Major cis-regulatory elements for rice bidirectional promoter activity reside in the 5'-untranslated regions

Abstract

Bidirectional promoters are defined as those that regulate adjacent genes organized in a divergent fashion (head to head orientation) and separated by <1 kb. In order to dissect bidirectional promoter activity in a model plant, deletion analysis was performed for seven rice promoters using promoter-reporter gene constructs, which identified three promoters to be bidirectional. Regulatory elements located in or close to the 5'-untranslated regions (UTR) of one of the genes (divergent gene pair) were found to be responsible for their bidirectional activity. DNA footprinting analysis identified unique protein binding sites in these promoters. Deletion/alteration of these motifs resulted in significant loss of expression of the reporter genes on either side of the promoter. Changes in the motifs at both the positions resulted in a remarkable decrease in bidirectional activity of the reporter genes flanking the promoter. Based on our results, we propose a novel mechanism for the bidirectionality of rice bidirectional promoters.

Keywords: (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid); 5′-Untranslated region; 6-carboxyfluorescein; 6FAM; Agrobacterium mediated transient expression assay; AmTEA; BDPs; BSA; Bidirectional promoters; Bovine serum albumin; CREs; Cis regulatory elements; Degree centigrade; Enhanced green fluorescent protein gene; HEPES; HEX; Hexachloro-fluorescein; INTS7; Integrator complex subunit 7; MS; Microliter; Micromolar; Milliliter; Murashige and Skoog; OD; Optical density; PBS; Phosphate buffered saline; Protein binding site; Quantitative polymerase chain reaction; RPM; RT-PCR; Red fluorescent protein gene; Reporter genes; Reverse transcription polymerase chain reaction; Revolutions per minute; Rice; TSS; Transcriptional start site; UTR; Untranslated region; egfp; gus; mL; qPCR; rfp; °C; β-glucuronidase gene; μL; μM.