(A) Domains and regions as identified in (Bartosik et al., 2004; Kusiak et al., 2011). (B) ParB is thought to be anchored at parS (grey) via the HtH motif found in the CDBD (red). ParB protomers …
(A–C) Schematics of different domain arrangements for dimeric B. subtilis ParB. Whilst it is known that ParB is dimeric in solution, the primary dimerisation interface is unclear and possibly …
(A) Representative TBM-EMSAs for wild type ParB and ParBR149G monitoring binding of parS-containing or non-specific 147 bp dsDNA. (B) Schematic of the magnetic tweezer assay used to monitor …
(A) SDS-PAGE of wild type ParB and mutant protein preparations. (B) Non-specific DNA binding isotherms for wild type ParB and ParBR149G measured using a PIFE assay. The data were fitted to the Hill …
(A) Ensemble overlay of the 14 lowest-energy CTD structures. Red and blue depict separate monomers within the dimer. (B) Secondary structure elements are identified. α1 indicates the N-terminus of …
NMR assignment, structure calculation and validation statistics.
acommonly assigned groups (i.e. excluding OH, Asp/Glu side-chain carbonyl, Lys amide and Arg guanidinium groups as well as tertiary aromatic carbons); bresidues 229–282; cvalues reported by ARIA 2.3 (Rieping et al., 2007); dordered residues (230-254, 259-278) as calculated by PSVS 1.5 (Bhattacharya et al., 2007); evalues reported by Procheck (Laskowski et al., 1993); fvalue reported by PDB validation software; g residues in secondary structure (231-245, 249-254, 257-264, 267-277). The structural validation programs used were as follows: CING (Doreleijers et al., 2012), Verify3D (Lüthy et al., 1992), Prosa II (Sippl, 1993), Procheck (Laskowski et al., 1993), MolProbity (Lovell et al., 2003).
(A) Assigned 1H-15N HSQC spectra of CTD. Arginine ε and η peaks are aliased once and twice, respectively. Starred peaks indicate additional (usually minor) species. (B) The C-terminal domain of B. …
(A–C) TBE-EMSAs for the titration of full length ParB and CTD against 147 bp DNA. Wild type and mutant proteins, K255A + K257A and K252A + K255A + K259A, are indicated. (D) Native mass spectrometry. …
(A) TBE-EMSA showing binding of the CTD to the 10 bp hairpin DNA substrate used for NMR experiments. (B) Assigned 1H-15N HSQC spectra of the CTD prior (blue) and after (red) titration with a 10 bp …
(A) Mean force-extension curves of DNA molecules co-incubated with ParB variants at the indicated concentrations. Non-condensed (protein-free) DNA data is fitted to the worm-like chain model. Solid …
(A) Representative traces of condensation assays monitored by magnetic tweezers. The expected DNA extension in the absence of ParB at each force is indicated by the dashed lines. (i), (ii) and (ii) …
(A) Mean force-extension curves for DNA molecules co-incubated with 1 µM ParB2 in the presence or absence of 5 µM CTD2. Non-condensed (protein-free) DNA data is fitted to the worm-like chain model. …
(A) Representative traces of condensation assays for ParB in the presence and absence of excess free CTD. The expected DNA extension in the absence of ParB at each force is indicated by the dashed …
(A) A working model for ParB network formation and concomitant DNA condensation incorporating ideas from this work and other published studies (see main text for details). At least two DNA:protein …
(A) Variant ParB-GFP mutants form abnormal foci in B. subtilis. Cells were grown overnight in slow growth conditions before dilution (1:100) into fast growth media, and were allowed to achieve at …
(A–B) Western blot analysis. Expression levels of ParBL270D+L274D and ParBK252A+K255A+K259A with a C-terminal gfp fusion were similar to wild type ParB. DivIVA is an abundant cell division protein …
Plasmid | Genotype | Genome location |
---|---|---|
oqAKPCR3 | 5’-AGCCGGATTGATCAAACATC-3’ | 359.32° |
oqAKPCR4 | 5’-AGAGCCGATCAGACGAAAAC-3’ | 359.32° |
oqAKPCR5 | 5’-GAGGCAAGCAAAGCTCACTC-3’ | 359.45° |
oqAKPCR6 | 5’-TGCCATGACAGAGCTGAAAC-3’ | 359.45° |
oqAKPCR7 | 5’-CTTTTCCAAGGCCTTTAGCC-3’ | 359.22° |
oqAKPCR8 | 5’-TCACGGAAAACCCATCATTT-3’ | 359.22° |
oqAKPCR9 | 5’-TATTGGCCTGCTTCATACCC-3’ | 359.65° |
oqAKPCR10 | 5’-TGGAGATTCTGTCCACGAAA-3’ | 359.65° |
oqPCR9 | 5’-AAAAAGTGATTGCGGAGCAG-3’ | 359.16° |
oqPCR10 | 5’-AGAACCGCATCTTTCACAGG-3’ | 359.16° |
oqPCR25 | 5’-TCCATAATCGCCTCTTGGAC-3’ | 359.37° |
oqPCR26 | 5’-AAGCGCATGCTTATGCTAGG-3’ | 359.37° |
oqPCR31 | 5’-GATCCGAAGGTCTGTCTACG-3’ | 359.76° |
oqPCR32 | 5’-CGATTGCGATTGTACGGTTG-3’ | 359.76° |
oqPCR57 | 5’-TTTGCATGAACTGGGCAATA-3’ | 146.52° |
oqPCR58 | 5’-TCCGAACATGTCCAATGAGA-3’ | 146.52° |