In eukaryotic organisms, genes encoding messenger RNA are transcribed by RNA polymerase II (Pol II) with the help of general transcription factors (GTFs) (1
). To initiate transcription, the TATA-binding protein (TBP) first binds to DNA. Next, TFIIA and TFIIB bind to TBP and the core promoter, followed by TFIIF and Pol II. Finally, TFIIE and TFIIH join to complete the assembly of a Pre-Initiation Complex (PIC) (2
). In addition, transcription of most genes requires activators, because the formation of chromatin makes the transcriptional ground state restrictive (4
). There are two general mechanisms by which activators facilitate transcription: directly through interacting with members of the Pol II entourage or indirectly through altering chromatin structure (5
). In either case, the location at which the activator binds to DNA determines which gene is activated. Therefore, a transcription activator requires a minimum of two domains, a DNA-binding domain and an activation domain. According to the recruitment model, the target of an activation domain is likely to be either a GTF or a subunit of the Pol II complex. Among the GTFs, TBP and TFIIB are most strongly implicated as the targets of activators (5
Although the general scheme of transcriptional activation by recruitment has been delineated in broad outline, certain important details remain elusive due to experimental difficulties. For example, an activator often interacts with multiple GTFs, and its effect on a single factor is therefore difficult to isolate; artificial recruitment of a single factor through fusion to a DNA-binding domain does not yield any information about the site or sites on the factor contacted by activators (5
). Many protein activators share a common amino-acid composition rather than exhibiting similarity in sequence or structure (7
); many RNA sequences have been isolated based on their capability to activate transcription, but the mechanistic basis for this activity is unknown (8
). Both observations raised questions regarding the specific features of surface topography that are essential for the function of an activation domain.
An understanding of the mechanism underlying a phenomenon should enable the design and construction of different systems that are able to reproduce that phenomenon. Therefore, deliberate creation of novel molecules with explicitly and strictly defined biological function is a reliable way to test our current knowledge. Following this principle, in the present study we implemented the mechanism of transcription activation by recruitment of a GTF using an RNA molecule assembled from refined and standardized parts, especially those derived from aptamers. To explore specificity inherent to both sides of the activator–target interface, we made use of a well-characterized site-specific aptamer as the activation domain of a synthetic activator.
RNA aptamers are generated in an in vitro
process emulating Darwinian evolution (10
). For many proteins, aptamers with a dissociation constant in the nanomolar range have been isolated. Because selection of an aptamer based on affinity for its target is performed outside the cellular and organismal milieu, the aptamer often interferes with the function of the protein when introduced into a living system (12
). Consequently, aptamers are routinely used as inhibitors of protein activity. Here we attempted to rationally convert this passive role of aptamers into an active one by placing an aptamer in a designed molecular context, in which it functions as one of several intentionally chosen interacting sites.
In particular, we constructed a ‘transcription activator RNA (taRNA)’ in the yeast Saccharomyces cerevisiae
, analogous to a protein-based activator. Using a set of modular parts in a combinatorial manner, we specifically implemented the mechanism of transcriptional activation by recruiting TFIIB to the promoter of reporter genes in the chromatin environment. For this purpose, an RNA aptamer for TFIIB (13
), which is a potent inhibitor of transcription by default, was converted into the activation domain of the taRNA by design. With the help of several other constructs originally designed for the yeast three-hybrid system (14
), we were able to show that this synthetic RNA molecule activated transcription at a level comparable to a protein activator. Comparing the results obtained by creating new RNA-based factors with those obtained by reorganizing existing protein-based factors allowed us to highlight some critical features of this mechanism.