Abstract
Nucleoid-associated proteins play an important role in condensing chromosomal DNA and regulating gene expression. We report here the characterization of the nucleoid-associated protein YejK, which was detected in a yeast two-hybrid screen using the ParE subunit of topoisomerase IV as bait. The purified protein likely exists in a monomer-dimer equilibrium in solution and can form tetramers. Cross-linking of the protein bound to DNA suggests that the active form could be either a dimer or tetramer. YejK, which is present at about 24,000 copies of monomer per mid-log phase cell, binds double-stranded DNA with a site size of 12-14 base pairs/monomer, does not display a significant preference for either bent compared with straight DNA or supercoiled compared with relaxed DNA, and untwists DNA somewhat as it binds. YejK binds RNA, but not single-stranded DNA, with 65% of the avidity with which it binds DNA. However, cells deleted for yejK do not show defects in either RNA or protein synthesis. YejK interacts with all the subunits of both DNA gyrase and topoisomerase IV and has measurable effects on their activities. In the presence of YejK, relaxation of negatively supercoiled DNA by topoisomerase IV becomes distributive, whereas relaxation of positively supercoiled DNA is stimulated. Relaxation of negatively supercoiled DNA by DNA gyrase is inhibited, whereas the extent of supercoiling of relaxed DNA is limited. A YejK-GFP chimera is an effective marker for the nucleoid in live cell imaging.
Highlights
We report here the characterization of the nucleoid-associated protein YejK, which was detected in a yeast two-hybrid screen using the ParE subunit of topoisomerase IV as bait
YejK, which is present at about 24,000 copies of monomer per mid-log phase cell, binds double-stranded DNA with a site size of 12–14 base pairs/monomer, does not display a significant preference for either bent compared with straight DNA or supercoiled compared with relaxed DNA, and untwists DNA somewhat as it binds
Two-hybrid interactions of DNA gyrase and topoisomerase IV (Topo IV) subunits with Gal4-YejK
Summary
To fit within the nucleoid, the chromosomal DNA of Escherichia coli has to be compacted by at least 400-fold in a manner that does not compromise either DNA replication or transcription This functional compaction is achieved by a combination of supercoiling of the DNA, macromolecular crowding, and the binding of (generally) small proteins that help shape and organize the DNA [1]. Fis, for example, can affect global supercoiling because it regulates the transcription of topoisomerase I and both subunits of DNA gyrase in a growth rate-dependent manner [3, 4] These two topoisomerases are responsible for homeostatically maintaining the optimal balance of chromosomal DNA supercoiling in E. coli [5]. Sloankettering.edu. 2 The abbreviations used are: NAP, nucleoid-associated protein; bp, base pair(s); (ϩ)sc, positively supercoiled; (Ϫ)sc, negatively supercoiled; Topo IV, topoisomerase IV; nt, nucleotide
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