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The presence and synthesis of transfer RNA (tRNA) is highly conserved in all organisms and a lot of genetic material is dedicated to its synthesis. tRNA contains a large number of modified nucleosides and several diverse functions have been found but much about their function is still unknown. By using a novel frameshifting system to select for tRNA modification mutants, new mutations were isolated and subsequently analyzed. This thesis examines the synthesis and function of a subset of tRNA modifications that have a sulfur (thio) -group as part of the modification. The isc operon encodes for proteins synthesizing iron sulfur centers ([Fe-S]) that are a part of the active site of many key enzymes in the cell and the thiolated nucleosides are dependant on a functional iron sulfur gene (iscS) for their synthesis. By studying thiolated tRNA it is not only possible to learn more about the synthesis of the modifications themselves, but also about the synthesis of [Fe-S] clusters. Based on an analysis of mutations in three of the isc operon genes (iscS, iscU, and iscA), a two-model pathway is proposed for the synthesis of Salmonella enterica Serovar Typhimurium thiolated tRNA modifications. The interactions of IscS with other proteins in the tRNA modification thiolation pathways suggest a more complex sulfur relay than had previously been envisioned. Some of the specificities and the effect of an iscA mutant on the levels of tRNA modifications lead to an examination of the role of IscA in [Fe-S] formation and its importance for tRNA modifications.