Synergistic activation of transcription by bacteriophage lambda cI protein and E. coli cAMP receptor protein.

Two heterologous prokaryotic activators, the bacteriophage lambda cI protein (lambda cI) and the Escherichia coli cyclic AMP receptor protein (CRP), were shown to activate transcription synergistically from an artificial promoter bearing binding sites for both proteins. The synergy depends on a functional activation (positive control) surface on each activator. These results imply that both proteins interact directly with RNA polymerase and thus suggest a precise mechanism for transcriptional synergy: the interaction of two activators with two distinct surfaces of RNA polymerase.

DNA-binding specificity is a major determinant of the activity and toxicity of zinc-finger nucleases.

The engineering of proteins to manipulate cellular genomes has developed into a promising technology for biomedical research, including gene therapy. In particular, zinc-finger nucleases (ZFNs), which consist of a nonspecific endonuclease domain tethered to a tailored zinc-finger (ZF) DNA-binding domain, have proven invaluable for stimulating homology-directed gene repair in a variety of cell types. However, previous studies demonstrated that ZFNs could be associated with significant cytotoxicity due to cleavage at off-target sites. Here, we compared the in vitro affinities and specificities of nine ZF DNA-binding domains with their performance as ZFNs in human cells. The results of our cell-based assays reveal that the DNA-binding specificity--in addition to the affinity--is a major determinant of ZFN activity and is inversely correlated with ZFN-associated toxicity. In addition, our data provide the first evidence that engineering strategies, which account for context-dependent DNA-binding effects, yield ZFs that function as highly efficient ZFNs in human cells.

DNA-binding Specificity Is a Major Determinant of the Activity and Toxicity of Zinc-finger Nucleases.

The engineering of proteins to manipulate cellular genomes has developed into a promising technology for biomedical research, including gene therapy. In particular, zinc-finger nucleases (ZFNs), which consist of a nonspecific endonuclease domain tethered to a tailored zinc-finger (ZF) DNA-binding domain, have proven invaluable for stimulating homology-directed gene repair in a variety of cell types. However, previous studies demonstrated that ZFNs could be associated with significant cytotoxicity due to cleavage at off-target sites. Here, we compared the in vitro affinities and specificities of nine ZF DNA-binding domains with their performance as ZFNs in human cells. The results of our cell-based assays reveal that the DNA-binding specificity-in addition to the affinity-is a major determinant of ZFN activity and is inversely correlated with ZFN-associated toxicity. In addition, our data provide the first evidence that engineering strategies, which account for context-dependent DNA-binding effects, yield ZFs that function as highly efficient ZFNs in human cells.

Identifying and modifying protein-DNA and protein-protein interactions using a bacterial two-hybrid selection system.

A bacterial two-hybrid system based on transcriptional activation in E. coli has recently been described. A variety of different protein-DNA and protein-protein interactions from bacteria, yeast, and humans have been studied using this bacterial-based system. The method, because it is based in bacteria, offers significant advantages relative to its yeast counterpart including the ability to analyze complex libraries > 10(8) in size, ease of use, and speed. The ability to easily and rapidly process very large libraries make this system a powerful tool for identifying, modifying, or optimizing protein-DNA and protein-protein interactions.

Activation of prokaryotic transcription through arbitrary protein-protein contacts.

Many transcriptional activators in prokaryotes are known to bind near a promoter and contact RNA polymerase, but it is not clear whether a protein-protein contact between an activator and RNA polymerase is enough to activate gene transcription. Here we show that contact between a DNA-bound protein and a heterologous protein domain fused to RNA polymerase can elicit transcriptional activation; moreover, the strength of this engineered protein-protein interaction determines the amount of gene activation. Our results indicate that an arbitrary interaction between a DNA-bound protein and RNA polymerase can activate transcription. We also find that when the DNA-bound 'activator' makes contact with two different components of the polymerase, the effect of these two interactions on transcription is synergistic.

Synergistic activation of transcription by Escherichia coli cAMP receptor protein.

Activation of gene expression in eukaryotes generally involves the action of multiple transcription factors that function synergistically when bound near a particular target gene. Such effects have been suggested to occur because multiple activators can interact simultaneously with one or more components of the basal transcription machinery. In prokaryotes, examples of synergistic effects on transcription are much more limited and can often be explained by cooperative DNA binding. Here we show that the Escherichia coli cAMP receptor protein (CRP) functions synergistically to activate transcription from a derivative of the lac promoter that bears a second CRP-binding site upstream of the natural binding site. We present evidence indicating that cooperative DNA binding of two CRP dimers does not account for the magnitude of the observed cooperative activation. We suggest, instead, that the two dimers stimulate transcription directly by contacting two distinct surfaces of RNA polymerase simultaneously. Thus, synergistic activation by CRP may provide a relatively simple model for examining the molecular basis of such effects in higher organisms.

A bacterial two-hybrid selection system for studying protein-DNA and protein-protein interactions.

We have developed a bacterial "two-hybrid" system that readily allows selection from libraries larger than 10(8) in size. Our bacterial system may be used to study either protein-DNA or protein-protein interactions, and it offers a number of potentially significant advantages over existing yeast-based one-hybrid and two-hybrid methods. We tested our system by selecting zinc finger variants (from a large randomized library) that bind tightly and specifically to desired DNA target sites. Our method allows sequence-specific zinc fingers to be isolated in a single selection step, and thus it should be more rapid than phage display strategies that typically require multiple enrichment/amplification cycles. Given the large library sizes our bacterial-based selection system can handle, this method should provide a powerful tool for identifying and optimizing protein-DNA and protein-protein interactions.

Genetic strategy for analyzing specificity of dimer formation: Escherichia coli cyclic AMP receptor protein mutant altered in its dimerization specificity.

Many transcriptional regulators function in homo- or heterodimeric combinations. The same protein can carry out distinct regulatory functions depending on the partner with which it associates. Here, we describe a mutant of the Escherichia coli cAMP receptor protein (CRP) that has an altered dimerization specificity; that is, mutant/mutant homodimers form preferentially over wild-type/mutant heterodimers. CRP dimerization involves the formation of a parallel coiled-coil structure, and our CRP mutant bears an amino acid substitution affecting the first "d" position residue within the alpha-helix that mediates CRP dimerization. The genetic strategy we used to isolate this CRP altered dimerization specificity (ADS) mutant is generalizable and could be utilized to isolate ADS mutants of other dimeric transcriptional regulators.