Two-dimensional NMR and molecular dynamics simulations have been used to determine the three-dimensional structures of two hairpin DNA structures: d-CTAGAG GATCCUTTTGGATCCT (abbreviated as U1-hairpin) and d-CTAGAGGATCCTTUTGGATCCT (abbreviated as U3-hairpin). The 1 H resonances of both of these hairpin structures have been assigned almost completely. NMR restrained molecular dynamics and energy minimization procedures have been used to describe the three-dimensional structures of these hairpins. This study and concurrent NMR structural studies on two other d-CTAGAGGA TCCTUTTGGATCCT (abbreviated as U2-hairpin) and d-CTAGAGGATCCTTTUGGATCCT (abbreviated as U4-hairpin) have shed light upon various interactions reported between Echerichia coli uracil DNA glycosylase (UDG) and uracil-containing DNA. The backbone torsion angles, which partially influence the local conformation of U12 and U14 in U1 and U3-hairpins, respectively, are probably locked in the trans conformation as in the case of U 13 in the U2-hairpin. Such a stretched-out backbone conformation in the vicinity of U 12 and U 14 is thought to be the reason why the K m value is poor for U1-and U3-hairpins as it is for the U2-hairpin. Furthermore, the bases U 12 and U 14 in both U1-and U3-hairpins adopt an anti conformation, in contrast with the base conformation of U 13 in the U2-hairpin, which adopts a syn conformation. The clear discrepancy observed in the U-base orientation with respect to the sugar moieties could explain why the V max value is 10-to 20-fold higher for the U1-and U3-hairpins compared with the U2-hairpin. Taken together, these observations support our interpretation that the unfavourable backbone results in a poor K m value, whereas the unfavourable nucleotide conformation results in a poor V max value. These two parameters therefore make the U1-and U3-hairpins better substrates for UDG compared with the U2-hairpin, as reported earlier [Kumar, N.

Two-dimensional NMR and molecular dynamics simulations have been used to determine the three-dimensional structures of two hairpin DNA structures: d-CTAGAG GATCCUTTTGGATCCT (abbreviated as U1-hairpin) and d-CTAGAGGATCCTTUTGGATCCT (abbreviated as U3-hairpin). The 1 H resonances of both of these hairpin structures have been assigned almost completely. NMR restrained molecular dynamics and energy minimization procedures have been used to describe the three-dimensional structures of these hairpins. This study and concurrent NMR structural studies on two other d-CTAGAGGA TCCTUTTGGATCCT (abbreviated as U2-hairpin) and d-CTAGAGGATCCTTTUGGATCCT (abbreviated as U4-hairpin) have shed light upon various interactions reported between Echerichia coli uracil DNA glycosylase (UDG) and uracil-containing DNA. The backbone torsion angles, which partially influence the local conformation of U12 and U14 in U1 and U3-hairpins, respectively, are probably locked in the trans conformation as in the case of U 13 in the U2-hairpin. Such a stretched-out backbone conformation in the vicinity of U 12 and U 14 is thought to be the reason why the K m value is poor for U1-and U3-hairpins as it is for the U2-hairpin. Furthermore, the bases U 12 and U 14 in both U1-and U3-hairpins adopt an anti conformation, in contrast with the base conformation of U 13 in the U2-hairpin, which adopts a syn conformation. The clear discrepancy observed in the U-base orientation with respect to the sugar moieties could explain why the V max value is 10-to 20-fold higher for the U1-and U3-hairpins compared with the U2-hairpin. Taken together, these observations support our interpretation that the unfavourable backbone results in a poor K m value, whereas the unfavourable nucleotide conformation results in a poor V max value. These two parameters therefore make the U1-and U3-hairpins better substrates for UDG compared with the U2-hairpin, as reported earlier [Kumar, N.

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.

No abstract