Uniform 13C/15N-Labeling of DNA by Tandem Repeat Amplification

Werner, M.D.; Gupta, Vineet; Lambert, Lester J.; Nagata, Takashi

doi:10.1016/s0076-6879(02)38225-9

Cited by 19 publications

(17 citation statements)

References 27 publications

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“…Others produce site specifically labeled nucleotides from E.coli (Latham et al 2005; Johnson et al 2006), and still others produce these labels using the pentose phosphate and de novo purine synthetic pathways (Schultheisz et al 2008). Similar methods for the production of uniform isotopically labeled deoxynucleosides have been proposed (Zimmer and Crothers 1995; Louis et al 1998; Masse et al 1998; Werner et al 2001; Nelissen et al 2009). Previously, 13 C-formate added to an unlabeled glucose minimal medium enabled selective labeling of purine C8 positions (Latham et al 2005), and use of E. coli deficient in the G6PDH gene enabled the site-labeling of pyrimidine C5 or C6 positions as well as various ribose carbon positions (Johnson et al 2006).…”

Section: Resultsmentioning

confidence: 94%

Site-specific labeling of nucleotides for making RNA for high resolution NMR studies using an E. coli strain disabled in the oxidative pentose phosphate pathway

Dayie

Thakur

2010

J Biomol NMR

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Escherichia coli (E. coli) is a versatile organism for making nucleotides labeled with stable isotopes (13C, 15N, and/or 2H) for structural and molecular dynamics characterizations. Growth of a mutant E. coli strain deficient in the pentose phosphate pathway enzyme glucose-6-phosphate dehydrogenase (K10-1516) on 2-13C-glycerol and 15N-ammonium sulfate in Studier minimal medium enables labeling at sites useful for NMR spectroscopy. However, 13C-sodium formate combined with 13C-2-glycerol in the growth media adds labels to new positions. In the absence of labeled formate, both C5 and C6 positions of the pyrimidine rings are labeled with minimal multiplet splitting due to 1JC5C6 scalar coupling. However, the C2/C8 sites within purine rings and the C1′/C3′/C5′ positions within the ribose rings have reduced labeling. Addition of 13C-labeled formate leads to increased labeling at the base C2/C8 and the ribose C1′/C3′/C5′ positions; these new specific labels result in two- to three-fold increase in the number of resolved resonances. This use of formate and 15N-ammonium sulfate promises to extend further the utility of these alternate site specific labels to make labeled RNA for downstream biophysical applications such as structural, dynamics and functional studies of interesting biologically relevant RNAs.

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Section: Resultsmentioning

confidence: 94%

Site-specific labeling of nucleotides for making RNA for high resolution NMR studies using an E. coli strain disabled in the oxidative pentose phosphate pathway

Dayie

Thakur

2010

J Biomol NMR

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“…The proteins were expressed with an extra His6-tail and a thrombin cleavage site at the N-terminus in Escherichia coli , BL21(DE3) pLysS, at 37°C in minimal medium (M9) containing 1 g/l [ 15 N] ammonium chloride, 3 g/l [ 13 C] glucose (for 15 N and 13 C-labeled proteins), vitamins, mononucleotides, metals, 100 mg/l carbenicillin and 34 mg/l chloramphenicol (38). Cells were grown to OD 600 ∼ 0.5 and induced with 1 mM iso-propylthio-β-D-galactopyranoside (IPTG).…”

Section: Methodsmentioning

confidence: 99%

Structure of Musashi1 in a complex with target RNA: the role of aromatic stacking interactions

Ohyama¹,

Nagata²,

Tsuda³

et al. 2011

Nucleic Acids Research

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142

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Mammalian Musashi1 (Msi1) is an RNA-binding protein that regulates the translation of target mRNAs, and participates in the maintenance of cell ‘stemness’ and tumorigenesis. Msi1 reportedly binds to the 3′-untranslated region of mRNA of Numb, which encodes Notch inhibitor, and impedes initiation of its translation by competing with eIF4G for PABP binding, resulting in triggering of Notch signaling. Here, the mechanism by which Msi1 recognizes the target RNA sequence using its Ribonucleoprotein (RNP)-type RNA-binding domains (RBDs), RBD1 and RBD2 has been revealed on identification of the minimal binding RNA for each RBD and determination of the three-dimensional structure of the RBD1:RNA complex. Unique interactions were found for the recognition of the target sequence by Msi1 RBD1: adenine is sandwiched by two phenylalanines and guanine is stacked on the tryptophan in the loop between β1 and α1. The minimal recognition sequences that we have defined for Msi1 RBD1 and RBD2 have actually been found in many Msi1 target mRNAs reported to date. The present study provides molecular clues for understanding the biology involving Musashi family proteins.

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“…Full‐length PEA‐15 was cloned into a pQE‐9 vector (Qiagen), which produces the recombinant protein with a hexahistidine (His 6 ) sequence at the N‐terminus. Uniformly 15 N‐ and 15 N/ 13 C‐labeled proteins were overexpressed in Escherichia coli BL21 cells by adaptive control fermentation (Werner et al ., 2001) in minimal medium containing 15 NH 4 Cl and/or [ 13 C]glucose as the sole nitrogen and carbon sources, respectively. Cells were grown at 37°C to an optical density ( A 600 nm ) of ∼3.0 and then induced with 1 mM isopropyl‐β‐ D ‐thiogalactopyranoside (IPTG) for 4 h. The cells were suspended in 50 mM Tris buffer pH 8.0, 1 M NaCl, 30 mM imidazole and 10 mM benzamidine hydrochloride, lysed with a French press, and centrifuged.…”

Section: Methodsmentioning

confidence: 99%

Recognition of ERK MAP kinase by PEA-15 reveals a common docking site within the death domain and death effector domain

Hill¹

2002

The EMBO Journal

122

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PEA-15 is a multifunctional protein that modulates signaling pathways which control cell proliferation and cell death. In particular, PEA-15 regulates the actions of the ERK MAP kinase cascade by binding to ERK and altering its subcellular localization. The three-dimensional structure of PEA-15 has been determined using NMR spectroscopy and its interaction with ERK de®ned by characterization of mutants that modulate ERK function. PEA-15 is composed of an N-terminal death effector domain (DED) and a C-terminal tail of irregular structure. NMR footprinting' and mutagenesis identi®ed elements of both the DED and tail that are required for ERK binding. Comparison of the DED-binding surface for ERK2 with the death domain (DD)-binding surface of Drosophila Tube revealed an unexpected similarity between the interaction modes of the DD and DED motifs in these proteins. Despite a lack of functional or sequence similarity between PEA-15 and Tube, these proteins utilize a common surface of the structurally similar DD and DED to recognize functionally diverse targets. Keywords: death domain/MAP kinase/NMR/ three-dimensional structure IntroductionThe death effector domain (DED) is one of several small protein recognition modules that mediate the assembly of complexes required for signal transduction in programmed cell death. DEDs found in the adaptor protein FADD and the proforms of the initiator caspases, caspase-8 (FLICE, MACH) and caspase-10, play a pivotal role in the initiation of death receptor-mediated apoptosis, whereas DEDs in viral or cellular FLICE-inhibitory proteins (FLIPs) have the ability to block apoptosis (Ashkenazi and Dixit, 1998;Krammer, 2000). The DED, together with the structurally related death domain (DD) and caspase recruitment domain (CARD), are members of the death motif superfamily characterized by a conservedsix a-helix bundle structure (Aravind et al., 1999;Hofmann, 1999;Fesik, 2000). In addition to a common three-dimensional (3D) fold, these protein domains typically associate via homotypic interactions (DD±DD, DED± DED or CARD±CARD) with complementary domains in their binding partners. Surprisingly, no common protein interaction surface has been discernible for these structurally related motifs (Jeong et al., 1999;Qin et al., 1999;Xiao et al., 1999;Fesik, 2000).DED-containing proteins are involved in other cellular signaling events besides the regulation of apoptosis. For example, the phosphoprotein enriched in astrocytes (PEA-15) activates the extracellular signal receptor-activated kinases (ERK1/2), members of the MAP kinase family Formstecher et al., 2001). PEA-15 is a small protein (15 kDa) that was ®rst identi®ed as an abundant phosphoprotein in brain astrocytes (Araujo et al., 1993) and subsequently was shown to be widely expressed in human tissues and highly conserved among mammals (Danziger et al., 1995;Estelle Âs et al., 1996;Ramos et al., 1998). Several studies have shown that PEA-15 regulates multiple cellular functions including Fas-and tumor necrosis factor-a (TNF-a)-induced apopt...

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Uniform 13C/15N-Labeling of DNA by Tandem Repeat Amplification

Cited by 19 publications

References 27 publications

Site-specific labeling of nucleotides for making RNA for high resolution NMR studies using an E. coli strain disabled in the oxidative pentose phosphate pathway

Site-specific labeling of nucleotides for making RNA for high resolution NMR studies using an E. coli strain disabled in the oxidative pentose phosphate pathway

Structure of Musashi1 in a complex with target RNA: the role of aromatic stacking interactions

Recognition of ERK MAP kinase by PEA-15 reveals a common docking site within the death domain and death effector domain

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