Although named for a role in the production of thiamin, the enzyme ThiI catalyzes the transfer of sulfur originating in cysteine to uridine-8 of tRNA to make 4-thiouridine, which acts as a photosensor for near -UV light in bacteria. Absorption of near –UV light triggers the crosslinking of 4-thiouridine and cytidine-13, which prevents an amino acid attachment to tRNA. The resulting stalled ribosomes activate the stringent response, which causes growth arrest, a protective mechanism to prevent mutation induced by UV light. This process is the basis of a UV screening method that allows assessment of ThiI function (whether it can produce the photosensor). The two proposed mechanisms for s4U generation both invoke persulfide formation on Cys-456 of ThiI by transfer of sulfur by the enzyme IscS. Both mechanisms also include unspecified general acid and base residues, which this work seeks to identify. Sequence alignments suggest five candidate amino acid residues for the mechanistically important acid/base groups. The polymerase chain reaction (PCR) with primers mismatched at the codons of interest allows site-specific mutation within the plasmid encoding ThiI. The mutagenesis has been completed for the five positions, as confirmed by DNA sequencing. After transformations into thiI- E coli, UV screenings allow determination of a position’s significance in ThiI function. Inactive variants do not make s4U and lack near –UV sensitivity because no s4U is made. After critical residues are identified, the altered ThiI will be overexpressed. Structural integrity can be assessed using CD spectroscopy and in vitro activity assays that monitor the incorporation of 35S into RNA will be employed to measure the activity of the variants. Through the use of mutagenesis, UV screening, and protein overexpression, the mechanistically important acid/base residues will be found.

Identification of acid/base residues important to the mechanism of ThiI

249th National Meeting (2015)

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