In general, native transactivating domains are relatively large, dozens of amino acids in length. However, fragments of native activator proteins have been described. An 11-amino-acid-long acidic domain from the RelA subunit of NFκ-B is capable of minimal activating behavior when fused to the Gal4 DNA-binding domain (Gal4BD) . The sequence, when joined together in tandem arrays, is as potent as the wild type RelA transactivator. Wu et al. showed that fragments of the Gal4 activation region between 17 and 41 amino acids in length provide activation functions of a magnitude in rough proportion to their length . This result suggests that short sequences are capable of stimulating transcription when complexed with promotor elements and that their effect is amplified by increasing the total content of such short sequences.
A series of papers has explored properties of random sequences, including random peptides, fused to DNA binding domains in the context of transcriptional activation assays similar to those used in the two-hybrid system. Ma and Ptashne isolated a set of E. coli DNA fragments that, when fused to Gal4BD, promote transcription . All (15/15) active sequences examined by the authors encode predicted polypeptides that are acidic, and a peptide as small as 12 amino acids was seen. One sequence (B42), 72 residues long, was shown to function as an activator when transferred to the lexA DNA binding domain. Yang et al. serendipitously discovered among a set of retinoblastoma-binding peptides three activators of length 16 residues that function as Gal4BD C-terminal fusions . All three are acidic, with charge -3 or -4. Giniger and Ptashne found that a designed acidic, amphipathic alpha helix of 15 amino acids activates transcription when fused to the Gal4BD . Using in vitro transcription assays, Gerber et al. demonstrated that homopolymeric stretches as short as 10 residues of glutamine or proline fused to the Gal4BD function as activators . Finally, Lu et al. screened for C-terminal Gal4BD peptides, 8 amino acids long. Only one basic residue was observed among the set of activator peptides, and no peptide had a net predicted positive charge .
An NMR structure for the lexA DNA-binding domain has been presented . The structure is compact and globular, and probably interferes only minimally with fused transactivating sequences. In addition, the success of the yeast two-hybrid technique suggests that the geometric constraints on transactivation are not severe (i.e., the DNA-binding elements and the transactivating portion of the molecule (or complex) do not require precise positioning with respect to each other). Because Gal4-GFP fusions are unstable, we were unable to analyze the GFP/peptide insertions identified in the lexA hybrid proteins in the context of the Gal4BD (not shown). However, a peptide selected in the context of the amino terminal portion of GFP functioned effectively when this GFP segment was removed and the peptide was grafted directly onto lexA. Due to differences in expression levels among C-terminal and internal GFP peptide constructs, we did not consider it useful to pursue more quantitative transactivation studies [22, 27].
Activator peptides were present in the two libraries at remarkably high frequencies – 10-3 in the case of the C-terminal library, approximately the same frequency observed by Lu et al. . The 5-fold lower frequency of activators in the lexA-GFP/peptide library may indicate a role for the C-terminal negative charge, the specific geometry, or the conformational flexibility of the fused (vs. inserted) peptides. Considerably more than half of the activator sequences we observed had a large excess of negative charge, a finding that is consistent with the involvement of acidic residues in the activation process . However, we observed several examples of charge neutral and basic peptides (Table 3). As a group, the sequences did not display obvious patterns of amphipathic helicity based on the lack of 3, 4 or 7 repeat units. Glutamine and proline residues were not significantly over-represented in the sequence set. Instead, in addition to glutamate, large hydrophobic amino acids were abundant. This result accords with mutagenesis experiments that have pinpointed acidic residues and phenylalanine as special contributors to transactivation [23, 28].
Two questions are raised by our and other studies of peptide activators: (i) what is the minimum size of an activator? and, (ii) what is the significance of the extreme sequence diversity evidenced by transactivating peptides? We identified and tested an 11-residue-long activator that functioned in two different contexts: at the C-terminus of a truncated GFP molecule and attached directly to lexA. Because we did not direct our screen at smaller peptides, we suspect that a deliberate effort to identify smaller activators would be fruitful. Indeed, Lu et al. found 8-mer activators, though none was tested for modularity . Nonetheless, the tendency for C-terminal activators to be longer than the average library peptide suggests a preference for length in the transactivation function .
The recruitment model for transcriptional activation stipulates that eukaryotic transcription is driven mainly by recruitment of preformed RNA polymerase holoenzyme to specific sites on the DNA . According to this model, peptides may bind one of numerous proteins that guide holoenzyme to the lexA binding site. Such proteins include components of holoenzyme and perhaps other factors [see ref. ]. Short peptides can bind proteins with reasonably high affinity (see ref. , for review). Because transactivation is a process that involves protein-protein interactions, it is not surprising that peptides linked to DNA-binding domains function as transactivators by bridging interactions that promote transcription.
The wide range of primary sequences and sequence features described to date among peptide activators supports the recruitment model. Given the diversity of proteins that comprise holoenzyme, this model implies that many sequences of different types would be competent activators; their sole requirement would be to bind proteins within holoenzyme . The acidic peptides may interact in a manner akin to native activators . But the basic ones may, for example, bind other proteins that contain acidic regions, thereby forming a bridge to holoenzyme. Alternatively, these basic peptides may bind different regions of the holoenzyme component(s) recognized by acidic peptides, or entirely different subunits .
Apart from scientific interest, these findings may have value in creating tools for drug and drug target discovery using the yeast two-hybrid system. Isolation of a panel of small peptides with different structural properties could improve the performance of the system by providing an option for multiple screens using a variety of peptides. This option might decrease false negatives and false positives if applied intelligently. For example, a series of positively charged, negatively charged and neutral peptides may enhance the accuracy of the method. In addition, short activating sequences may prove useful in the context of isolating peptides that interact with other proteins. Peptides that bind specific proteins of therapeutic interest in the yeast two-hybrid system have a variety of applications in drug development. These peptides could be used as lead-ins for creation of peptido-mimetics. Alternatively, they might provide reagents for high-throughput displacement screens . Typically, such peptides are isolated from libraries on the activation domain of the two hybrid system . By substituting small pepticals for the activation function, it may be possible to define binding sites with higher resolution in the context, for instance, of the reverse two-hybrid approach ; Caponigro et al., unpublished). Small peptides active in the yeast two-hybrid system may be synthesized by Merrifield procedures, labeled, and used directly in screens for small molecules . This approach might avoid laborious testing of hybrid protein/peptides to determine which peptides bind independent of their fusion partner. Finally, non-peptidic molecules that bind in a sequence-specific manner to DNA have been fused to 20- and 16-residue peptides. These hybrid molecules activate transcription [9, 31]. It may be possible to produce small-molecule activators (or repressors) that bind DNA specifically, but also activate transcription of adjacent genes through a non-peptidic, more drug-like moiety.