We propose to purify and characterize the transfer RNA (tRNA) sulfurtransferases of Eschrichia coli. These enzymes catalyze the transfer of sulfur from cysteine to specific nucleotide acceptors during the processing of precursor tRNA. A major obstacle to purifying these enzymes previously was the lack of a proper substrate. We will overcome this diffuculty by the use of tRNA which is lacking the specific thionucleotide for each sulfurtransferase. The pure tRNA substrates will be isolated from sulfurdeficient tRNA mixtures, produced by E. coli HfrC, re1A, cys-Met-, Lamda after cysteine starvation. For example, the substrate for the 4-thiouridine and 2-methylthio-N-isopentenyladenosine sulfurtransferases will be tRNAtyr, purified by aminoacylation followed by BD-cellulose chromatography. the substrates for the 2-thiocytidine and 5-methylaminomethyl-2-thiouridine sulfurtransferases will be tRNSarg and tRNAglu, respectively. These substrates will be aminoacylated, a napthoxyacetyl grup attached to the aminoacyl-tRNA, and the derivatized aminoacyl-tRNA is separated from the bulk of free tRNA by chromatography on BD-cellulose. We will determine the missing thionucleotide for each of these substrates. Purification of the sulfurtransferases will be carried out using a novel affinity matrix, sulfur-deficient tRNA-Sepharose. This column will separate the sulfurtransferases from proteins which do not bind to tRNA. Final purification and resolution of the specific sulfurtransferases will be carried out by ion-exchange methods such as phosphocellulose of DEAE-cellulose chromatography. Specific sulfurtransferases will be detected by measuring sulfur transfer from [35S]cysteine to the tRNA isoacceptor lacking that thionucleotide. Our purification scheme utilizes a minimum number of steps and methods which have been successful in purifying other tRNA modification enzymes. Once the sulfurtransferases have reached a suitable stage of purification we can determine the required reaction components and kinetic parameters which each substrate. We will identify all of the reaction products and determine the mechanism of the sulfur transfer reaction. Activators and inhibitors of the enzymes will be sought and the regulation of enzyme activity will be investigated. Finally, functional changes in the underthiolated tRNA substrates before and after sulfurtransferase treatment will be studied. In this way, we may obtain information on the role of thionucleotides in tRNA, in addition to information about a previously uncharacterized group of enzymes.
| Harris, C L; Kolanko, C J (1995) Aminoacyl-tRNA synthetase complex in Saccharomyces cerevisiae. Biochem J 309 ( Pt 1):321-4 |
| Harris, C L (1990) High-molecular-weight forms of aminoacyl-tRNA synthetases and tRNA modification enzymes in Escherichia coli. J Bacteriol 172:1798-803 |
| Harris, C L; Kolanko, C J (1989) A rapid diethylaminoethyl paper disk assay for transfer RNA sulfurtransferase. Anal Biochem 176:57-62 |
| Harris, C L (1987) An aminoacyl-tRNA synthetase complex in Escherichia coli. J Bacteriol 169:2718-23 |
