RIT1, A TRANSFER-RNA BACKBONE-MODIFYING ENZYME THAT MEDIATES INITIATOR AND ELONGATOR TRANSFER-RNA DISCRIMINATION

Using a genetic screen in yeast aimed at identifying cellular factors involved in initiator and elongator methionine tRNA discrimination in the translational process, we have identified a mutation that abolish the requirement for elongator methionine tRNA. The gene affected, whic h we call the ribosylation of the initiator tRNA gene or RIT1, encodes a 2'-O-ribosyl phosphate transferase. This enzyme modifies exclusivel y the initiator tRNA in position 64 using 5-phosphoribosyl-1'-pyrophos phate as the modification donor. As the initiator tRNA participates bo th in the initiation and elongation of translation in a rit1 strain, w e conclude that the 2'-O-ribosyl phosphate modification discriminates the initiator tRNAs from the elongator tRNAs during protein synthesis. The modification enzyme was shown to recognize the stem-loop IV regio n that is unique in eukaryotic cytoplasmic initiator tRNAs.