| Abstract
| - We have improved the in vitro assay for 4-thiouridine (s4U) biosynthesis in EscherichiacolitRNA by substituting an unmodified tRNA transcript as substrate and including recombinant ThiI protein,a known factor required for s4U synthesis. Using this assay, we have purified an enzyme from wild-typeE. coli that is able to provide sulfur for s4U synthesis in vitro. The purified protein has a molecularweight of 45 kDa and contains pyridoxal phosphate as a cofactor. This protein catalyzes sulfur transferfrom cysteine to tRNA and is analogous to factor C previously reported (Lipsett, M. N. (1972) J. Biol.Chem. 247, 1458−1461). UV spectroscopy and HPLC analysis of thiolated tRNA and its digests confirmthat the product of the in vitro reaction is s4U. N-Terminal sequence analysis of the purified proteinidentifies it as IscS, a recently characterized NifS-like cysteine desulfurase that mobilizes sulfur for thesynthesis of [Fe−S] clusters. We have cloned and overexpressed iscS and show that the recombinantprotein displayed tRNA sulfurtransferase activity equal to that of the native protein. We also show that,of the multiple proteins in E. coli with cysteine desulfurase activity as observed by native gel staining,only IscS is able to mobilize the sulfur for transfer to tRNA. Our identification of IscS as a tRNAsulfurtransferase provides support for this activity in vivo and further expands the role for NifS proteinsas versatile sulfur-carrying enzymes.
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