L. James Maher

Crystal structure of NF-κB (p50)2 complexed to a high-affinity RNA aptamer

We have recently identified an RNA aptamer for the transcription factor NF-κB p50 homodimer [(p50)2], which exhibits little sequence resemblance to the consensus DNA target for (p50)2, but binds (p50)2 with an affinity similar to that of the optimal DNA target. We describe here the 2.45-Å resolution x-ray crystal structure of the p50 RHR/RNA aptamer complex. The structure reveals that two RNA molecules bind independent of each other to the p50 N-terminal Ig-like domains. The RNA secondary structure is comprised of a stem and a stem–loop separated by an internal loop folded into a kinked helix because of the cross-strand stacking of three internal loop guanines. These guanines, placed at the edge of the 3′ helix, together with the major groove of the irregular 3′ helix, form the binding surface for p50. Each p50 monomer uses the same surface to recognize the distorted RNA major groove as observed in the κB DNA/p50 RHR complex, resulting in strikingly similar interfaces. The structure reveals how the aptamer specifically selects p50 and discriminates against p65. We also discuss the physiological implications of RNA binding by (p50)2.

Yeast genetic selections to optimize RNA decoys for transcription factor NF-κB

In vitro-selected RNA aptamers are potential inhibitors of disease-related proteins. Our laboratory previously isolated an RNA aptamer that binds with high affinity to human transcription factor NF-κB. This RNA aptamer competitively inhibits DNA binding by NF-κB in vitro and is recognized by its target protein in vivo in a yeast three-hybrid system. In the present study, yeast genetic selections were used to optimize the RNA aptamer for binding to NF-κB in the eukaryotic nucleus. Selection for improved binding to NF-κB from RNA libraries encoding (i) degenerate aptamer variants and (ii) sequences present at round 8 of 14 total rounds of in vitro selection yielded RNA aptamers with dramatically improved in vivo activity. Furthermore, we show that an in vivo-optimized RNA aptamer exhibits specific “decoy” activity, inhibiting transcriptional activation by its NF-κB target protein in a yeast one-hybrid assay. This decoy activity is enhanced by the expression of a bivalent aptamer. The combination of in vitro and in vivo genetic selections was crucial for obtaining RNA aptamers with in vivo decoy activity.

Mechanisms of DNA bending

Genome packaging and gene regulation require DNA bending. Recent developments in the elucidation of the mechanisms involved in DNA bending include new X-ray structures (most notably that of the mammalian nucleosome) wherein DNA is bent, controversy surrounding interpretation of DNA-bending experiments with basic-leucine zipper proteins, studies of electrostatic effects in DNA bending, and the design of artificial DNA-bending ligands.

HMG Proteins and DNA Flexibility in Transcription Activation

ABSTRACT The relative stiffness of naked DNA is evident from measured values of longitudinal persistence length (∼150 bp) and torsional persistence length (∼180 bp). These parameters predict that certain arrangements of eukaryotic transcription activator proteins in gene promoters should be much more effective than others in fostering protein-protein interactions with the basal RNA polymerase II transcription apparatus. Thus, if such interactions require some kind of DNA looping, DNA loop energies should depend sensitively on helical phasing of protein binding sites, loop size, and intrinsic DNA curvature within the loop. Using families of artificial transcription templates where these parameters were varied, we were surprised to find that the degree of transcription activation by arrays of Gal4-VP1 transcription activators in HeLa cell nuclear extract was sensitive only to the linear distance separating a basal promoter from an array of bound activators on DNA templates. We now examine the hypothesis that this unexpected result is due to factors in the extract that act to enhance apparent DNA flexibility. We demonstrate that HeLa cell nuclear extract is rich in a heat-resistant activity that dramatically enhances apparent DNA longitudinal and torsional flexibility. Recombinant mammalian high-mobility group 2 (HMG-2) protein can substitute for this activity. We propose that the abundance of HMG proteins in eukaryotic nuclei provides an environment in which DNA is made sufficiently flexible to remove many constraints on protein binding site arrangements that would otherwise limit efficient transcription activation to certain promoter geometries.

Mechanism of DNA flexibility enhancement by HMGB proteins

The mechanism by which sequence non-specific DNA-binding proteins enhance DNA flexibility is studied by examining complexes of double-stranded DNA with the high mobility group type B proteins HMGB2 (Box A) and HMGB1 (Box A+B) using atomic force microscopy. DNA end-to-end distances and local DNA bend angle distributions are analyzed for protein complexes deposited on a mica surface. For HMGB2 (Box A) binding we find a mean induced DNA bend angle of 78°, with a standard error of 1.3° and a SD of 23°, while HMGB1 (Box A+B) binding gives a mean bend angle of 67°, with a standard error of 1.3° and a SD of 21°. These results are consistent with analysis of the observed global persistence length changes derived from end-to-end distance measurements, and with results of DNA-stretching experiments. The moderately broad distributions of bend angles induced by both proteins are inconsistent with either a static kink model, or a purely flexible hinge model for DNA distortion by protein binding. Therefore, the mechanism by which HMGB proteins enhance the flexibility of DNA must differ from that of the Escherichia coli HU protein, which in previous studies showed a flat angle distribution consistent with a flexible hinge model.

Novel insights from hybrid LacI/GalR proteins: family-wide functional attributes and biologically significant variation in transcription repression

LacI/GalR transcription regulators have extensive, non-conserved interfaces between their regulatory domains and the 18 amino acids that serve as ‘linkers’ to their DNA-binding domains. These non-conserved interfaces might contribute to functional differences between paralogs. Previously, two chimeras created by domain recombination displayed novel functional properties. Here, we present a synthetic protein family, which was created by joining the LacI DNA-binding domain/linker to seven additional regulatory domains. Despite ‘mismatched’ interfaces, chimeras maintained allosteric response to their cognate effectors. Therefore, allostery in many LacI/GalR proteins does not require interfaces with precisely matched interactions. Nevertheless, the chimeric interfaces were not silent to mutagenesis, and preliminary comparisons suggest that the chimeras provide an ideal context for systematically exploring functional contributions of non-conserved positions. DNA looping experiments revealed higher order (dimer–dimer) oligomerization in several chimeras, which might be possible for the natural paralogs. Finally, the biological significance of repression differences was determined by measuring bacterial growth rates on lactose minimal media. Unexpectedly, moderate and strong repressors showed an apparent induction phase, even though inducers were not provided; therefore, an unknown mechanism might contribute to regulation of the lac operon. Nevertheless, altered growth correlated with altered repression, which indicates that observed functional modifications are significant.

DNA mimicry by a high-affinity anti-NF-κB RNA aptamer

The binding of RNA molecules to proteins or other ligands can require extensive RNA folding to create an induced fit. Understanding the generality of this principle involves comparing structures of RNA before and after complex formation. Here we report the NMR solution structure of a 29-nt RNA aptamer whose crystal structure had previously been determined in complex with its transcription factor target, the p502 form of NF-κB. The RNA aptamer internal loop structure has pre-organized features that are also found in the complex, including non-canonical base pairing and cross-strand base stacking. Remarkably, the free RNA aptamer structure possesses a major groove that more closely resembles B-form DNA than RNA. Upon protein binding, changes in RNA structure include the kinking of the internal loop and distortion of the terminal tetraloop. Thus, complex formation involves both pre-formed and induced fit binding interactions. The high affinity of the NF-κB transcription factor for this RNA aptamer may largely be due to the structural pre-organization of the RNA that results in its ability to mimic DNA.

DNA bending by a phantom protein

BACKGROUND Despite its stiffness, duplex DNA is extensively bent and folded during packaging and gene expression in biological systems. Modulation of the electrostatic repulsion between phosphates in the DNA backbone may be important in the bending of DNA by proteins. Here, we analyze the shape of DNA molecules that have been modified chemically to mimic the electrostatic consequences of a bound protein. RESULTS We have simulated salt bridges between DNA phosphates and cationic amino acid sidechains of a phantom protein by tethering ammonium cations to one face of the DNA helix. Tethered ammonium cations, but not neutral acetylated controls, induce DNA to bend toward its neutralized surface. CONCLUSIONS The shape of DNA molecules bearing a laterally-asymmetric distribution of tethered cations agrees qualitatively with theoretical predictions and with results previously obtained using neutral phosphate analogs. These data suggest principles that might be applied to the design of artificial DNA-bending proteins.

Selection and characterization of anti-NF-κB p65 RNA aptamers

NF-kappaB transcription factors include a group of five mammalian proteins that form hetero- or homodimers and regulate hundreds of target genes involved in acute inflammation, HIV-1 transcription activation, and resistance to cancer therapy. We previously used in vitro selection to develop a small RNA aptamer (anti-p50) that binds the DNA-binding domain of NF-kappaB p50(2) with low nanomolar affinity but does not bind NF-kappaB p65(2). Here, we report the in vitro selection of anti-NF-kappaB p65 RNA aptamers using parallel in vitro selections with either a fully randomized RNA library or a degenerate RNA library based on the primary sequence of the 31-nucleotide anti-p50 RNA aptamer. We report the characterization of these aptamers with respect to NF-kappaB target specificity, affinity, minimal sequence requirements, secondary structure, and competition with DNA kappaB sites. These results expand opportunities for artificial inhibition of NF-kappaB transcription factor dimers containing p65 subunits.

DNA bridging and looping by HMO1 provides a mechanism for stabilizing nucleosome-free chromatin.

The regulation of chromatin structure in eukaryotic cells involves abundant architectural factors such as high mobility group B (HMGB) proteins. It is not understood how these factors control the interplay between genome accessibility and compaction. In vivo, HMO1 binds the promoter and coding regions of most ribosomal RNA genes, facilitating transcription and possibly stabilizing chromatin in the absence of histones. To understand how HMO1 performs these functions, we combine single molecule stretching and atomic force microscopy (AFM). By stretching HMO1-bound DNA, we demonstrate a hierarchical organization of interactions, in which HMO1 initially compacts DNA on a timescale of seconds, followed by bridge formation and stabilization of DNA loops on a timescale of minutes. AFM experiments demonstrate DNA bridging between strands as well as looping by HMO1. Our results support a model in which HMO1 maintains the stability of nucleosome-free chromatin regions by forming complex and dynamic DNA structures mediated by protein–protein interactions.