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The C‐terminal domain of Histone‐like protein from Mycobacterium smegmatis participates in DNA binding
Author(s) -
Mukherjee Anirban,
Grove Anne
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.4.a78-d
Subject(s) - dna supercoil , microbiology and biotechnology , biology , hmg box , electrophoretic mobility shift assay , dna , mycobacterium smegmatis , transcription (linguistics) , protein–dna interaction , nucleoid , dna binding protein , biochemistry , dna replication , transcription factor , gene , mycobacterium tuberculosis , escherichia coli , tuberculosis , medicine , linguistics , pathology , philosophy
Histone‐like proteins are nucleoid‐associated architectural proteins that are ubiquitously found in eubacteria. The best characterized and most abundant proteins of this family are HU, IHF, H‐Ns and Fis. HU from different bacterial species has been shown to be involved in DNA compaction, oriC‐ dependent replication, recombination and transcription. Upregulation of a homologue of HU has been reported in Mycobacterium smegmatis , the non‐pathogenic model for Mycobacterium tuberculosis , under cold shock and oxidative stress. We show that this 212 residue protein, which has a basic C‐terminal tail with PAAK and PAKK repeats, has high affinity for linear and supercoiled DNA as shown by electrophoretic mobility shift assays (EMSA). Two mutant proteins were constructed by PCR‐based mutagenesis, one without the basic tail, 168 residues long, and one 90 amino acids long, which resembles normal HU in the number of residues. The DNA binding affinity of the wild type protein is greater and binds to 76 bp linear DNA with high affinity, K d = 0.92 ± 0.13 nM, as compared to the repeat‐less mutant protein, K d ≅ 172 nM. It does not supercoil DNA like most other HU proteins do, but prevents relaxation of supercoiled DNA by Topoisomerase I. The wild type protein shows no preference in binding supercoiled plasmid DNA over linear plasmid DNA. The wild type protein, at 0.33μM, represses transcription in vitro but the repeat‐less mutant protein does not show any significant repression of transcription. These data indicate that the Lysine‐rich repeat‐containing C‐terminus participates in DNA interactions. Research supported by NSF.