A Gradient-Enhanced HCCH-TOCSY Experiment for Recording Side-Chain 1H and 13C Correlations in H2O Samples of Proteins

Kay, Lewis E.; Xu, Guangyi; Singer, A.U.; Muhandiram, D. R.; Forman‐Kay, Julie D.

doi:10.1006/jmrb.1993.1053

Cited by 488 publications

(358 citation statements)

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“…Under these conditions, the HNCA triple resonance experiment [42] yielded 30 correlations. Ή-^Ν TOCSY-HSQC [43] and 1 H-13 C HCCH-TOCSY [44] spectra gave even fewer transfers. Acceptable triple-resonance spectra could only be obtained after 70% deuteration of the protein.…”

Section: Biophysical Properties Of Pl9mentioning

confidence: 96%

NMR structural characterization of the CDK inhibitor p19^INK4d

et al. 1997

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Section: Biophysical Properties Of Pl9mentioning

confidence: 96%

NMR structural characterization of the CDK inhibitor p19^INK4d

et al. 1997

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“…The substrate RNA and the ribozyme RNA were prepared enzymatically from a synthetic DNA template by using T7 RNA polymerase and either unlabeled rNTPs or 13 C, 15 N-labeled rNTPs (9,10). Each transcribed RNA was purified by Prepcell (Bio-Rad).…”

Section: Rna Sample Preparationmentioning

confidence: 99%

“…The cleaved RNA product (31 nt and 10 nt) was gel purified on 15% polyacrylamide͞7 M urea gels, because the Prepcell had only about 5-nt resolution. NMR samples were 0.5 mM for a fully 13 C, 15 N-labeled RNA strand and 2 mM for an unlabeled RNA strand in 0.01 mM EDTA͞10 mM sodium phosphate (pH 6.5). Purple membrane (PM, Munich Innovative Biomaterials, Marburg, Germany) was added to 2 mg͞ml for the measurement of residual dipolar couplings (11,12).…”

Section: Rna Sample Preparationmentioning

confidence: 99%

Structural features of an influenza virus promoter and their implications for viral RNA synthesis

Bae

Cheong

Lee

et al. 2001

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

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The influenza A virus, a severe pandemic pathogen, has a segmented RNA genome consisting of eight single-stranded RNA molecules. The 5 and 3 ends of each RNA segment recognized by the influenza A virus RNA-dependent RNA polymerase direct both transcription and replication of the virus's RNA genome. Promoter binding by the viral RNA polymerase and formation of an active open complex are prerequisites for viral replication and proliferation. Here we describe the solution structure of this promoter as solved by multidimensional, heteronuclear magnetic resonance spectroscopy. Our studies show that the viral promoter has a significant dynamic nature and reveal an unusual displacement of an adenosine that forms a novel (A-A)⅐U motif and a C-A mismatch stacked in a helix. The characterized structural features of the promoter imply that the specificity of polymerase binding results from an internal RNA loop. In addition, an unexpected bending (46 ؎ 10°) near the initiation site suggests the existence of a promoter recognition mechanism similar to that of DNA-dependent RNA polymerase and a possible regulatory function for the terminal structure during open complex formation.T he influenza A virus, a member of the Orthomyxoviridae family, has a genome consisting of eight single-stranded RNA molecules of negative polarity. These eight RNA segments encode ten proteins, including three RNA-dependent RNA polymerase (RdRp) proteins (PA, PB1, PB2) and one nucleoprotein (NP). The transcription and replication of the viral genome are performed in the nucleus of infected cells by a ribonucleoprotein (RNP) complex that is composed of the three polymerase proteins, NP, and the viral RNA (vRNA). Replication of the vRNA produces a full-length copy of positive-sense RNA (cRNA). The cRNA is then used in the formation of another RNP complex, which serves to generate newly synthesized vRNA. Transcription is also performed by the same RNP complex; however, initiation occurs by the pirating of a 7-methyl guanosine cap structure from a host mRNA. Viral transcription produces an mRNA molecule that is 15 to 22 nucleotides (nt) shorter than cRNA and contains a poly(A) tail (1).The influenza A virus RdRp binds specifically to the partial duplex promoter (also referred to as the panhandle RNA), which is made from the 5Ј and 3Ј termini of each RNA genome segment. Within this partial duplex, 13 nt at the 5Ј end and 12 nt at the 3Ј end are highly conserved among most influenza A virus variants (Fig. 1A). All of the necessary signals for replication and genome packaging appear to reside in these terminal sequences (2), and several pieces of evidence imply a regulatory role for the terminal structure in viral transcription initiation (3-6), termination, and polyadenylation (7,8).The RdRps are for the most part virus-encoded polymerases that synthesize RNA from an RNA template without a DNA intermediate. Despite the unique features of RdRps, little has been deciphered about their mechanisms of promoter recognition and RNA synthesis. The promoter of the...

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“…The sequence used an HSQC module to improve resolution in the "N dimension ) and gradient water suppression (Kay et al, 1993). The spectrum was recorded on a "Nlabeled sample at 2.29 m M , with a mixing time of 80 ms and sign discrimination by States-TPPI in both indirect dimensions.…”

Section: Nmr Data Collection and Processingmentioning

confidence: 99%

Refined structure of villin 14T and a detailed comparison with other actin‐severing domains

1997

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Villin 14T is the amino terminal actin monomer binding domain from the actin-severing and bundling protein villin. Its structure has been determined in solution using heteronuclear multidimensional nuclear magnetic resonance (NMR) spectroscopy (Markus MA, Nakayama T, Matsudaira P, Wagner G. 1994. Solution structure of villin 14T, a domain conserved among actin-severing proteins. Protein Science 3:70-81). An additional nuclear Overhauser effect (NOE) spectroscopy data set, acquired using improved gradient techniques, and further detailed analysis of existing data sets, produced an additional 601 NOE restraints for structure calculations. The overall fold does not change significantly with the additional NOE restraints but the definition of the structure is improved, as judged by smaller deviations among an ensemble of calculated structures that adequately satisfy the NMR restraints. Some of the side chains, especially those in the hydrophobic core of the domain, are much more defined. This improvement in the detail of the solution structure of villin 14T makes it interesting to compare the structure with the crystal structure of gelsolin segment 1, which shares 58% sequence identity with villin 14T, in an effort to gain insight into villin 14T's weaker affinity for actin monomers. Villin 14T has smaller side chains at several positions that make hydrophobic contacts with actin in the context of gelsolin segment 1. The structure is also compared with the structure of the related actin-severing domain, severin domain 2.

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A Gradient-Enhanced HCCH-TOCSY Experiment for Recording Side-Chain 1H and 13C Correlations in H2O Samples of Proteins

Cited by 488 publications

References 0 publications

NMR structural characterization of the CDK inhibitor p19^INK4d

NMR structural characterization of the CDK inhibitor p19^INK4d

Structural features of an influenza virus promoter and their implications for viral RNA synthesis

Refined structure of villin 14T and a detailed comparison with other actin‐severing domains

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A Gradient-Enhanced HCCH-TOCSY Experiment for Recording Side-Chain 1H and 13C Correlations in H2O Samples of Proteins

Cited by 488 publications

References 0 publications

NMR structural characterization of the CDK inhibitor p19INK4d

NMR structural characterization of the CDK inhibitor p19INK4d

Structural features of an influenza virus promoter and their implications for viral RNA synthesis

Refined structure of villin 14T and a detailed comparison with other actin‐severing domains

Contact Info

Product

Resources

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NMR structural characterization of the CDK inhibitor p19^INK4d

NMR structural characterization of the CDK inhibitor p19^INK4d