期刊
BIOCHEMISTRY
卷 47, 期 33, 页码 8744-8753出版社
AMER CHEMICAL SOC
DOI: 10.1021/bi800010s
关键词
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资金
- National Science Foundation [MCB-0414875, MCB-0744240 to A.G.]
Histone-like proteins (such as HU, H-NS, and Fis) participate in nucleoid organization and in DNA replication, recombination, and transcription. Cold shock and anoxia upregulates a homologue of HU (Hlp) in Mycobacterium smegmatis, the nonpathogenic model of Mycobacterium tuberculosis. We show using electrophoretic mobility shift assays that Hlp, which in addition to the HU fold has a basic C-terminal tail containing multiple PAKK and PAAK repeats, has very high affinity for DNA. The affinity of Hlp for 76 bp linear DNA is higher, K(d) = 0.037 +/- 0.001 nM, compared to art Hlp variant without the C-terminal repeats, K(d) = 2.5 +/- 0.1 nM and the isolated C-terminal repeat domain, K(d) = 0.8 +/- 0.2 nM, where Kd in all cases reflects an aggregate affinity for the DNA probes, not the affinity for binding to a single site. Hlp lacking the entire C-terminal domain binds DNA only poorly. These data indicate that both Hlp domains contribute to high-affinity DNA binding. Hlp promotes DNA end-joining in the presence of T4 DNA ligase, and this property is mediated by the C-terminal repeats. At < 100 nM concentration, Hlp represses transcription by T7 RNA polymerase in vitro whereas the individual N- and C-terminal domains do not, even when present together. Notably, while DNA end-joining can be achieved by the isolated C-terminal domain, transcriptional repression requires for both domains to be present on a single polypeptide. Given the low cellular concentration of Hlp, our data suggest that its primary functional role may be in DNA-dependent responses to environmental stress rather than in nucleoid organization.
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