Crystal Structures of Two Plasmid Copy Control Related RNA Duplexes:  An 18 Base Pair Duplex at 1.20 Å Resolution and a 19 Base Pair Duplex at 1.55 Å Resolution

Klosterman, P. S.; Shah, Sapan A.; Steitz, Thomas A.

doi:10.1021/bi9912793

Cited by 80 publications

(75 citation statements)

References 32 publications

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“…Sulfate ions can mimic the phosphate of the nucleotide backbone and provide clues to the potential ligandbinding site. Therefore, a 19-mer RNA duplex (40) was docked onto the receptor by aligning a phosphate from the duplex backbone onto each of the sulfate ions in turn (Fig. 3 A and B).…”

Section: Resultsmentioning

confidence: 99%

The molecular structure of the Toll-like receptor 3 ligand-binding domain

Bell

Botos

Hall

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

347

275

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Innate immunity is the first line of defense against invading pathogens. Toll-like receptors (TLRs) act as sentinels of the innate immune system, sensing a variety of ligands from lipopolysaccharide to flagellin to dsRNA through their ligand-binding domain that is composed of leucine-rich repeats (LRRs). Ligand binding initiates a signaling cascade that leads to the up-regulation of inflammation mediators. In this study, we have expressed and crystallized the ectodomain (ECD) of human TLR3, which recognizes dsRNA, a molecular signature of viruses, and have determined the molecular structure to 2.4-Å resolution. The overall horseshoe-shaped structure of the TLR3-ECD is formed by 23 repeating LRRs that are capped at each end by specialized non-LRR domains. The extensive ␤-sheet on the molecule's concave surface forms a platform for several modifications, including insertions in the LRRs and 11 N-linked glycans. The TLR3-ECD structure indicates how LRR loops can establish distinct pathogen recognition receptors.dsRNA ͉ innate immunity ͉ pathogen recognition receptor

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Section: Resultsmentioning

confidence: 99%

The molecular structure of the Toll-like receptor 3 ligand-binding domain

Bell

Botos

Hall

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

347

275

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“…Guanosine and cytidine are natural nucleosides, while isoguanosine and isocytidine are unnatural nucleosides. The GC pair was extracted from the crystal structure of 1QC0 (94). Transposing the carbonyl and amino groups of G and C creates iG and iC, respectively.…”

Section: Resultsmentioning

confidence: 99%

“…The calculated electrostatic potential surfaces of 5′CG/3′GC vs 5′iCiG/3′iGiC (top), 5′GC/ 3′CG vs 5′iGiC/3′iCiG (middle), and 5′GG/3′CC vs 5′iGiG/3′iCiC (bottom). Structures for 5′CG/3′GC, 5′GC/3′CG, and 5′GG/3′CC were extracted from the crystal structure of 1QC0 (94), and the iso structures were created using the program Insight II (Biosym Technologies, San Diego, CA) by transposing the amino and carbonyl groups. The potential surfaces were created with the program GRASP (77).…”

Section: Resultsmentioning

confidence: 99%

RNA Challenges for Computational Chemists

Yildirim

Turner

2005

Biochemistry

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Some experimental results for the thermodynamics of RNA folding cannot be explained by simple pairwise hydrogen-bonding models. Such effects include the stabilities of isoguanosineisocytidine (iG-iC) base pairs and of various 2 × 2 nucleotide internal loops. Presumably, these results can be explained by base stacking effects, which can be partitioned into Coulombic and overlap effects. We review experimental measurements that provide benchmarks for testing the approximations and theories used for modeling nucleic acids. Quantitative agreement between experiment and theory will indicate understanding of the interactions determining RNA stability and structure.An understanding of the physical-chemical interactions underlying RNA folding would allow predictions of structure and perhaps function from sequence. The large number of atoms in an RNA molecule necessitates the use of approximate methods, rather than the rigorous equations of quantum mechanics. Many approaches are possible, including coarsegrained potentials (1), which use residue-centered force fields; molecular mechanics, which uses atom-centered force fields such as AMBER (2), CHARMM (3, 4), and GROMOS (5, 6); approximate quantum mechanics, which considers interactions of electrons and nuclei (7,8); and QM/MM, which combines quantum mechanics with molecular mechanics (9-16). Depending on the property to be predicted, the size of the RNA, and the domain of interest, different approaches will provide acceptable approximations.The secondary structure of RNA can sometimes be deduced by sequence comparison, which relies on the Watson-Crick rules for base pairing and the assumption that secondary structures are more conserved than sequences for function (17). Often, however, there are not enough sequences to determine a definitive secondary structure. For these cases, freeenergy minimization with a nearest neighbor model is the most popular method for predicting secondary structures (18-38). In this method, possible secondary structure motifs are assigned free-energy parameters, and these values are added to predict the total free energy of forming a secondary structure. The structure with the lowest free energy is assumed to dominate in solution. Rigorously, however, the concentrations of the various possible structures are predicted to be weighted by a Boltzmann factor, exp(−ΔG°/RT), where ΔG° is the free energy change for folding, R is the gas constant, 1.987 cal K −1 mol −1 , and T is the temperature in kelvins. Understanding intermolecular interactions such as hydrogen bonding, stacking, and so forth would allow accurate prediction of ΔG° and therefore secondary structure for an RNA. † This work was supported by NIH Grant GM22939 (D.H.T. Watson-Crick base pairs are the most common and most extensively studied motif in RNA structures. Configurations with the same base compositions but different permutations of base pairs generally have different free energies. For example, the duplexes (5′CGCG3′) 2 and (5′GGCC3′) 2 both have four GC base pairs, bu...

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“…PCR fragments were digested with EcoRI and NcoI and cloned into plasmid pZS*32. After transformation into Escherichia coli XL-1 Blue (Stratagene), 2⅐10 8 colonies were obtained (library 1). A smaller library with positions 14 and 18 randomized (library 2) was constructed by using splice overlap extension-PCR (15) and primer pairs Rndm2b 5Ј-GAG TTT CTC CAG AAG CGT TAA TGT MNN GCT TCT GAT MNN GCG GGC CAT GTT AAG GGC-3, RopPstIf 5Ј-ATG AAC TGC AGA TAA TCC CCC TTA CAC GGA GGC ATC AGT GAC C-3Ј and RopBamHIb 5Ј-GCT GCA TGT GGA TCC TCA GAG GTT TTC ACC GTC ATC ACC-3Ј, Rndmf 5Ј-CAT TAA CGC TTC TGG AGA AAC TC-3Ј (M ϭ A͞C).…”

Section: Methodsmentioning

confidence: 99%

“…The protein-RNA interaction has been studied by using biochemical (7), crystallographic (8) and NMR (9, 10) approaches, but the details are unknown. Earlier work with alanine-scanning mutagenesis (11,12) identified some of the key residues in the RNA-binding site (7).…”

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confidence: 99%

Identification of functional similarities between proteins using directed evolution

Christ

Winter²

2003

Proc. Natl. Acad. Sci. U.S.A.

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Protein sequences are often highly redundant and evolution can change them beyond recognition. It can therefore be difficult to identify proteins with functional or structural similarities by inspection of their sequences. Here we have used an experimental evolutionary approach to detect hidden similarities between the antisense RNA-binding protein Rop and other proteins. We created an envelope of functional Rop mutants by combinatorial mutagenesis, used the compilation of mutant sequences to search a database of protein structures, and thereby identified a segment of the enzyme valyl-tRNA-synthetase (ValRS). Further inspection revealed that the structures of the RNA-binding sites of both proteins are highly related, as indeed are the RNA ligands. From the known 3D structure of the ValRS in complex with tRNA, we were able to build a model of an RNA hairpin pair in complex with Rop that has proved to be consistent with the biochemical and NMR data for the interaction between Rop and RNA hairpins. We suggest that this approach (mutational envelope scanning), by generating sequence information de novo, can help uncover hidden similarities between proteins.

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Crystal Structures of Two Plasmid Copy Control Related RNA Duplexes: An 18 Base Pair Duplex at 1.20 Å Resolution and a 19 Base Pair Duplex at 1.55 Å Resolution

Cited by 80 publications

References 32 publications

The molecular structure of the Toll-like receptor 3 ligand-binding domain

The molecular structure of the Toll-like receptor 3 ligand-binding domain

RNA Challenges for Computational Chemists

Identification of functional similarities between proteins using directed evolution

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