Molecular flexibility of Mycobacterium tuberculosis ribosome recycling factor and its functional consequences: An exploration involving mutants.

Internal mobility of the two domain molecule of ribosome recycling factor (RRF) is known to be important for its action. Mycobacterium tuberculosis RRF does not complement E. coli for its deficiency of RRF (in the presence of E. coli EF-G alone). Crystal structure had revealed higher rigidity of the M. tuberculosis RRF due to the presence of additional salt bridges between domains. Two inter-domain salt bridges and one between the linker region and the domain containing C-terminal residues were disrupted by appropriate mutations. Except for a C-terminal deletion mutant, all mutants showed RRF activity in E. coli when M. tuberculosis EF-G was also co-expressed. The crystal structures of the point mutants, that of the C-terminal deletion mutant and that of the protein grown in the presence of a detergent, were determined. The increased mobility resulting from the disruption of the salt bridge involving the hinge region allows the appropriate mutant to weakly complement E. coli for its deficiency of RRF even in the absence of simultaneous expression of the mycobacterial EF-G. The loss of activity of the C-terminal deletion mutant appears to be partly due to the rigidification of the molecule consequent to changes in the hinge region.

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 discuss hypotheses related to the origin of metE in M. tuberculosis.

Rapid and reliable site directed mutagenesis using Kunkel's approach.

Oligonucleotide based site directed mutagenesis (SDM) is an invaluable technique in molecular biology. Among the various methods developed for SDM, the PCR-based approach, Kunkel's and the Eckstein's procedures are widely used. The Kunkel's method, on account of its cost effectiveness and simplicity, is preferred by many a scientist. However, a general drawback of this method is the high background due to persistence of the parent template resulting in low efficiency of mutagenesis. In this report, we describe a modification of the Kunkel's method to increase the efficiency of selecting against the wild type strand. We have used Sequenase for the extension reaction, and introduced an in vitro UDG step to enhance the biological selection against the parent strand. Consequently, the efficiency of the modified method is enhanced to allow screening of the mutants directly by DNA sequencing. A step by step single tube protocol which is over in less than three hours makes it a method of choice for efficient and cost-effective site directed mutagenesis.