Detailed Architecture of the Barley ChloroplastpsbD-psbC Blue Light-responsive Promoter

Minkyun Kim,Karen E Thum,Daryl T Morishige,John E Mullet

doi:10.1074/jbc.274.8.4684

Abstract

The photosystem II reaction center chlorophyll protein D2, is encoded by the chloroplast gene psbD. PsbD is transcribed from at least three different promoters, one which is activated by high fluence blue light. Sequences within 130 base pairs (bp) of the psbD blue light-responsive promoter (BLRP) are highly conserved in higher plants. In this study, the structure of the psbD BLRP was analyzed in detail using deletion and site-directed mutagenesis and in vitro transcription. Deletion analysis showed that a 53-bp DNA region of the psbD BLRP, from -57 to -5, was sufficient for transcription in vitro. Mutation of a putative prokaryotic -10 element (TATTCT) located from -7 to -12 inhibited transcription from the psbD BLRP. In contrast, mutation of a putative prokaryotic -35 element, had no influence on transcription. Mutation of a TATATA sequence located between the barley psbA -10 and -35 elements significantly reduced transcription from this promoter. However, site-directed mutation of sequences located between -35 and -10 had no effect on transcription from the psbD BLRP. Transcription from the psbD BLRP was previously shown to require a 22-bp sequence, termed the AAG-box, located between -36 and -57. The AAG-box specifically binds the protein complex AGF. Site-directed mutagenesis identified two different sequence motifs in the AAG-box that are important for transcription in vitro. Based on these results, we propose that positive factors bind to the AAG-box and interact with the chloroplast-encoded RNA polymerase to promote transcription from the psbD BLRP. Transcription from the psbD BLRP is thus similar to type II bacterial promoters that use activating proteins to stimulate transcription. Transcription of the psbD BLRP was approximately 6. 5-fold greater in plastid extracts from illuminated versus dark-grown plants. This suggests that light-induced activation of this promoter in vivo involves factors interacting with the 53-bp psbD BLRP in vitro.

Highlights

The photosystem II reaction center chlorophyll protein D2, is encoded by the chloroplast gene psbD
We propose that positive factors bind to the AAG-box and interact with the chloroplast-encoded RNA polymerase to promote transcription from the psbD blue light-responsive promoter (BLRP)
These results indicate that the ϳ53-bp DNA region from Ϫ57 to Ϫ5 is sufficient for transcription from the psbD BLRP in vitro

Summary

Introduction

The photosystem II reaction center chlorophyll protein D2, is encoded by the chloroplast gene psbD. We propose that positive factors bind to the AAG-box and interact with the chloroplast-encoded RNA polymerase to promote transcription from the psbD BLRP. These results indicate that the Ϫ10 sequence is required for transcription from the psbD BLRP in vitro, whereas the Ϫ35 and Ϫ10 elements are both required for transcription from the rbcL and psbA promoters.

Results

Conclusion