Reid C. Johnson

Solution structure of the HMG protein NHP6A and its interaction with DNA reveals the structural determinants for non-sequence-specific binding.

NHP6A is a chromatin‐associated protein from Saccharomyces cerevisiae belonging to the HMG1/2 family of non‐specific DNA binding proteins. NHP6A has only one HMG DNA binding domain and forms relatively stable complexes with DNA. We have determined the solution structure of NHP6A and constructed an NMR‐based model structure of the DNA complex. The free NHP6A folds into an L‐shaped three α‐helix structure, and contains an unstructured 17 amino acid basic tail N‐terminal to the HMG box. Intermolecular NOEs assigned between NHP6A and a 15 bp 13C, 15N‐labeled DNA duplex containing the SRY recognition sequence have positioned the NHP6A HMG domain onto the minor groove of the DNA at a site that is shifted by 1 bp and in reverse orientation from that found in the SRY–DNA complex. In the model structure of the NHP6A–DNA complex, the N‐terminal basic tail is wrapped around the major groove in a manner mimicking the C‐terminal tail of LEF1. The DNA in the complex is severely distorted and contains two adjacent kinks where side chains of methionine and phenylalanine that are important for bending are inserted. The NHP6A–DNA model structure provides insight into how this class of architectural DNA binding proteins may select preferential binding sites.

Control of transcription by nucleoid proteins

Nucleoid proteins are a group of abundant DNA binding proteins that modulate the structure of the bacterial chromosome. They have been recruited as specific negative and positive regulators of gene transcription and their fluctuating patterns of expression are often exploited to impart an additional level of control with respect to environmental conditions.

The shape of the DNA minor groove directs binding by the DNA-bending protein Fis.

The bacterial nucleoid-associated protein Fis regulates diverse reactions by bending DNA and through DNA-dependent interactions with other control proteins and enzymes. In addition to dynamic nonspecific binding to DNA, Fis forms stable complexes with DNA segments that share little sequence conservation. Here we report the first crystal structures of Fis bound to high- and low-affinity 27-base-pair DNA sites. These 11 structures reveal that Fis selects targets primarily through indirect recognition mechanisms involving the shape of the minor groove and sequence-dependent induced fits over adjacent major groove interfaces. The DNA shows an overall curvature of approximately 65 degrees , and the unprecedented close spacing between helix-turn-helix motifs present in the apodimer is accommodated by severe compression of the central minor groove. In silico DNA structure models show that only the roll, twist, and slide parameters are sufficient to reproduce the changes in minor groove widths and recreate the curved Fis-bound DNA structure. Models based on naked DNA structures suggest that Fis initially selects DNA targets with intrinsically narrow minor grooves using the separation between helix-turn-helix motifs in the Fis dimer as a ruler. Then Fis further compresses the minor groove and bends the DNA to generate the bound structure.

Identification of two functional regions in Fis: the N-terminus is required to promote Hin-mediated DNA inversion but not lambda excision.

The Fis protein of E. coli binds to a recombinational enhancer sequence that is required to stimulate Hin‐mediated DNA inversion. Fis is also required for efficient lambda prophase excision in vivo. The properties of mutant Fis proteins were examined in vivo and in vitro with respect to their stimulatory effects on these two different site‐specific DNA recombination reactions. Both recombination reactions are dramatically affected by mutations altering a helix‐turn‐helix DNA binding motif located near the Fis C‐terminus (residues 74–93). These mutations invariably decrease DNA binding affinity and some cause reduced DNA bending. Mutations in the Fis N‐terminal region reduce or abolish the stimulation of Hin‐mediated DNA recombination by Fis, but have little or no effect on DNA binding or lambda excision. We conclude that there are at least two functionally distinct domains in Fis: a C‐terminal DNA binding region that is required for promoting both DNA recombination reactions and an N‐terminal region that is uniquely required for Hin‐mediated inversion.

The S. cerevisiae architectural HMGB protein NHP6A complexed with DNA: DNA and protein conformational changes upon binding

NHP6A is a non-sequence-specific DNA-binding protein from Saccharomyces cerevisiae which belongs to the HMGB protein family. Previously, we have solved the structure of NHP6A in the absence of DNA and modeled its interaction with DNA. Here, we present the refined solution structures of the NHP6A-DNA complex as well as the free 15bp DNA. Both the free and bound forms of the protein adopt the typical L-shaped HMGB domain fold. The DNA in the complex undergoes significant structural rearrangement from its free form while the protein shows smaller but significant conformational changes in the complex. Structural and mutational analysis as well as comparison of the complex with the free DNA provides insight into the factors that contribute to binding site selection and DNA deformations in the complex. Further insight into the amino acid determinants of DNA binding by HMGB domain proteins is given by a correlation study of NHP6A and 32 other HMGB domains belonging to both the DNA-sequence-specific and non-sequence-specific families of HMGB proteins. The resulting correlations can be rationalized by comparison of solved structures of HMGB proteins.

Concentration-dependent exchange accelerates turnover of proteins bound to double-stranded DNA

The multistep kinetics through which DNA-binding proteins bind their targets are heavily studied, but relatively little attention has been paid to proteins leaving the double helix. Using single-DNA stretching and fluorescence detection, we find that sequence-neutral DNA-binding proteins Fis, HU and NHP6A readily exchange with themselves and with each other. In experiments focused on the Escherichia coli nucleoid-associated protein Fis, only a small fraction of protein bound to DNA spontaneously dissociates into protein-free solution. However, if Fis is present in solution, we find that a concentration-dependent exchange reaction occurs which turns over the bound protein, with a rate of kexch = 6 × 104 M−1s−1. The bacterial DNA-binding protein HU and the yeast HMGB protein NHP6A display the same phenomenon of protein in solution accelerating dissociation of previously bound labeled proteins as exchange occurs. Thus, solvated proteins can play a key role in facilitating removal and renewal of proteins bound to the double helix, an effect that likely plays a major role in promoting the turnover of proteins bound to DNA in vivo and, therefore, in controlling the dynamics of gene regulation.

Mechanism for specificity by HMG-1 in enhanceosome assembly.

ABSTRACT Assembly of enhanceosomes requires architectural proteins to facilitate the DNA conformational changes accompanying cooperative binding of activators to a regulatory sequence. The architectural protein HMG-1 has been proposed to bind DNA in a sequence-independent manner, yet, paradoxically, it facilitates specific DNA binding reactions in vitro. To investigate the mechanism of specificity we explored the effect of HMG-1 on binding of the Epstein-Barr virus activator ZEBRA to a natural responsive promoter in vitro. DNase I footprinting, mutagenesis, and electrophoretic mobility shift assay reveal that HMG-1 binds cooperatively with ZEBRA to a specific DNA sequence between two adjacent ZEBRA recognition sites. This binding requires a strict alignment between two adjacent ZEBRA sites and both HMG boxes of HMG-1. Our study provides the first demonstration of sequence-dependent binding by a nonspecific HMG-box protein. We hypothesize how a ubiquitous, nonspecific architectural protein can function in a specific context through the use of rudimentary sequence recognition coupled with cooperativity. The observation that an abundant architectural protein can bind DNA cooperatively and specifically has implications towards understanding HMG-1s role in mediating DNA transactions in a variety of enzymological systems.

Variation of the folding and dynamics of the Escherichia coli chromosome with growth conditions

We examine whether the Escherichia coli chromosome is folded into a self‐adherent nucleoprotein complex, or alternately is a confined but otherwise unconstrained self‐avoiding polymer. We address this through in vivo visualization, using an inducible GFP fusion to the nucleoid‐associated protein Fis to non‐specifically decorate the entire chromosome. For a range of different growth conditions, the chromosome is a compact structure that does not fill the volume of the cell, and which moves from the new pole to the cell centre. During rapid growth, chromosome segregation occurs well before cell division, with daughter chromosomes coupled by a thin inter‐daughter filament before complete segregation, whereas during slow growth chromosomes stay adjacent until cell division occurs. Image correlation analysis indicates that sub‐nucleoid structure is stable on a 1 min timescale, comparable to the timescale for redistribution time measured for GFP–Fis after photobleaching. Optical deconvolution and writhe calculation analysis indicate that the nucleoid has a large‐scale coiled organization rather than being an amorphous mass. Our observations are consistent with the chromosome having a self‐adherent filament organization.

Molecular anatomy of a transcription activation patch: FIS–RNA polymerase interactions at the Escherichia coli rrnB P1 promoter

FIS, a site‐specific DNA binding and bending protein, is a global regulator of gene expression in Escherichia coli. The ribosomal RNA promoter rrnB P1 is activated 3‐ to 7‐fold in vivo by a FIS dimer that binds a DNA site immediately upstream of the DNA binding site for the C‐terminal domain (CTD) of the α subunit of RNA polymerase (RNAP). In this report, we identify several FIS side chains important specifically for activation of transcription at rrnB P1. These side chains map to positions 68, 71 and 74, in and flanking a surface‐exposed loop adjacent to the helix–turn–helix DNA binding motif of the protein. We also present evidence suggesting that FIS activates transcription at rrnB P1 by interacting with the RNAP αCTD. Our results suggest a model for FIS‐mediated activation of transcription at rrnB P1 that involves interactions between FIS and the RNAP αCTD near the DNA surface. Although FIS and the transcription activator protein CAP have little structural similarity, they both bend DNA, use a similarly disposed activation loop and target the same region of the RNAP αCTD, suggesting that this is a common architecture at bacterial promoters.

The DNA Architectural Protein HMGB1 Displays Two Distinct Modes of Action That Promote Enhanceosome Assembly

ABSTRACT HMGB1 (also called HMG-1) is a DNA-bending protein that augments the affinity of diverse regulatory proteins for their DNA sites. Previous studies have argued for a specific interaction between HMGB1 and target proteins, which leads to cooperative binding of the complex to DNA. Here we propose a different model that emerged from studying how HMGB1 stimulates enhanceosome formation by the Epstein-Barr viral activator Rta on a target gene, BHLF-1. HMGB1 stimulates binding of individual Rta dimers to multiple sites in the enhancer. DNase I and hydroxyl radical footprinting, electrophoretic mobility shift assays, and immobilized template assays failed to reveal stable binding of HMGB1 within the complex. Furthermore, mutational analysis failed to identify a specific HMGB1 target sequence. The effect of HMGB1 on Rta could be reproduced by individual HMG domains, yeast HMO1, or bacterial HU. These results, combined with the effects of single-amino-acid substitutions within the DNA-binding surface of HMGB1 domain A, argue for a mechanism whereby DNA-binding and bending by HMGB1 stimulate Rta-DNA complex formation in the absence of direct interaction with Rta or a specific HMGB1 target sequence. The data contrast with our analysis of HMGB1 action on another BHLF-1 regulatory protein called ZEBRA. We discuss the two distinct modes of HMGB1 action on a single regulatory region and propose how HMGB1 can function in diverse contexts.