Book of Abstracts: Albany 2005
Single-molecule Studies of the Compactosome: A Nucleosome-like Structure of Bacterial Chromosomes
One of the important elements in the formation of the bacterial chromosome is the strong compaction of the single, circularized DNA molecule comprising the genome. In bacteria such as E. coli, this compaction is of the order of 1000 times. A group of about ten different proteins collectively referred to as "histone-like" contribute to the compaction of the genome, in addition to many important roles they play in DNA transactions such as in the control of gene expression and replication.
I will present the results of an experimental study of DNA interacting with HU, an abundant histone-like protein that shows no affinity to particular DNA sequences, using two single-molecule methodologies. Fluorescence resonance energy transfer measurements at the level of both large ensembles of complexes (ensemble-FRET), and single complexes (sp-FRET) are used to probe the small-scale structure of HU-DNA complexes formed from short (30-60 bp) labeled double-stranded oligonucleotides. The elastic behavior of single twist-relaxed and supercoiled HU-DNA complexes formed from long DNA molecules (∼10kbp) is studied using magnetic tweezers, in order to understand the large scale structure of such complexes.
Our results show that for small but increasing HU concentrations, HU compacts DNA strongly in a salt-dependent fashion, and affects differently the elastic behavior of positive and negatively supercoiled substrates. Above a threshold however, further increase in HU concentration causes a decrease in both HU-induced compaction and its effects on supercoiling. This non-monotonic behavior cannot be explained by the binding of single HU dimers. Evidence is presented showing that the non-monotonic behavior is a result of the formation of high-order nucleoprotein complexes. We propose a structural model of the HU/DNA complex, the compactosome, which shares some features in common with eukaryotic chromosomes. The model is based on the cooperative interaction of a number of HU dimers that bind in phase with the DNA helical pitch, bending DNA in concert.
1Department of Physics of Complex Systems