The DNA Binding Protein Lsr2 from Mycobacterium tuberculosis

Summers, Emma Louise

The DNA Binding Protein Lsr2 from Mycobacterium tuberculosis

dc.contributor.advisor	Arcus, Vickery L.
dc.contributor.advisor	Cursons, Raymond T.
dc.contributor.author	Summers, Emma Louise
dc.date.accessioned	2012-09-05T03:21:45Z
dc.date.available	2012-09-05T03:21:45Z
dc.date.issued	2012
dc.date.updated	2012-08-31T03:00:43Z
dc.description.abstract	Lsr2 is a small, basic DNA binding protein that is highly conserved in mycobacteria and related actinomycetes. Lsr2 is essential for growth in Mycobacterium tuberculosis and previous studies have shown that Lsr2 is involved in down-regulating a wide range of genes involved in cell wall synthesis and metabolic functions. This regulatory function is likely to influence bacterial growth and survival. This research investigated the biochemistry and 3D structure of Lsr2 from M. tuberculosis. Transmission electron microscopy (TEM) analysis of Lsr2 in complex with DNA revealed a regular fibril-like arrangement of protein coating double-stranded DNA. In addition, it was shown that Lsr2 physically protected DNA from DNase activity. The structure of the C-terminal DNA binding domain of Lsr2, determined by others part way through this research, prompted site directed mutagenesis of residues proposed to interact with DNA. Modification of arginine residues significantly reduced the binding of Lsr2 to DNA and fibril-like structures were not observed using TEM, for arginine mutants. The first crystal structure of the N-terminal domain of Lsr2 is reported here. Two high resolution structures in monoclinic and hexagonal space groups were solved using X-ray crystallography and ab initio phasing. Proteolytic processing of the N-terminus of Lsr2 was revealed by the structure in P2₁ and this process was recreated using the protease trypsin which resulted in crystal formation in a P3₁21 space group. Both structures show linear chains of dimeric N-terminal Lsr2, as shown by crystallographic symmetry, linked by overlapping anti-parallel β-sheets, revealing a mechanism of protein oligomerisation. Oligomerisation only occurs after the removal of the first three residues M1, A2 and K3. In solution, protein oligomerisation was recreated with trypsin, resulting in the formation of large protein complexes. A change in DNA topology after the addition of trypsin to full-length Lsr2/DNA complexes was observed using TEM. This mechanism is likely to be important to M. tuberculosis under “stress” conditions where proteases are known to be upregulated and where cross-linking and condensation of DNA is critical.
dc.format.mimetype	application/pdf
dc.identifier.citation	Summers, E. L. (2012). The DNA Binding Protein Lsr2 from Mycobacterium tuberculosis (Thesis, Doctor of Philosophy (PhD)). University of Waikato, Hamilton, New Zealand. Retrieved from https://hdl.handle.net/10289/6628	en
dc.identifier.uri	https://hdl.handle.net/10289/6628
dc.language.iso	en
dc.publisher	University of Waikato
dc.rights	All items in Research Commons are provided for private study and research purposes and are protected by copyright with all rights reserved unless otherwise indicated.
dc.subject	DNA binding protein
dc.subject	Mycobacterium tuberculosis
dc.subject	Tuberculosis
dc.subject	Lsr2
dc.title	The DNA Binding Protein Lsr2 from Mycobacterium tuberculosis	en
dc.type	Thesis
pubs.place-of-publication	Hamilton, New Zealand	en_NZ
thesis.degree.grantor	University of Waikato
thesis.degree.level	Doctoral
thesis.degree.name	Doctor of Philosophy (PhD)