Architecture and transcriptional activity of the initiator element of the TATA-less RPL21 gene

Abstract

The nuclear RPL21 gene coding for the plastid ribosomal protein L21 is a TATA-less gene that is overexpressed in a leaf-dependent manner by the specific usage of a strong initiator called P1. We have previously shown that the RPL21 core promoter spanning from -23 to +104 relative to P1 start site activates transcription in the same manner as does the full promoter. Here, we present results of experiments aimed at deciphering the RPL21 core promoter architecture. Results of transient expression using various 5' deletions of the core promoter fused to a chloramphenicol acetyl transferase (CAT) reporter gene show that 34 bp encompassing the P1 initiation site (from -23 to +11) are required for full transcription activation. Gel-shift analysis shows that five DNA/protein complexes (C1-C5) are formed on this 34-bp fragment with protein extracts from green tissues. C1 is the major complex present during seed germination. The other complexes are present in young leaf tissues suggesting a role in transcription activation. Linker scanning mutagenesis experiments show that the five complexes form two independent groups: I (C1-C3) and II (C4 and C5), with a common binding site located on P1. Using transgenic plants, we show that three nucleotides encompassing the P1 start site and three trinucleotides necessary for group I binding are determinant for RPL21 activation. These results identify an unusually compact core structure, which is centred on P1 initiation site and is responsible for transcription activation. A model of the architecture of this region is presented.