The Interactions of 2‐Thiopyrimidine Bases with Hydroxymercurybenzene Sulfonate

Scheit, K. H.; Faerber, Peter

doi:10.1111/j.1432-1033.1973.tb02714.x

Cited by 15 publications

(16 citation statements)

References 10 publications

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“…The assay was measured for the incorporation of 35 S from L-[ 35 S]cysteine into tRNA that was specifically bound on the discs, and the rates were assessed in the linear range of the progress curve at Ͻ10% of the product formation. Under these conditions, unmodified tRNA Phe gave an initial rate constant (k obs ) of 1.0 min Ϫ1 at 20 M (Table I).…”

Section: Resultsmentioning

confidence: 99%

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Substrate Specificity for 4-Thiouridine Modification in Escherichia coli

Lauhon

Erwin

Ton

2004

Journal of Biological Chemistry

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The biosynthesis of 4-thiouridine (s 4 U) in Escherichia coli tRNA requires the action of both the thiamin pathway enzyme ThiI and the cysteine desulfurase IscS. IscS catalyzes sulfur transfer from L-cysteine to ThiI, which utilizes Mg-ATP to activate uridine 8 in tRNA and transfers sulfur to give s 4 U. In this work, we show through deletion analysis of unmodified E. coli tRNA Phe that the minimum substrate for s 4 U modification is a mini-helix comprising the stacked acceptor and T stems containing an internal bulged region. The size of the bulged loop must be at least 4 nucleotides and contain the target uridine as the first nucleotide. Replacement of the T loop sequence with a tetraloop in the deletion substrate increases activity and shows that the T⌿C primary sequence is not a recognition element. An unmodified tRNA Phe transcript in which the 3 -terminal ACCA sequence is removed to give a blunt terminus has <0.1% activity, although the addition of a single overhanging base essentially restores activity. In addition, reducing the distance of the 3 terminus relative to U8 by as little as 1 bp severely impairs activity. By dissecting a minimal RNA substrate in the T loop region, a two-piece system consisting of a substrate RNA and a "guide" RNA is efficiently modified. Our results indicate that outside of the modified U8, there is no primary sequence requirement for substrate recognition. However, the secondary and tertiary structure restrictions appear sufficient to explain why s 4 U modification is limited in the cell to tRNA.

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Section: Resultsmentioning

confidence: 99%

“…This assay measures the incorporation of 35 4 U formation at 10 and 20 M were the same within error. Reactions were allowed to proceed for 6 min at 37°C.…”

Section: Methodsmentioning

confidence: 99%

Substrate Specificity for 4-Thiouridine Modification in Escherichia coli

Lauhon

Erwin

Ton

2004

Journal of Biological Chemistry

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“…1) has four mercury atoms bonded to a central carbon producing a compact structure (4) having 326 electrons in a sphere of -3.5 A radius. Mercury is known to bind strongly and selectively to the sulfur of 4-thiouridine residues in various Escherichia coli tRNAs (5)(6)(7)(8) and to poly[d(A-s4T)] (9), poly(s2C), and poly(s2U) (10). As demonstrated by the present work, TAMM is soluble at neutral pH in the presence of N,N-dimethyl-2-aminoethanethiol.…”

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confidence: 50%

Tetrakis(acetoxymercuri)methane: a polymetallic reagent for labeling sulfur in nucleic acids.

Strothkamp¹,

Lehmann²,

Lippard³

1978

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

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Tetrakis(acetoxymercuri)methane binds to the sulfur atom of 6-thioguanosine and also to the 4-thiouridine residue of Escherichia coli tRNAVal. A 4:1 complex is formed between 6-thioguanosine and tetrakis(acetoxymercuri)methane. Addition of 3 equivalents of NN-dimethyl-2-aminoethanethiol hydrochloride to tetrakis(acetoxymercuri)methane effectively blocks three of the four mercury atoms, rendering the compound monofunctional toward 6-thioguanosine. Under appropriate conditions, tetrakis(acetoxymercuri)methane, in the presence or absence of NN-dimethyl-2-aminoethanethiol hydrochloride, binds to the 4-thiouridine residue in E. coli tRNAval without forming intermolecular crosslinks. These results suggest that tetrakis(acetoxymercuri)methane will be a useful polymetallic reagent for labeling sulfur sites in polynucleotides. It may also prove to be a valuable reagent for preparing heavy metal derivatives of proteins for x-ray crystallographic study. The selective attachment of heavy metal atoms to nucleic acids can facilitate their study by electron microscopy (1). Recently, platinum was shown to bind selectively to the sulfur atoms of phosphorothioate groups enzymatically incorporated into a polynucleotide adjacent to specific bases (2). This method of achieving selective labeling has the advantage that the same chemical group can serve as the heavy metal binding site at any of the bases. Moreover, it is possible to use different metal atoms, provided that they have a high affinity for sulfur.A polymetallic reagent would, by virtue of its greater electron content, be more readily detected in the electron microscope than a single metal atom. The desirable properties of such a reagent are that it (i) show a strong preference for binding sulfur, (ii) be stable in aqueous solution near neutral pH, and (iii) have only one reactive metal atom to prevent crosslinking. The compound tetrakis(acetoxymercuri)methane (TAMM) (3, 4) appears to meet these requirements. This compound (Fig.

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“…The other potential donor atoms of nucleic acids all have more class a (12), or "hard" (13), character compared to sulfur. The selectivity of heavy metals for sulfur in a polynucleotide has been previously demonstrated by the specific binding of mercury to the 4-thiouridine residues of several Escherichia coli tRNAs (14)(15)(16) and the thiolated nucleosides of polyd(A-s4T) (17), poly(s2C), and poly(s2U) (18). The present report describes the platinum binding properties of the phosphorothioate group in mono-and polynucleotides.…”

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confidence: 93%

“…Glassware was cleaned with chromic acid, rinsed thoroughly with deionized water, allowed to dry at room temperature, and then baked at 3000 for at least 18 preparing its solutions. Because of uncertainty about the fourth ligand in the coordination sphere-water, hydroxide or nitrate all being possibilities-this compound will be referred to as "platinum terpyridine nitrate".…”

mentioning

confidence: 99%

Platinum binds selectively to phosphorothioate groups in mono- and polynucleotides: a general method for heavy metal staining of specific nucleotides.

Strothkamp¹,

Lippard²

1976

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

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Platinum binding to nucleoside phosphorothioates has been examined to determine their suitability as heavy metal labeling sites for the potential electron microscopic sequencing of nucleic acids. The complex platinum terpyridine nitrate forms a 1:1 adduct with either adenosine or uridine monophosphorothioate. Spectroscopic evidence strongly indicates the presence of platinum-sulfur bonds. Both platinum terpyridine nitrate and chloroterpyridineplatinum(II) bind to poly(sA-U), a polymer prepared from adenosine 5'-O-(1-thiotriphosphate) and UTP. Binding to the sulfur atoms of the phosphorothioate groups is quantitative, as shown by double label experiments using [35S]poly(sA-U) and [3H]chloroterpyridine-platinum(II). Similar experiments with [14C]poly(A-U) indicated no platinum binding. No evidence of nicking or loss of sulfur from poly(sA-U) could be detected after platinum binding. The phosphorothioate group is a strong, highly selective binding site for platinum in polynucleotides. Previous studies have demonstrated quantitative enzymatic incorporation of phosphorothioate groups into a polynucleotide adjacent to a specific base [Matzura, H. & Eckstein, F. (1968) Eur. J. Biochem. 3, 448-452]. The use of heavy metal-labeled phosphorothioate groups for the sequencing of nucleic acids by electron microscopy therefore appears feasible.

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The Interactions of 2‐Thiopyrimidine Bases with Hydroxymercurybenzene Sulfonate

Cited by 15 publications

References 10 publications

Substrate Specificity for 4-Thiouridine Modification in Escherichia coli

Substrate Specificity for 4-Thiouridine Modification in Escherichia coli

Tetrakis(acetoxymercuri)methane: a polymetallic reagent for labeling sulfur in nucleic acids.

Platinum binds selectively to phosphorothioate groups in mono- and polynucleotides: a general method for heavy metal staining of specific nucleotides.

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