(A) Patch and dispersed locations of K to Q mutations. In A5, the five phosphorylation sites were changed to alanine, producing a constitutively unphosphorylated H1. Starting with A5, in Q5A5, five lysines in the region of the phosphorylation sites were changed to glutamines, reducing the positive charge without introducing any negatively charged molecules that resemble phosphates. Q5A5-1, -2, and -3 are like Q5A5 except that five lysines on the N-terminal side of the phosphorylation region, at the immediate C-terminal side of the phosphorylation region and in the C-terminal region of H1, were changed to glutamines. In 5Q, five lysines (≈30 aa apart) were changed to glutamines. The net charges of the Q5A5, Q5A5-1, -2, and –3, and 5Q versions of H1 were the same, similar to that of E5. (B) Patch and dispersed locations of neutral to K mutations. In E5, the five phosphorylation sites were changed to glutamic acids, mimicking a constitutively phosphorylated H1. Starting with E5, in K5E5 five neutral residues in the region of the phosphorylation sites were changed to lysines, introducing positive charges. K5E5-1, -2, and -3 are like K5E5 except that five neutral residues at the N-terminal side of the phosphorylation region, at the immediate C-terminal side of the phosphorylation region, and in the C-terminal region of H1 were changed to lysines. In 5K, five neutral residues (≈30 aa apart) were changed to lysines. The net charges of the K5E5, K5E5-1, -2, and –3, and 5K versions of H1 were the same, similar to that of A5.

The nucleation-propagation model of H1-DNA interactions. H1 is proposed to be constantly exchanging, with bound and unbound fractions in equilibrium (30, 31). For all forms of H1, dissociation is nucleated from the region with the lowest positive charge and then propagated to other parts of the molecule. Similarly, association is nucleated from the region with the highest positive charges. In WT cells with fully dephosphorylated H1 or in A5 cells, nucleation of dissociation is relatively low, whereas nucleation of reassociation is high. When WT H1 is phosphorylated or the phosphorylation sites are mutated to glutamic acid (E5), the phosphorylation region is negatively charged and becomes the nucleation center for dissociation. In the Q5A5 series, reducing positive charges creates new nucleation centers in other parts of H1, again shifting the equilibrium toward dissociation. In the K5E5 series, although a nucleation site is produced by the five Es at the phosphorylation sites, we hypothesize that the newly introduced positive charge patches could produce a nucleation center that increases the association rate or that the newly introduced positive charges could produce a kinetic barrier to propagation. In 5Q and 5K cells, the introduced charges are spread out throughout the whole molecule. Because no new nucleation center was created, they behave like A5 or E5 cells, respectively.

(A) Circular permutation of the charge patch. Wild-type (WT) and mutant (A and E) charge patches, from residues 35 to 54, were moved to different regions of H1 in a circularly permutated way. In N, the patch was moved to the extreme N terminus of the protein, from position 4 to 30. In M, the patch was moved near the center of H1, from residues 84 to 110. In C, the patch was moved to the C terminus, to positions 124–150. (C|N) is the junction of the C and N terminus if the ends of H1 were ligated. (B) The Charge patch functions at different sites in H1. Whole-cell RNAs (10 μg) were isolated from WT-N, M, and C; A-N, M, and C; and E-N, M, and C strains at 0, 6, and 12 h of starvation. They were analyzed by Northern blots probed with CyP1 and ngoA genes. 26s rRNA was used as loading control.

Total RNA (10 μg) isolated from cells after 0, 6, and 12 h of starvation, was analyzed by Northern blotting using CyP1 and ngoA specific probes. 26S rRNA was used as loading control. (A) Q5A5-1, Q5A5-2, Q5A5-3, and 5Q strains. (B) K5E5-1, K5E5-2, K5E5-3, and 5K strains.