Cloning and restriction mapping of the trmA gene coding for transfer ribonucleic acid (5-methyluridine)-methyltransferase in Escherichia coli K-12

Abstract

A hybrid plasmid from the Clarke and Carbon collection was isolated. This plasmid carries the trmA gene of E. coli, which is necessary for the formation of 5-methyluridine (m5U, ribothymidine) present in all tRNA chains of the organism so far sequenced. A restriction map of the argCBH-trmA regions is presented. By using cloning in vitro, the trmA gene was located on a 2.9-kilobase pair DNA fragment. These results and comparison with .lambda.dargECBH transducing phages established the gene order: argECBH trmA bfe in the 88 min region of the E. coli chromosomal map. Plasmids carrying this 2.9 kilobase pair DNA fragment overproduce the enzyme tRNA(M5U)methyltransferase (EC 2.1.1.35) 20-40 times. When this 2.9 kilobase pair chromosomal DNA fragment was expressed in a minicell system, a polypeptide of a MW of 42,000 was synthesized. This polypeptide was tentatively identified as the tRNA(m5U)methyltransferase. These results support the earlier suggestion that the trmA gene is the structural gene for the tRNA(m5U)methyltransferase.