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Charged residues in the H-NS linker drive DNA binding and gene silencing in single cells

  1. Linda J. Kenneya,c,d,1
  1. aMechanobiology Institute, National University of Singapore, Singapore 117411;
  2. bDepartment of Physics, National University of Singapore, Singapore 117411;
  3. cJesse Brown Veterans Administration Medical Center, Chicago, IL 60607;
  4. dDepartment of Microbiology, University of Illinois at Chicago, Chicago, IL 60612
  1. Edited by Sankar Adhya, National Cancer Institute, National Institutes of Health, Bethesda, MD, and approved October 20, 2017 (received for review September 29, 2017)

Significance

H-NS is a nucleoid-associated protein that plays a major role in silencing pathogen genes. We discovered that the unstructured linker region connecting the N-terminal oligomerization and C-terminal DNA binding domains plays an important and surprising role in promoting DNA binding. Superresolution imaging identified H-NS foci that required DNA binding for their formation and were associated with the nucleoid. Removing the linker led to the disappearance of foci and a substantially lower affinity for DNA. It was proposed that H-NS compacts DNA, but decreasing DNA binding in cells did not lead to a relaxation of the nucleoid, suggesting H-NS does not play a major role in nucleoid compaction. Molecular dynamic simulations suggested that target acquisition by H-NS may involve sliding along the DNA.

Abstract

Nucleoid-associated proteins (NAPs) facilitate chromosome organization in bacteria, but the precise mechanism remains elusive. H-NS is a NAP that also plays a major role in silencing pathogen genes. We used genetics, single-particle tracking in live cells, superresolution microscopy, atomic force microscopy, and molecular dynamics simulations to examine H-NS/DNA interactions in single cells. We discovered a role for the unstructured linker region connecting the N-terminal oligomerization and C-terminal DNA binding domains. In the present work we demonstrate that linker amino acids promote engagement with DNA. In the absence of linker contacts, H-NS binding is significantly reduced, although no change in chromosome compaction is observed. H-NS is not localized to two distinct foci; rather, it is scattered all around the nucleoid. The linker makes DNA contacts that are required for gene silencing, while chromosome compaction does not appear to be an important H-NS function.

Footnotes

  • ?1To whom correspondence should be addressed. Email: kenneyl{at}uic.edu.
  • Author contributions: Y.G., Y.H.F., R.S.W., Q.T., J.Y., and L.J.K. designed research; Y.G., Y.H.F., R.S.W., and Q.T. performed research; Y.G., Y.H.F., R.S.W., Q.T., J.Y., and L.J.K. analyzed data; and Y.G., Y.H.F., R.S.W., Q.T., J.Y., and L.J.K. wrote the paper.

  • The authors declare no conflict of interest.

  • This article is a PNAS Direct Submission.

  • This article contains supporting information online at www.danielhellerman.com/lookup/suppl/doi:10.1073/pnas.1716721114/-/DCSupplemental.

Published under the PNAS license.

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