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Characterization of the initiator tRNA gene locus and identification of a strong promoter from Mycobacterium tuberculosis

Vasanthakrishna, M and Kumar, NV and Varshney, U (1997) Characterization of the initiator tRNA gene locus and identification of a strong promoter from Mycobacterium tuberculosis. In: Microbiology, 143 (11). pp. 3591-3598.

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An initiator tRNA gene, metA, and a closely linked fragment of a second initiator-tRNA-like sequence, metB, from Mycobacterium tuberculosis H37Ra have been cloned and characterized. The promoter region of metA shows the presence of conserved sequence elements, TAGCCT and TTGGCG, with resemblance to -10 and -35 promoter regions. The deduced sequence of the mature tRNA contains the three unique features of the eubacterial initiator tRNAs represented by (i) a C:U mismatch at position 1:72, (ii) three consecutive base pairs, 29-31G:C39-41 in the anticodon stem, and (iii) a purine:pyrimidine (A:U) base pair at position 11:24 in the dihydrouridine stem. A putative hairpin structure consisting of an 11 bp stem and a three-base loop found in the 3' flanking region is followed by a stretch of T residues and may serve as a transcription terminator. Analysis of the expression of metA and of its promoter using chloramphenicol acetyltransferase fusion constructs in Mycobacterium smegmatis shows that metA is a functional gene driven by a strong promoter. Furthermore, the overexpressed transcripts are fully processed and formylated in vivo. The metB clone shows the presence of sequences corresponding to those downstream of position 30 of the tRNA. However, the CCA sequence at the 3' end has been mutated to CCG. Interestingly, the 3' flanking sequences of both the genes are rich in CCT repeats. The metB locus also harbours a repeat element, IS6110. A method to prepare total RNA from mycobacteria (under acidic conditions) to analyse in vivo status of tRNAs is described.

Item Type: Journal Article
Publication: Microbiology
Publisher: Society for General Microbiology
Additional Information: Copyright of this article belongs to Society for General Microbiology.
Keywords: Mycobacteria;initiator tRNA;promoter;repeats;IS6110.
Department/Centre: Division of Biological Sciences > Molecular Reproduction, Development & Genetics
Date Deposited: 13 Mar 2009 08:40
Last Modified: 19 Sep 2010 05:24
URI: http://eprints.iisc.ac.in/id/eprint/18683

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