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The C‐terminal domain of Histone‐like protein from Mycobacterium smegmatis mediates DNA binding and transcription repression
Author(s) -
Mukherjee Anirban,
Grove Anne
Publication year - 2007
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.21.6.a1032
Subject(s) - dna supercoil , dna , microbiology and biotechnology , nucleoid , biology , transcription (linguistics) , hmg box , electrophoretic mobility shift assay , protein–dna interaction , mycobacterium smegmatis , histone , plasmid , topoisomerase , dna binding protein , dna replication , transcription factor , biochemistry , gene , mycobacterium tuberculosis , escherichia coli , tuberculosis , linguistics , philosophy , medicine , pathology
Histone‐like proteins (such as HU, IHF, H‐NS and Fis) are nucleoid‐associated architectural proteins involved in DNA compaction, oriC‐ dependent replication, recombination and transcription, and some are ubiquitous in eubacteria. Cold shock and oxidative stress upregulates of a homologue of HU (Hlp) in Mycobacterium smegmatis , the non‐pathogenic model for M. tuberculosis . We show that Hlp, 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. Three mutant proteins were constructed, one without the C‐terminal repeats (RL‐Hlp), one deleted for the entire C‐terminal domain (TL‐Hlp) and a third representing the C‐terminal repeat sequence (CTR). The binding affinity of Hlp to 76 bp linear DNA is greater, K d = 0.033 ± 0.001 nM, compared to RL‐Hlp, K d ≅ 172 nM, and TL‐Hlp (barely detectectable complex). The CTR binds to 76 bp DNA with high affinity, Kd =0.82 ±0.17 nM. Hlp does not supercoil DNA, but prevents relaxation of supercoiled DNA by Topoisomerase I. Hlp does not prefer supercoiled plasmid DNA over linear plasmid DNA and, at 0.33μM, represses transcription in vitro whereas the Rl‐Hlp fails to repress transcription at equivalent concentrations. Hlp does not bend DNA. These data indicate that the Lysine‐rich repeat‐containing C‐terminus mediates DNA interactions. Research supported by NSF.