Dr. V. NAGARAJA
Professor
Ph.D. (Indian Institute of Science, Bangalore. India)
Room No. 351-A
Phone: 293 2598; 360 0668
E_mail :vraj@mcbl.iisc.ernet.in
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Our research interests continue to be in the area of protein: nucleic acid interactions and structure-function studies with the proteins involved in such interactions. The major molecular events investigated are enzymology of DNA supercoiling, regulation of gene expression and R-M systems.

DNA gyrase and topoisomerase I from mycobacteria: The function of DNA topo-isomerases is essential for cellular processes such as replication, transcription, recombination and chromosomal segregation. Contrasting activities of different kinds of topoisomerases ensure to regulate the levels of supercoiling inside the cells. One of our major thrusts is to understand the structure, function and in vivo roles of different topoisomerases in mycobacteria. Both type I (topoisomerase I) and type II (DNA gyrase) enzymes are being studied in this context and also to develop strategies to design new therapeutics. DNA gyrase from M. smegmatis is shown to be an efficient decatenase suggesting a role in DNA segregation. In addition to unraveling the unique features of transcriptional regulation, we demonstrate that gyr operon mRNA is highly stable, a characteristic correlating well with growth rate and other macromolecular events of the organism.  Based on comparative studies, we have classified DNA gyrases into two groups.  These studies have culminated into development of peptide inhibitors specific to mycobacterial gyrase with a novel mechanism of action. Further, GyrI, a chromosomally encoded inhibitor of DNA gyrase of E. coli has been characterized. The protein appears to be a repair factor against chromosomal lesions caused by gyrase poisons.

Previously, we demonstrated the unique properties of mycobacterial topoisomerase I, distinct from other prokaryotic and eukaryotic topoisomerases. The enzyme lacks the zinc fingers characteristic of other type IA topoisomerases. High-resolution foot printing studies have revealed that mycobacterial topoisomerase I contacts a much larger substrate DNA and interacts in an asymmetric fashion.  After nicking the DNA, the enzyme holds DNA sequences on either side of the nick, thus following “enzyme bridged” mechanism for DNA relaxation. Enzyme contains two distinct DNA binding domains for specific and non-specific interactions. The activities of type IA topoisomerases are stimulated by SSB without direct protein: protein interactions.  The latter observations highlight the importance of functional cooperation between different DNA transaction proteins.

Biology of R-M systems: In addition to isolating new restriction enzymes, we have carried out detailed molecular interaction studies with KpnI restriction endonuclease and methyltransferase.  These enzymes are dimeric and bind to the same target sequence in contrasting fashion. These differences in the recognition pattern reflect the different chemistry employed in their reactions.

Regulation of gene expression-transcription termination: Intrinsic transcription termin-ation signals provide an extremely economical mechanism for the termination since they can function in the absence of any proteins. The text book definition of transcription terminators is based on E. coli paradigm.  Now, we have demonstrated using both in silico and experi-mental approach that intrinsic terminators of various types exist and function in mycobacteria. We have developed an efficient, versatile algorithm, GeSTer, for finding terminators in genomewide search.  In addition to the classical E. coli type terminators, the algorithm identifies several new kinds of structures as putative terminators. By in vitro and in vivo experiments, these are shown to function as effective terminators. The different kinds of terminators are concentrated within the first 50 nucleotides downstream of coding region in most bacterial genomes pointing to the conserved mechanism.  Now, we are in the process of elucidating the process of factor dependent termination and the possible connection between the two mechanisms.

Regulation of mom gene expression: I had presented a model for a novel mechanism of transcription activation of mom gene of phage Mu by transactivator C protein. The model is now verified by experiments which demonstrate asymmetric unwinding of promoter elements by C protein. Reorientation of promoter elements by C mediated unwinding leads into polymerase recruitment to the promoter. We also show that an intrinsic DNA distortion in the spacer region of the promoter acts in cis as a negative element in mom operon transcription.  The C protein has 3-helix bundle helix turn helix motif with an architecture similar to NarL family of proteins and prd homeodomain of Drosophila. The direct interaction of the protein with transcription machinery is being investigated to elucidate its role in subsequent steps of promoter: polymerase interactions in influen-cing transcription activation.

To conclude, different types of "DNA transaction" processes which play key roles in important cellular functions, are being investig-ated. These studies form the basis for our understanding of many molecular events and open up avenues for specific applications.

 

 

SELECTED PUBLICATIONS

Sikder, D., Unniraman, S., Bhaduri, T. and Nagaraja, V. (2001) Functional cooperation between topoisomerase I and single strand DNA binding protein. J. Mol. Biol. 306, 669-679.

Sikder, D. and Nagaraja, V. (2001) A novel bipartite mode of binding of M. smegmatis topoisomerase 1 to its recognition sequence.   J. Mol. Biol. 312, 347-357.

Unniraman, S., Prakash, R. and Nagaraja, V. (2001) Alternate paradigm for intrinsic transcription termination in eubacteria. J. Biol. Chem. 276, 41850-41855.

Basak, S. and Nagaraja, V. (2001) DNA unwinding mechanism for the transcriptional activation of momP1 promoter by the transactivator protein C of bacteriophage Mu. J. Biol. Chem. 276, 46941-46945.

Chatterji, M. and Nagaraja, V. (2002) GyrI, a counter defensive strategy against protein-aeous inhibitors of DNA gyrase. EMBO Rep. 3, 261-267.

Manjunatha, U. H., Dalal, M., Chatterji, M., Radha, D. R., Visweswariah, S. S. and Nagaraja, V. (2002) Functional characteri-zation of Mycobacterium smegmatis DNA gyrase: a potent decatenase. Nucleic Acids Res. 30, 2144-2153.