the latter positioned precisely in the catalytic pocket. Mutants of cGAS dsDNA-binding or catalytic pocket residues exhibit reduced or abrogated activity. Our studies have identified c[G(2',5')pA(3',5')p] as a founding member of a family of metazoan 2',5'-containing cyclic heterodinucleotide second messengers distinct from bacterial 3',5' cyclic dinucleotides., (Copyright © 2013 Elsevier Inc. All rights reserved.)

We recently reported that Drosophila Insensitive (Insv) promotes sensory organ development and has activity as a nuclear corepressor for the Notch transcription factor Suppressor of Hairless [Su(H)]. Insv lacks domains of known biochemical function but contains a single BEN domain (i.e., a "BEN-solo" protein). Our chromatin immunoprecipitation (ChIP) sequencing (ChIP-seq) analysis confirmed binding of Insensitive to Su(H) target genes in the Enhancer of split gene complex [E(spl)-C]; however, de novo motif analysis revealed a novel site strongly enriched in Insv peaks (TCYAATHRGAA). We validate binding of endogenous Insv to genomic regions bearing such sites, whose associated genes are enriched for neural functions and are functionally repressed by Insv. Unexpectedly, we found that the Insv BEN domain binds specifically to this sequence motif and that Insv directly regulates transcription via this motif. We determined the crystal structure of the BEN-DNA target complex, revealing homodimeric binding of the BEN domain and extensive nucleotide contacts via α helices and a C-terminal loop. Point mutations in key DNA-contacting residues severely impair DNA binding in vitro and capacity for transcriptional regulation in vivo. We further demonstrate DNA-binding and repression activities by the mammalian neural BEN-solo protein BEND5. Altogether, we define novel DNA-binding activity in a conserved family of transcriptional repressors, opening a molecular window on this extensive gene family.

Riboswitches are mRNA regions that regulate the expression of genes in response to various cellular metabolites. These RNA sequences, typically situated in the untranslated regions of mRNAs, possess complex structures that dictate highly specific binding to certain ligands, such as nucleobases, coenzymes, amino acids, and sugars, without protein assistance. Depending on the presence of the ligand, metabolite-binding domains of riboswitches can adopt two alternative conformations, which define the conformations of the adjacent sequences involved in the regulation of gene expression. In order to understand in detail the nature of riboswitch-ligand interactions and the molecular basis of riboswitch-based gene expression control, it is necessary to determine the three-dimensional structures of riboswitch-ligand complexes. This chapter outlines the techniques that are employed to prepare riboswitch-ligand complexes for structure determination using X-ray crystallography. The chapter describes the principles of construct design, in vitro transcription, RNA purification, complex formation, and crystallization screening utilized during the successful crystallization of several riboswitches.

The core ribosomal protein S8 binds to the central domain of 16S rRNA independently of other ribosomal proteins and is required for assembling the 30S subunit. It has been shown with E. coli ribosomes that a short rRNA fragment restricted by nucleotides 588-602 and 636-651 is sufficient for strong and specific protein S8 binding. In this work, we studied the complexes formed by ribosomal protein S8 from Thermus thermophilus and Methanococcus jannaschii with short rRNA fragments isolated from the same organisms. The dissociation constants of the complexes of protein S8 with rRNA fragments were determined. Based on the results of binding experiments, rRNA fragments of different length were designed and synthesized in preparative amounts in vitro using T7 RNA-polymerase. Stable S8-RNA complexes were crystallized. Crystals were obtained both for homologous bacterial and archaeal complexes and for hybrid complexes of archaeal protein with bacterial rRNA. Crystals of the complex of protein S8 from M. jannaschii with the 37-nucleotide rRNA fragment from the same organism suitable for X-ray analysis were obtained.

Three 5S rRNA-binding ribosomal proteins (L5, L18, TL5) of extremely thermophilic bacterium Thermus thermophilus have earlier been isolated. Structural analysis of their complexes with rRNA requires identification of their binding sites in the 5S rRNA. Previously, a TL5-binding site has been identified, a TL5-RNA complex crystallized, and its structure determined to 2.3 A. The sites for L5 and L18 were characterized, and two corresponding 5S rRNA fragments constructed. Of these, a 34-nt fragment specifically interacted with L5, and a 55-nt fragment interacted with L5, L18, and with both proteins. The 34-nt fragment-L5 complex was crystallized; the crystals are suitable for high-resolution X-ray analysis.

In this work we show for the first time that the overproduced N-terminal fragment (residues 1-91) of ribosomal protein TL5 binds specifically to 5S rRNA and that the region of this fragment containing residues 80-91 is a necessity for its RNA-binding activity. The fragment of Escherichia coli 5S rRNA protected by TL5 against RNase A hydrolysis was isolated and sequenced. This 39 nucleotides fragment contains loop E and helices IV and V of 5S rRNA. The isolated RNA fragment forms stable complexes with TL5 and its N-terminal domain. Crystals of TL5 in complex with the RNA fragment diffracting to 2.75 A resolution were obtained.

Binding of Escherichia coli and Thermus thermophilus ribosomal proteins S15 to a 16S ribosomal RNA fragment from T. thermophilus (nt 559-753) has been investigated in detail by extensive deletion analysis, filter-binding assays, gel mobility shift, structure probing, footprinting with chemical, enzymatic, and hydroxyl radical probes. Both S15 proteins recognize two distinct sites. The first one maps in the bottom of helix 638-655/717-734 (H22) and in the three-way junction between helix 560-570/737-747 (H20), helix 571-600/606-634 (H21), and H22. The second is located in a conserved purine-rich region in the center of H22. The first site provides a higher contribution to the free energy of binding than the second one, and both are required for efficient binding. A short RNA fragment of 56 nt containing these elements binds S15 with high affinity. The structure of the rRNA is constrained by the three-way junction and requires both magnesium and S15 to be stabilized. A 3D model, derived by computer modeling with the use of experimental data, suggests that the bound form adopts a Y-shaped conformation, with a quasi-coaxial stacking of H22 on H20, and H21 forming an acute angle with H22. In this model, S15 binds to the shallow groove of the RNA on the exterior side of the Y-shaped structure, making contact with the two sites, which are separated by one helix turn.

This communication describes a novel highly sensitive method for measuring the affinity of a monoclonal antibody for its antigen. It is based on a radioimmunoassay in which the antigen is labeled with radioactivity. It is therefore particularly well adapted to the study of trace amounts of radiolabeled polypeptide chains produced either in vivo, or in vitro by a cell free protein synthesis system or by chemical radiolabeling. It offers several advantages over previously described methods. Though making use of insolubilized antibody, it does measure the true affinity constant of the monoclonal antibody in solution for the antigen. It can be used even when the antigen is present at concentrations far below the dissociation constant of the antibody/antigen complex. It does not require the antigen or the antibody to be purified. In most cases, it requires no sophisticated equipment. This method could be easily adapted to the determination of the equilibrium constant of any type of protein/ligand system.