Bledta II: Synthesis of a new tumor‐visualizing derivative of CO(III)‐bleomycin

DeRiemer, Leslie H.; Meares, Claude F.; Goodwin, David A.; Diamanti, Carol I.

doi:10.1002/jlcr.2580181017

Cited by 58 publications

(28 citation statements)

References 19 publications

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“…Gene 32 mRNA fragment was transcribed in vitro from a PCR-generated DNA template using T7 RNA polymerase and gel purified, all as described (Hüttenhofer & Noller, 1992)+ Anticodon stem-loop analogs of tRNA were also transcribed in vitro using T7 RNA polymerase+ During transcription, a phosphorothioate was introduced at the 59 end of the ASLs by transcribing in the presence of a fivefold molar excess of 59-guanosine-phosphorothioate over each NTP, essentially as described (Joseph et al+, 1997;Joseph & Noller, 1996)+ The 59-GMPS-ASLs were derivatized with Fe(II)BABE (DeRiemer et al+, 1981;Rana & Meares, 1990) and purified (Joseph et al+, 1997)+ Similarly, tRNA Phe was transcribed from plasmid p67CF10 (Sampson et al+, 1989) and derivatized with Fe(II)BABE as described (Joseph & Noller, 1996) …”

Section: In Vitro Transcription Of Gene 32 Mrna and Aslsmentioning

confidence: 99%

Calculation of the relative geometry of tRNAs in the ribosome from directed hydroxyl-radical probing data

Joseph¹,

Whirl²,

Kondo³

et al. 2000

RNA

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The many interactions of tRNA with the ribosome are fundamental to protein synthesis. During the peptidyl transferase reaction, the acceptor ends of the aminoacyl and peptidyl tRNAs must be in close proximity to allow peptide bond formation, and their respective anticodons must base pair simultaneously with adjacent trinucleotide codons on the mRNA. The two tRNAs in this state can be arranged in two nonequivalent general configurations called the R and S orientations, many versions of which have been proposed for the geometry of tRNAs in the ribosome. Here, we report the combined use of computational analysis and tethered hydroxyl-radical probing to constrain their arrangement. We used Fe(II) tethered to the 59 end of anticodon stem-loop analogs (ASLs) of tRNA and to the 59 end of deacylated tRNA Phe to generate hydroxyl radicals that probe proximal positions in the backbone of adjacent tRNAs in the 70S ribosome. We inferred probe-target distances from the resulting RNA strand cleavage intensities and used these to calculate the mutual arrangement of A-site and P-site tRNAs in the ribosome, using three different structure estimation algorithms. The two tRNAs are constrained to the S configuration with an angle of about 458 between the respective planes of the molecules. The terminal phosphates of 39CCA are separated by 23 Å when using the tRNA crystal conformations, and the anticodon arms of the two tRNAs are sufficiently close to interact with adjacent codons in mRNA.

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Section: In Vitro Transcription Of Gene 32 Mrna and Aslsmentioning

confidence: 99%

Calculation of the relative geometry of tRNAs in the ribosome from directed hydroxyl-radical probing data

Joseph¹,

Whirl²,

Kondo³

et al. 2000

RNA

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“…Preparation of BABE was done as described (10) (13). Kethoxal probing was used to control for assembly of S4 as described (7), and poly(U)-dependent binding of tRNAPhe, performed as described (22), was used as an assay for the activity of reconstituted 30S subunits.…”

Section: Methodsmentioning

confidence: 99%

“…The binding of S4 to 16S rRNA has been extensively characterized (4)(5)(6)(7)(8); it is one of six small-subunit ribosomal proteins that bind specifically to the RNA in the absence of the other proteins (9). Fe(II) is tethered to the unique cysteine residue at position 31 of protein S4 via 1-(p-bromoacetamidobenzyl)-EDTA (BABE), a reagent that has successfully been used to map intramolecular proximities in proteins (10,11). Iron-derivatized protein S4 ([Fe-Cys3t]S4) is then bound to 16S rRNA, either alone, or in a fully assembled 30S ribosomal subunit.…”

mentioning

confidence: 99%

Directed hydroxyl radical probing of 16S rRNA using Fe(II) tethered to ribosomal protein S4.

Heilek

Marusak

Meares

et al. 1995

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

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Localized hydroxyl radical probing has been used to explore the rRNA neighborhood around a unique position in the structure of the Escherichia coli 30S ribosomal subunit. Fe(II) was attached to ribosomal protein S4 at Cys-31 via the reagent 1-(p-bromoacetamidobenzyl)-EDTA. [Fe-Cys31] S4 was then complexed with 16S rRNA or incorporated into active 30S ribosomal subunits by in vitro reconstitution with 16S rRNA and a mixture of the remaining 30S subunit proteins. Hydroxyl radicals generated from the tethered Fe resulted in cleavage of the 16S rRNA chain in two localized regions of its 5' domain. One region spans positions [419][420][421][422][423][424][425][426][427][428][429][430][431][432] and is close to the multihelix junction previously placed at the RNA binding site of S4 by chemical and enzymatic protection (footprinting) and crosslinking studies. A second site of directed cleavage includes nucleotides 297-303, which overlap a site that is protected from chemical modification by protein S16, a near neighbor of S4 in the ribosome. These results provide useful information about the three-dimensional organization of 16S rRNA and indicate that these two regions of its 5' domain are in close spatial proximity to Cys-31 of protein S4.Understanding the molecular mechanism of translation depends on detailed knowledge of the three-dimensional structure of the ribosome. In the absence of an x-ray crystal structure, a wide variety of alternative biochemical and physical approaches have been devised to obtain information concerning the relative locations of ribosomal proteins and rRNA and other macromolecular components of translation in the ribosome (1). Indeed, even with a well-resolved electron density map in hand, information of this kind will likely be essential for its interpretation.In the studies presented here, we describe a biochemical method for obtaining information about the three-dimensional structure of RNA-protein complexes such as the ribosome. It consists of generating hydroxyl radicals locally from Fe(II) tethered to a single position in the ribosome, which results in cleavage of the rRNA backbone at positions that are in close proximity to the Fe(II) ion. Because of the short lifetime of hydroxyl radicals in aqueous solution, cleavage is usually restricted to positions in the RNA that are within about 10 A of the Fe(II) ion (2, 3). We use ribosomal protein S4 as a model system for these studies. The binding of S4 to 16S rRNA has been extensively characterized (4-8); it is one of six small-subunit ribosomal proteins that bind specifically to the RNA in the absence of the other proteins (9). Fe(II) is tethered to the unique cysteine residue at position 31 of protein S4 via 1-(p-bromoacetamidobenzyl)-EDTA (BABE), a reagent that has successfully been used to map intramolecular proximities in proteins (10, 11). Iron-derivatized protein S4 S4) is then bound to 16S rRNA, either alone, or in a fully assembled 30S ribosomal subunit. Hydroxyl radicals are generated after assembly of the ribonucleopro...

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“…As the preferred conformation of the ligand is important in the context of the complexation, a detailed study of the 1 …”

Section: Methodsmentioning

confidence: 99%

“…Vicinal diamines are an important class of organic compounds; they have been widely used as chelating agents in radiopharmaceuticals [1,2], as ligands in analytical chemistry, and for the synthesis of compounds with pharmaceutical importance. Compounds include precursors in the synthesis of azamacrocycles [3] and heterocyclic compounds [4], intermediates in the synthesis of analgesics.…”

Section: Introductionmentioning

confidence: 99%

Synthesis of a series of vicinal diamines with potential biological activity

Kurteva

Lyapova

2004

Open Chemistry

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A broad range of vicinal diamines based on styrene oxide are synthesised via mixtures of regioisomeric amino alcohols. The ring opening of the intermediate aziridinium ions by primary amines proceeds with high regioselectivity, leading to the target diamines as single regioisomers for all reaction series. The compounds are of potential biological interest as ligands for cisplatin analogues. Anticancer activity tests of both groups of compounds are in progress.

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Bledta II: Synthesis of a new tumor‐visualizing derivative of CO(III)‐bleomycin

Cited by 58 publications

References 19 publications

Calculation of the relative geometry of tRNAs in the ribosome from directed hydroxyl-radical probing data

Calculation of the relative geometry of tRNAs in the ribosome from directed hydroxyl-radical probing data

Directed hydroxyl radical probing of 16S rRNA using Fe(II) tethered to ribosomal protein S4.

Synthesis of a series of vicinal diamines with potential biological activity

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