The RITE assay: identifying effectors that target the transcription machinery using phage display technology.

We describe an approach using phage display to identify effectors (activators and repressors) of transcription based on the particular component of the general transcription machinery that they target. We refer to this approach as the reverse identification of transcriptional effectors (RITE) assay. A library of phages containing cDNA-encoded peptides displayed on their surfaces is screened using as the target a specific region of one of the general transcription factors (e.g., the C terminus of hTAFII135). The amino acid sequence encoded by the cDNA of an interacting phage is determined and analyzed in a database homology search to identify known or novel factors that may interact with the target protein. Candidate effectors from the homology search are synthesized from recombinant clones and tested for their abilities to bind to the target protein and to functionally modulate transcription in vivo when co-expressed with the transcriptional target protein. Because the RITE assay is a direct measure of the interactions between general transcription proteins and their effectors, it has an advantage over the well-known yeast two-hybrid system, which is not amenable to identifying transcription factor interactions.

Oligomerization properties of the viral oncoproteins adenovirus E1A and human papillomavirus E7 and their complexes with the retinoblastoma protein.

Human papillomavirus 16 E7 (HPV16 E7) and adenovirus 5 E1A (Ad5 E1A) are encoded by highly divergent viruses yet are functionally similar in their ability to bind the retinoblastoma (pRB) tumor suppressor protein, causing the aberrant displacement of E2F trancription factors. The amino acid residues of HPV16 E7 that are necessary for stability, for inhibition of pRB function, and for cell transformation are also necessary for E7 oligomerization. However, neither the specific oligomerization state of HPV16 E7 nor of Ad5 E1A as a function of pRB-binding has been characterized. To gain insight into HPV16 E7 and Ad5 E1A oligomerization properties, sedimentation equilibrium experiments were performed with recombinant HPV16 E7 and Ad5 E1A proteins. These studies reveal that, despite the overall functional similarities between these proteins, monomers, dimers, and tetramers of HPV16 E7 were detected while only reversible monomer-dimer association was identified for Ad5 E1A. The apparent K(d(monomer)-(dimer)) of HPV16 E7 is approximately 100-fold lower than that of a comparable region of Ad5 E1A, and it is concluded that under physiological protein concentrations HPV16 E7 exists primarily as a dimer. Sedimentation equilibrium experiments of pRB/Ad5 E1A and of pRB/HPV16 E7 complexes demonstrate that the tight association of pRB with the viral oncoproteins does not disturb their inherent oligomerization properties. Taken together, this study demonstrates significant differences between the Ad5 E1A and HPV16 E7 oligomerization states that are potentially related to their distinct structures and specific mechanisms of pRB-inactivation.

The transactivation domain of adenovirus E1A interacts with the C terminus of human TAF(II)135.

The CR3 activation domain of the human adenovirus E1A protein stimulates transcription by forming protein-protein interactions with DNA sequence-specific binding factors and components of the TFIID complex. Here, we demonstrate that CR3 can complex with the extreme C-terminal 105 amino acids of the human TATA box binding-factor-associated protein, hTAF(II)135. Furthermore, the C-terminal region of hTAF(II)135 can block transcriptional stimulation from an E1A-inducible promoter in vivo. This ability of the C terminus of hTAF(II)135 to bind CR3 and to inhibit E1A-inducible activation is highly specific. These results demonstrate for the first time that a discrete fragment of a mammalian TBP-associated factor which targets a specific activator can impair the stimulation of transcription.

The viral oncoproteins Ad5 E1A, HPV16 E7 and SV40 TAg bind a common region of the TBP-associated factor-110.

A function shared by the adenovirus E1A, papillomavirus E7 and SV40 TAg oncoproteins is their ability to interfere with normal cell growth by interacting with members of the retinoblastoma protein family. In this study, we show that each of these oncoproteins can also bind to the 921 amino acid TBP-associated factor-110 (TAF-110). The significance of the binding is underscored by the observation that each oncoprotein binds to the same 77 amino acid carboxyl region of TAF-110. In the case of E1A and TAg, this finding is consistent with their abilities to stimulate transcription initiation, in part, through their known interactions with TBP. While it is not clear whether E7 can also activate promoters through protein:protein interactions with components of the transcription initiation complex, our demonstration that E7 can bind to TAF-110, as well as TBP, suggests that E7 may modulate the expression of specific promoters which could contribute to the pathogenesis of human papillomavirus.

Mapping interactions between the catalytic domain of resolvase and its DNA substrate using cysteine-coupled EDTA-iron.

Single cysteine-substituted mutants of gamma delta resolvase have been covalently modified using a novel sulfhydryl-specific EDTA derivative, EDTA-2-aminoethyl 2-pyridyl disulfide (EPD). Iron, chelated by the coupled EDTA and in the presence of reducing agent, generates reactive oxygen species that result in localized cleavage of the DNA to which resolvase is bound. The procedure provides valuable information on two fronts. First, it allows the identification of regions or surfaces of the protein that are in close proximity to DNA even though they may not be part of the DNA-binding domain. Second, it allows identification of the portions of DNA that are closest to each EDTA-derivatized cysteine, since the DNA cleavages observed are highly localized and their efficiency drops rapidly as a function of the distance between the EDTA-Fe complex and the deoxyribose target. We have used the procedure to investigate the interaction of gamma delta resolvase with the three DNA binding sites that constitute its recombination substrate, res. The data indicate that the two N-terminal domains of a resolvase dimer interact symmetrically with site I, which contains the recombination cross-over point, but asymmetrically with the accessory sites, II and III. The patterns of DNA cleavage obtained with several different EDTA-coupled mutants have enabled us to propose a model for the interaction between resolvase and site I.

Interactions between the trp repressor and its operator sequence as studied by base analogue substitution.

A series of modified trp operator sequences has been prepared by the incorporation of seven different base analogues. Four of the analogues allow the site-specific deletion of functional groups present on the dA-dT and dT-dA base pairs at positions -4/+4 and -5/+5 in the trp operator. The remaining three analogues permit the incorporation of structural analogues of the native dA-dT or dG-dC base pairs. The duplex operator sequences all exhibit Tm values well above ambient temperature (48-70 degrees C), and these values generally correlate very well with the number of interstrand hydrogen bonds present. The affinity between the trp repressor and 14 modified operator sequences was examined using a recently developed alkaline phosphatase protection assay. The results from the analogue sequences used in this study suggest that the structure of the dA-dT or dT-dA base pairs at positions -4/+4 and -5/+5, respectively, has relatively little effect upon the solution binding by the trp repressor, but the protein is very sensitive to the orientation of the amino and carbonyl functional groups at the -4/+4 positions, which are involved in the formation of an interbase hydrogen bond present in the major groove. (The term structure in this case refers to the hydrogen bonding structure of the base pairs. We recognize that the introduction of conservative functional group deletions or reversals may affect other structural criteria such as hydration.) The deletion of individual functional groups from the operator sequence suggests that the carbonyl at dT+4 is critical for formation of the high-affinity sequence-specific complex. Additionally, the thymine methyl group at dT+4 and the N7 nitrogen of dA+5 appear to be critical contacts necessary for high-affinity binding by the repressor. The thymine carbonyl and the adenine N7 nitrogen are each responsible for approximately -1.5 kcal/mol of apparent free energy of binding. The thymine methyl provides a somewhat smaller contribution of -0.7 kcal/mol. Deletion of either of the adenine amino groups at dA-4 or dA+5 results in a sequence that binds to the repressor with a higher affinity than observed with the native sequence; this can be explained in that the functional groups lost are not critical for binding, and the resulting increased flexibility of the operator, or the creation of a more hydrophobic surface at these sites, enhances van der Waals contacts between the protein and the nucleic acid.

DNA curvature does not require bifurcated hydrogen bonds or pyrimidine methyl groups.

Short tracts of the homopolymer dA.dT confer intrinsic curvature on the axis of the DNA double helix. This phenomenon is assumed to be a consequence of such tracts adopting a stable B'-DNA conformation that is distinct from B-form structure normally assumed by other DNA sequences. The more stable B' structure of dA.dT tracts has been attributed to several possible stabilizing factors: (1) optimal base stacking interactions consequent upon the high propeller twist, (2) bifurcated hydrogen bonds between adjacent dA.dT base-pairs, (3) stacking interactions involving the dT methyl groups, and finally (4) a putative spine of ordered water molecules in the minor groove. DNA oligodeoxynucleotides have been synthesized that enable these hypotheses to be tested; of particular interest is the combination of effects due to bifurcation (2) and methylation of the pyrimidines nucleotides (3). The data indicate that neither bifurcated hydrogen bonds nor pyrimidine methyl groups nor both are essential for DNA curvature. The data further suggest that the influence of the minor groove spine of hydration on the B'-formation is small. The experiments favor the hypothesis that base stacking interactions are the dominant force in stabilizing the B'-form structure.