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

Abstract

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 approximately 40% 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 RPODmay be the only sigma factor gene in C. reinhardtii. The levels of RPODmRNA 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 RPODis under circadian clock control. These results suggest that regulation of RPODexpression contributes to the circadian clock's control of chloroplast transcription.

The sulfur acclimation SAC3 kinase is required for chloroplast transcriptional repression under sulfur limitation in Chlamydomonas reinhardtii

“…It was recently proposed that in Chlamydomonas, circadian rhythms of chloroplast transcription are regulated by transcriptional oscillations of RPOD (19). In contrast, the data presented here show a constant level of RPOD mRNA (Fig.…”

“…Although PTK has not been tested with Sig1, recombinant Sig1 can be phosphorylated in vitro by human casein kinase 2 (CK2) (data not shown), consistent with Sig1 containing multiple consensus CK2 recognition sites (as noted in ref. 19). We also note that alternative models for Sac3 function exist, including one where it is phosphorylated under ϩS conditions and dephosphorylated when activated (38).…”

“…PEP additionally requires a sigma factor for promoter recognition, which is encoded by a gene family in land plants (18) but by a single gene in Chlamydomonas. This gene, RPOD, encodes the protein termed Sig1 or CrRpoD (19,20). The fact that Chlamydomonas chloroplasts possess PEP but not NEP and that Sig1 apparently acts as the only specificity factor would appear to facilitate global control of chloroplast transcription activity.…”

Function of plastid sigma factors in higher plants: regulation of gene expression or just preservation of constitutive transcription?

“…In the unicellular algae Chlamydomonas reinhardtii no NEP is present in chloroplasts, only one single sigma factor seems to exist (Carter et al 2004;Bohne et al 2006) and regulation is limited mainly to the post-transcriptional level (Rochaix 2002;Choquet and Wollman 2002). The fact that chloroplasts of higher plants harbour six different sigma factors weakens the concept of mainly post-transcriptional regulation of plastid gene expression.…”

Real-Time Monitoring of Chloroplast Gene Expression by a Luciferase Reporter: Evidence for Nuclear Regulation of Chloroplast Circadian Period

“…The mRNA level of nucleus-encoded chloroplast factor oscillates in a circadian manner in several photosynthetic species (10,19,30). In Chlamydomonas, the mRNA levels of a large number of nucleus-encoded chloroplast ribosomal proteins are under circadian control (23), and cytoplasmic protein synthesis is required for transcription of several chloroplast genes during circadian peaks (22).…”