Organization and copy number of initiator tRNA genes in slow- and fast-growing mycobacteria

We have previously reported the isolation and characterization of a functional initiator tRNA gene, metA, and a second initiator tRNA-like sequence, metE, from Mycobacterium tuberculosis. Here we describe the fine mapping of the initiator tRNA gene locus of the avirulent (H37Ra) and virulent (H37Rv) strains of M. tuberculosis. The genomic blot analyses show that the 1.7 kb (harbouring metE) and the 6.0 kb Bamffi (harbouring metA) fragments are linked. Further, sequencing of a portion of the 6.0 kb fragment, in conjunction with the sequence of the 1.7 kb fragment confirmed the presence of an IS6110 element in the vicinity of metE. The IS element is flanked by inverted (28 bp, with 3 contiguous mismatches in the middle) and direct (3 bp) repeats considered to be the hallmarks of IS6110 integration sites. The organization of the initiator tRNA gene locus is identical in both the H37Ra and H37Rv strains and they carry a single copy of the functional initiator tRNA gene. Interestingly, the fast growing Mycobacterium smegmatis also bears a single initiator tRNA gene. This finding is significant in view of the qualitative differences in total tRNA poolsand the copy number of rRNA genes in the fast- and slow-growing mycobacteria. Finally, we discusshypotheses related to the origin of metE in M. tuberculosis. 1