MODIFIED NUCLEOTIDES IN BACILLUS-SUBTILIS TRANSFER RNA(TRP) HYPEREXPRESSED IN ESCHERICHIA-COLI

In the present study, modified nucleotides in the B.subtilis tRNA(Trp) Cloned and hyperexpressed in E. coli have been identified by TLC and HPLC analyses. The modification patterns of the two isoacceptors of cl oned B.subtilis tRNA(Trp) have been compared with those of native tRNA (Trp) from B.subtilis and from E.coli. The modifications of the A73 mu tant of B.subtilis tRNA(Trp), which is inactive toward its cognate Trp RS, were also investigated. The results indicate the formation of the modified nucleotides s4U8, Gm18, D20, Cm32, i6A/ms2i6A37, T54 and PSI5 5 on cloned B.subtilis tRNA(Trp). This modification pattern resembles the pattern of E.coli tRNA(Trp), except that m7G is missing from the c loned tRNA(Trp), probably on account of its short extra loop. In contr ast, the pattern departs substantially from that of native B.subtilis tRNA(Trp). Therefore, the cloned B.subtilis tRNA(TrP) has taken on lar gely the modification pattern of E.coli tRNA(Trp) despite the 26% sequ ence difference between the two species of tRNA, gaining in particular the Cm32 and Gm18 modifications from the E.coli host. A notable diffe rence between the isoacceptors of the cloned tRNA(Trp) was seen in the extent of modification of A37, which occurred as either the hypomodif ied i6A or the hypermodified MS2i6A form. Surprisingly, base substitut ion of guanosine by adenosine at position 73 of the cloned tRNA(Trp) h as led to the abolision of the 2'-O-methylation modification of the re mote G18 residue.