Transcriptional organization of the large and the small ATP synthase operons, atpI/H/F/A and atpB/E, in Arabidopsis thaliana chloroplasts

Abstract

The ATP synthase is a ubiquitous enzyme which is found in bacteria and eukaryotic organelles. It is essential in the photosynthetic and respiratory processes, by transforming the electrochemical proton gradient into ATP energy via proton transport across the membranes. In Escherichia coli, the atp genes coding for the subunits of the ATP synthase enzyme are grouped in the same transcriptional unit, while in higher plants the plastid atp genes are organized into a large (atpI/H/F/A) and a small (atpB/E) atp operon. By using the model plant Arabidopsis thaliana, we have investigated the strategy evolved in chloroplasts to overcome the physical separation of the atp gene clusters and to coordinate their transcription. We show that all the identified promoters in the two atp operons are PEP dependent and require sigma factors for specific recognition. Our results indicate that transcription of the two atp operons is initiated by at least one common factor, the essential SIG2 factor. Our data show that SIG3 and SIG6 also participate in transcription initiation of the large and the small atp operon, respectively. We propose that SIG2 might be the factor responsible for coordinating the basal transcription of the plastid atp genes and that SIG3 and SIG6 might serve to modulate plastid atp expression with respect to physiological and environmental conditions. However, we observe that in the sigma mutants (sig2, sig3 and sig6) the deficiency in the recognition of specific atp promoters is largely balanced by mRNA stabilization and/or by activation of otherwise silent promoters, indicating that the rate-limiting step for expression of the atp operons is mostly post-transcriptional.

Complex Processing Patterns of mRNAs of the Large ATP Synthase Operon in Arabidopsis Chloroplasts

“…Two bands (a′ and a) of around 1300 and 1500 nucleotides, respectively, were detected, that from the size could correspond to atpI mono-cistronic mRNAs. In our previous analyses we have observed that only the larger of the two bands (a) disappeared in the sig2 mutant [7]. These data suggested that the larger and very faint band (a) corresponds to the SIG2 dependent primary atpI transcripts (starting at position −229) while the smaller band (a′) corresponds to the processed atpI mRNAs (with the 5′ end at position −48, as based on our cRT-PCR data).…”

“…They were also revealed by DNA probes hybridizing to the atpH coding region (data not shown) or the 3′ UTR (Figure 2B, probe 2). Our previous analyses have shown that only the larger of these two bands (b) was SIG2 dependent, corresponding to the SIG2-dependent primary atpI/H co-transcripts (starting at position −229, see below), while the smaller band (b′) likely corresponds to the processed atpI/H mRNAs (with the 5′ end at position −48 on the basis of the cRT-PCR data in this manuscript) [7].…”

“…The small filled rectangles correspond to the sRNAs [3], [4]. Upward (with right tip) and downward arrows indicate the 5′ end positions of primary and processed transcripts, respectively, with negative numbers corresponding to the distance from the ATG as described [7]. Left and right-directed arrow heads correspond to primers for the cRT-PCR analysis.…”

“…Top panel: the schematic presentation of the atpI/H/F/A operon is as in Figures 1A and 2A. Downward and upward arrows indicate the position of 5′ and 3′ ends of the atp transcripts, respectively, on the bases of cRT-PCR (this manuscript) and primer-extension data [7]. For the 3′ ends of the atpI/H (b′), atpH (e) and atpH/F (f) mRNAs, both the distance from the corresponding stop codon and from the start codon of the downstream cistrons is indicated.…”

“…Recently, we have analyzed the transcriptional organization of the plastid large ATP synthase ( atp ) operon of Arabidopsis thaliana that consists of the genes atpI/H/F/A [7]. This operon is transcribed by the plastid-encoded RNA polymerase (PEP) starting from a promoter at −225/229 in front to the atpI coding sequence, which is dependent on the Sigma factor 2 (SIG2).…”