Poster: Rhythms

Abs # 380: Structure and circadian regulation of the unique sigma factor-like gene in Chlamydomonas reinhardtii

Presenter:	Carter, Matthew L., mattcarter@mail.utexas.edu
Authors	Carter, Matthew L. (A)   Herrin, David L (A)
Affiliations:	(A): Section of Molecular Cell and Developmental Biology, University of Texas at Austin

In higher plants, the transcription of plastid genes is mediated by at least two types of RNA polymerase (RNAP); a plastid-encoded eubacterial-type RNAP in which promoter specificity is conferred by sigma factors encoded by a nuclear multigene family, and a single-subunit bacteriophage-type RNAP encoded in the nucleus. In contrast, green algae such as Chlamydomonas reinhardtii appear to possess only the bacterial-type enzyme. Since transcription of much, if not most, of the chloroplast genome in C. reinhardtii is regulated by the circadian clock, we sought to identify sigma factor genes that may be responsible for this nuclear regulation. In this paper, we describe the characterization of a nuclear gene (RpoD) encoding a putative sigma factor. In addition to a predicted chloroplast transit peptide at the N-terminus, the C-terminal half of the RPOD protein contains 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 found in ~40% of sigma factors examined, indicating they may be of general significance. Genomic southern blots and BLAST searches of the nuclear genome (JGI version 2) and EST databases indicate there is only one sigma factor-like gene in C. reinhardtii, rather than the multiple genes found in other photosynthetic organisms. The expression of RpoD mRNA showed a circadian rhythm with the maximum level occurring immediately prior to or during the peak in chloroplast transcription. An antibody to a synthetic peptide of RPOD is being generated, and will be used to analyze levels of this protein during the L-D cycle.