1) Mechanism of transcription regulation. Adenine at -11 position of a TATA element a promoter is critical and sufficient to signal base pair deformation. The conserved A: T base pair at the -11 position of the promoters in Escherichia coli is very sensitive to substitutions. In vitro transcription with the galP1 promoter having a natural or unnatural base in either strand at position -11 showed that only a purine base with no side group at C2 in the nontemplate strand is transcriptionally potent; neither a purine with amino group at C2 nor a pyrimidine support transcription. The amino group at C6 in the omnipresent adenine at -11 does not play any role in promoting transcription. The nature of the base, complementary or noncomplementary, at -11 in the template strand also does not influence transcription. We proposed that the adenine, by becoming extrahelical, interacts with an amino acid(s) of the 2.3-2.4 region of sigma for which an unsubstituted C2 hydrogen is critical. 2) Effect of varying supercoiling on transcription and its regulation. The effect of superhelicity of DNA templates on transcription is well documented in several cases. However, the amount of supercoiling that is needed to bring about any changes and the steps at which such effects are exerted were not systemically studied. We investigated the effect of DNA supercoiling on transcription from a set of promoters present on a plasmid by using a series of topoisomers with different superhelical densities ranging from totally relaxed to more than physiological. In vitro transcription assays with these topoisomers in the absence and presence of gene regulatory proteins showed that the effect of negative supercoiling on intrinsic transcription varies from promoter to promoter. Some of the promoters, in which DNA superhelicity stimulated transcription, displayed specific optima of superhelical density while others did not. The results also showed that the amount of abortive RNA synthesis from two of the promoters decreased and full-length RNA increased with increasing supercoiling, indicating for the first time an inverse relationship between full-length and abortive RNA synthesis and supporting a role of DNA superhelicity in promoter clearance. DNA supercoiling might also influence the point of RNA chain termination. Furthermore, the effect of varying the amount of supercoiling on the action of gene regulatory proteins suggested the mode of action, which is consistent with previous results. Our results underscore the importance of DNA supercoiling in fine-tuning promoter activities, which should be relevant in cell physiology given that local changes in chromosomal supercoiling must occur in different environments. 3) Interaction of adenylate cyclase and cyclic AMP receptor protein. We have investigated the possibility that adenylate cyclase makes physical contact with the receptor protein for efficient delivery of cyclic AMP for activation of transcription from a promoter. By using purified His-tagged adenylate cyclase and CRP and employing Nickel column, we have demonstrated a physical interaction between the two proteins. This was further confirmed by co-immuno precipitation assays. We are currently investigating the biochemical properties of this complex. Bacterial Chromosome Structure. 1) A-tract cluster facilitates DNA condensation in chromosome. Molecular mechanisms of bacterial chromosome packaging are still unclear, as bacteria lack nucleosomes or other apparent basic elements of DNA compaction. Among the factors facilitating DNA condensation may be propensity of the DNA molecule for folding due to its intrinsic curvature. As suggested previously, the sequence correlations in genome reflect such a propensity [Trifonov and Sussman (1980) Proc. Natl Acad. Sci. USA, 77, 3816-3820].
| Semsey, Szabolcs; Krishna, Sandeep; Erdossy, Janos et al. (2009) Dominant negative autoregulation limits steady-state repression levels in gene networks. J Bacteriol 191:4487-91 |
| Zwieb, Christian; Adhya, Sankar (2009) Plasmid vectors for the analysis of protein-induced DNA bending. Methods Mol Biol 543:547-62 |
| Lia, Giuseppe; Semsey, Szabolcs; Lewis, Dale E A et al. (2008) The antiparallel loops in gal DNA. Nucleic Acids Res 36:4204-10 |
| Adhya, Sankar L; Court, Donald L; Friedman, David I et al. (2008) Amos Oppenheim (31 October 1934 to 24 September 2006). Mol Microbiol 67:685-6 |
| Lewis, Dale E A; Komissarova, Natalia; Le, Phuoc et al. (2008) DNA sequences in gal operon override transcription elongation blocks. J Mol Biol 382:843-58 |
| Rokney, Assaf; Kobiler, Oren; Amir, Amnon et al. (2008) Host responses influence on the induction of lambda prophage. Mol Microbiol 68:29-36 |
| Edgar, Rotem; Rokney, Assaf; Feeney, Morgan et al. (2008) Bacteriophage infection is targeted to cellular poles. Mol Microbiol 68:1107-16 |
| Court, Donald L; Oppenheim, Amos B; Adhya, Sankar L (2007) A new look at bacteriophage lambda genetic networks. J Bacteriol 189:298-304 |
| Guo, Fusheng; Adhya, Sankar (2007) Spiral structure of Escherichia coli HUalphabeta provides foundation for DNA supercoiling. Proc Natl Acad Sci U S A 104:4309-14 |
| Semsey, Szabolcs; Virnik, Konstantin; Adhya, Sankar (2006) Three-stage regulation of the amphibolic gal operon: from repressosome to GalR-free DNA. J Mol Biol 358:355-63 |
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