Molecular and cellular principles of genome folding and genome function

Inaugural lecture given by Prof.dr. R.T. Dame on the acceptance of his position as professor of Molecular and Cellular Biochemistry at the Universiteit Leiden on Monday May 10, 2021Macromolecular Biochemistr

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NWOVICI 016.160.613Macromolecular Biochemistr

The organization of bacterial genomes: towards understanding the interplay between structure and function

Genomes are arranged in a confined space in the cell, the nucleoid or nucleus. This arrangement is hierarchical and dynamic, and follows DNA/chromatin-based transactions or environmental conditions. Describing the interplay between local genome structure and gene activity is a long-standing quest in biology. Here, we focus on systematic studies correlating bacterial genome folding and function. Parallels on organizational similarities with eukaryotes are drawn. The biological relevance of hierarchical units in bacterial genome folding and the causal relationship between genome folding and its activity is unclear. We discuss recent quantitative approaches to tackle these questions. Moreover, we sketch a perspective of experiments necessary to iteratively and systematically build, test and improve structure–function models of bacterial chromatin.Macromolecular Biochemistr

Post-translational modification of nucleoid-associated proteins: an extra layer of functional modulation in bacteria?

Post-translational modification (PTM) of histones has been investigated in eukaryotes for years, revealing its widespread occurrence and functional importance. Many PTMs affect chromatin folding and gene activity. Only recently the occurrence of such modifications has been recognized in bacteria. However, it is unclear whether PTM of the bacterial counterparts of eukaryotic histones, nucleoid-associated proteins (NAPs), bears a comparable significance. Here, we scrutinize proteome mass spectrometry data for PTMs of the four most abundantly present NAPs in Escherichia coli (H-NS, HU, IHF and FIS). This approach allowed us to identify a total of 101 unique PTMs in the 11 independent proteomic studies covered in this review. Combined with structural and genetic information on these proteins, we describe potential effects of these modifications (perturbed DNA-binding, structural integrity or interaction with other proteins) on their function.Macromolecular Biochemistr

Single molecule FRET analysis of DNA binding proteins

The complex binding dynamics between DNA and proteins are often obscured by ensemble averaging effects in conventional biochemical experiments. Single-molecule fluorescence methods are powerful tools to investigate DNA–protein interaction dynamics in real time. In this chapter, we focus on using single-molecule Förster Resonance Energy Transfer (smFRET) to probe the binding dynamics of individual proteins on single DNA molecules. We provide a detailed discussion of total internal reflection fluorescence (TIRF) instrument design, nucleic acid labeling with fluorophores, flow cell surface passivation, and data analysis methods.Macromolecular Biochemistr

Chromosome organization in bacteria: mechanistic insights into genome structure and function

Macromolecular Biochemistr

JUMPING MODE ATOMIC FORCE MICROSCOPY ON GRANA MEMBRANES FROM SPINACH

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