Craig J. Benham
Professor, Depts of Mathematics and of Biomedical Engineering
UC Davis
cjbenham@ucdavis.edu
Separation of the two strands of the DNA duplex is a necessary step in the initiation of both replication and transcription. So the occurrences and locations of duplex strand openings must be stringently controlled in vivo. Although these initiation events commonly are regulated by enzymatic processes, anything that alters the stability of the duplex can affect the ease of opening, and hence serve a regulatory function. In particular, the topological constraint of negative superhelicity can destabilize the DNA duplex, causing its strands to separate at specific positions where its thermodynamic stability is low. Substantial amounts of unconstrained superhelicity, sufficient to drive strand opening, have been documented to occur in vivo in both prokaryotes and eukarotes.
We have developed computational methods to predict the destabilization properties of superhelical DNA molecules having any specified base sequence, including complete chromosomes. This phenomenon is context-dependent, hence complex to analyze, because superhelical stresses couple together the transition behaviors of all sites that experience them. The energy and conformational parameters used in this analysis are all taken from experimental measurements, so there are no free parameters. Yet when it is used to analyze specific DNA sequences, the results of this method are in quantitatively precise agreement with experimental measurements of the locations and extents of local strand separations. This justifies its use to predict the duplex destabilization properties of other DNA base sequences, on which experiments have not been performed.
We have analyzed the stress-induced duplex destabilization (SIDD) properties of many complete prokaryotic genomes, and of a wide variety of eukaryotic and viral sequences. Our results have illuminated the role of SIDD in a variety of regulatory processes, some of which will be described as time allows. I will focus in particular on one paradigm example of a previously unknown class of transcriptional regulatory mechanisms, which governs the ilvGMEDA promoter in E. coli. This mechanism coordinates the basal level of expression of this operon with the nutritional and environmental states of the cell through the effects of regulatory binding of the IHF protein of the SIDD properties of the region. In particular, IHF binding mediates a translocation of superhelical energy from an upstream supercoiling-induced DNA duplex destabilized (SIDD) site to the -10 region of the promoter, facilitating open complex formation and upregulating transcription.