Abstract
Reporter gene-based expression systems have been intensively used in plants for monitoring the activity of gene promoters. However, rRNA transcripts are unable to efficiently express a reporter gene due to a lack of a 5' cap. Because of this obstacle, plant rRNA gene promoters are less well characterized to this day. We developed a virus-based reporter system to characterize the Nicotiana benthamiana rRNA (NbrRNA) gene promoter. The system utilizes the cap-independent translation strategy of viral genomic mRNA and uses the virus-expressed green fluorescent protein (GFP) as an indicator of the rRNA gene promoter activity in virus-infected plants. Based on the reporter system, some characteristics of the N. benthamiana rRNA gene promoter were revealed. The results showed that the strength of the NbrRNA gene promoter was lower than that of the cauliflower mosaic virus (CaMV) 35S promoter, a well-characterized polymerase II promoter. The sequences between −77 and +42 are sufficient for the NbrRNA gene promoter-mediated transcription and the NbrRNA gene promoter may lack the functional upstream control element (UCE). Interestingly, NbrRNA gene promoter activity was increased when the 35S enhancer was introduced. An intron-excision mediated assay revealed that the NbrRNA gene promoter can be inefficiently used by RNA polymerase II in N. benthamiana cells. This virus-based reporter system is easier to operate and more convenient when compared with the previously Pol I promoter assays. And it offers a promising solution to analyzing the functional architecture of plant rRNA gene promoter.
Highlights
Transcription of nuclear DNA to RNA in eukaryotic cells is carried out by at least three distinct RNA polymerases (Roeder and Rutter, 1969)
RRNA gene promoters generally consist of a core promoter sequence, which directs basal transcription and contains sequences from about −40 to +10 relative to the transcription initiation site (TIS), and an upstream control element (UCE), which strengthens ribosomal RNAs (rRNAs) transcription and typically extends to approximately −150 (Russell and Zomerdijk, 2005)
The green fluorescence was observed at infiltrated zones with all 5'-deletion promoter constructs (Figure 1D), indicating the NbrRNA gene promoter can drive viral RNA-based vector to express green fluorescent protein (GFP) in N. benthamiana and this viral RNA-based reporter system could be used to measure rRNA gene promoter activity in plants
Summary
Transcription of nuclear DNA to RNA in eukaryotic cells is carried out by at least three distinct RNA polymerases (Roeder and Rutter, 1969). RRNA gene promoters generally consist of a core promoter sequence, which directs basal transcription and contains sequences from about −40 to +10 relative to the transcription initiation site (TIS), and an upstream control element (UCE), which strengthens rRNA transcription and typically extends to approximately −150 (Russell and Zomerdijk, 2005). The consensus sequence motif TATATA(A/G)GGG around TIS is highly conserved in plants (Doelling and Pikaard, 1996). The sequences between −55/−33 and +6 suffice to mediate transcription initiation of rRNA genes in Arabidopsis thaliana (Doelling and Pikaard, 1995). The data obtained from another study showed that the foreign rRNA gene promoter can be used by the host Pol II in an inefficient way and with an altered initiation site (Doelling and Pikaard, 1996)
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
