The complexity in the long BDP mechanism
The TATA box is a fundamental promoter element within eukaryotes, which is crucial in initiating transcription for all three RNA polymerases. The canonical TATA box sequence is typically recognized as 5’-TATAWAW-3’(Kwak et al. , 2013). However, a bidirectional TATA box (5’-TATATAT-3’) has been identified to possess transcriptional capabilities in both directions, demonstrating heightened transcriptional activity(Xu, Thali and Schaffner, 1991). Analyzing split tests for BDPs lacks specific guiding principles. In this study, we employed the TATA box as a pivotal marker for segmenting the PPik-H4 . The TATA box sequence at 462-469 bp in the Pik-H4 promoter is a bidirectional TATA box, comprising two bidirectional TATA boxes situated on both the sense strand and the antisense strand, respectively (Figure 2E). Mutation of this segment resulted in a significant reduction in downstream gene expression on both sides of the promoter. Examining the promoter activity of a 100 bp sequence encompassing the integrated bidirectional TATA box, along with an adjacent bidirectional TATA box, revealed that the sequence indeed possessed bidirectional activity. Nevertheless, it could not fully restore the transcriptional activity of Pik1-H4and Pik2-H4 in both directions back to the levels of NP (Figure 2D-F, BT1). Moreover, the recovery of expression intensity in downstream reporter genes was observed after adding the TSSs for both genes (Figure 2D-F, BT2), indicating that the 100 bp sequence primarily functions as a bidirectional regulatory element, with TSSs being indispensable for gene expression.
Transcriptional regulation is a complex process involving a multitude of factors, including TFBSs, cis elements, SNPs(Liu et al. , 2019), neighboring transcription units(Lee et al. , 2014) and various epigenetic modifications (Chen et al., 2014). In contrast to the human genome, where approximately 67% of BDPs are shorter than 300 bp (Trinklein et al., 2004), plant BDPs tend to be longer (Krom and Ramakrishna, 2008), introducing greater complexity to their regulatory mechanisms. Some reports on split BDPs focus on segments within 1500 bp (Banerjee et al., 2013; Rao and Virupapuram, 2021), and identify crucial promoter regions that contribute to the basal expression of BDGs under specific experimental conditions. However, these studies often overlook the intricate cis regulatory elements for gene regulation or specificity. In our research, even by combining the minimal core promoter regions of Pik1-H4 andPik2-H4 , we could not achieve BDP activity equivalent to NP levels (Figure 2A-C, B3). Intriguingly, this combination even increased GFP expression, indicating the intricate interplay of cis elements within PPik-H4 . Given that Pik BDPs encompass lengths of 2492 bp or 2542 bp, further assays to split and analyze promoters are necessary to unravel the underlying regulatory mechanisms of bidirectional NLR gene pairs. Unveiling the functionality of BDPs holds significant implications for synthetic biology, but it’s evident that more extensive research is needed to understand these processes comprehensively.