Structure, circadian regulation and bioinformatic analysis of the unique sigma factor gene in Chlamydomonas reinhardtii

In higher plants, the transcription of plastid genes is mediated by at least two types of RNA polymerase (RNAP); a plastid-encoded bacterial RNAP in which promoter specificity is conferred by nuclear-encoded sigma factors.. and a nuclear-encoded phage-like RNAP. Green algae. however, appear to possess only the bacterial enzyme. Since transcription of much, if not most. of the chloroplast genome in Chlamydomonas reinhardtii is regulated by the circadian clock and the nucleus. we sought to identify sigma factor genes that might be responsible for this regulation. We describe a nuclear gene (RPOD) that is predicted to encode an 80 kDa protein that, in addition to a predicted chloroplast transit peptide at the N-terminus. has the conserved motifs (2.1-4.2) diagnostic of bacterial sigma-70 factors. We also identified two motifs not previously recognized for sigma factors, adjacent PEST sequences and a leucine zipper. both suggested to be involved in protein-protein interactions. PEST sequences were also found in similar to40% of sigma factors examined, indicating they may be of general significance. Southern blot hybridization and BLAST searches of the genome and EST databases suggest that RPOD may be the only sigma factor gene in C. reinhardtii. The levels of RPOD mRNA increased 2-3-fold in the mid-to-late dark period of light-dark cycling cells. just prior to, or coincident with, the peak in chloroplast transcription. Also. the dark-period peak in RPOD mRNA persisted in cells shifted to continuous light or continuous dark for at least one cycle, indicating that RPOD is under circadian clock control. These results suggest that regulation of RPOD expression contributes to the circadian clock's control of chloroplast transcription.