Interaction of tRNAs and of phosphorothioate-substituted nucleic acids with an organomercurial. Probing the chemical environment of thiolated residues by affinity electrophoresis

Abstract

The interactions of 4-thiouridine and 5-[(methylamino)methyl]-2-thiouridine in tRNA and of phosphorothioate esters in nucleic acids with an organomercurial have been investigated. For this purpose, an affinity electrophoretic system has been developed in which the mercury derivative has been covalently immobilized in a standard polyacrylamide gel. The retardation of thiolated macromolecules was found to be sensitive to the chemical environment of the sulfur atom, giving characteristic interaction constants dependent on the nature of the modification and its accessibility to binding. The interaction could, in the case of tRNA, be abolished by conventional specific chemical modification of the thiolated bases, as well as by irradiation with 32P-derived beta-emission. Not only has the fractionation of sulfur-modified from unmodified species been attained but a quantitative application of the technique has made it possible to study the binding of mercury and, by competition, that of magnesium in terms of the conformation of tRNA.

The conserved Wobble uridine tRNA thiolase Ctu1–Ctu2 is required to maintain genome integrity

“…To see whether the 2-thiolation occurred specifically on tRNA LYS , we used the APM/PAGE band shift method, which relies on the strong retardation of thiolated tRNA in electrophoresis because of the affinity of the thio group with the mercuric compound (APM) in the gel (22). These experiments showed that the tRNA LYS , but not the tRNA MET , which has a C at position 34, was thiolated in vivo in S. pombe and C. elegans and that the thiolation was absent in a ctu1 mutant from either species (Fig.…”

Substrate Specificity for 4-Thiouridine Modification in Escherichia coli

“…T7 RNA polymerase was purified from strain BL21(pAR219), which was a generous gift of F. Studier. Acryloyl aminophenylmercury(II) chloride (APM) for affinity electrophoresis was prepared as described previously (31) and used as a stock solution of 1 mg/ml in formamide. L-[ 35 S]Cysteine was obtained from PerkinElmer Life Sciences.…”

“…APM Affinity Gel Assays with Sulfide as Substrate-For reactions with sulfide as sulfur source, a gel shift assay using APM polyacrylamide gels was used based on the work of Igloi (31). This technique incorporates APM into the polyacrylamide gel.…”

“…In vitro, we find that sulfide is an excellent source of sulfur for s 4 U synthesis but only at millimolar concentrations. We used 32 P-labeled tRNA Phe as substrate and measured the fraction of modified tRNA using affinity electrophoresis (31) and PhosphorImager analysis. Reaction times were 2 min in the linear range of the progress curve.…”

Inactivation of the Ribozyme with Fluorescent Dyes

“…2). [9][10][11] The mobility of RNA that carry thiophosphate monoester is diminished, compared with non-thiophosphated one. This is the evidence of the strong interaction between mercury and sulfur.…”