Chlamydomonas reinhardtii encodes a single sigma70-like factor which likely functions in chloroplast transcription.
Chlamydomonas reinhardtii EST clones encoding a protein highly similar to prokaryotic sigma factors and plant sigma-like factors (SLFs) were used to isolate a BAC clone containing the full-length gene CrRpoD. The gene is likely to be single-copy, in contrast to small gene families encoding SLFs in plants. The CrRpoD mRNA comprises 3,033 nt with an open reading frame of 2,256 nt, encoding a putative protein of 752 amino acids with a molecular mass of 80.2 kDa. The sequence contains conserved regions 2-4 typically found in sigma factors, and an unusually long amino terminal extension, which by in silico analysis has properties of a chloroplast transit peptide. Expression of CrRpoD was confirmed by immunodetection of a 85 kDa polypeptide in a preparation enriched for chloroplast proteins. To demonstrate functionality in transcription initiation, a recombinant CrRpoD-thioredoxin fusion protein was reconstituted with E. coli RNA polymerase core enzyme and tested in vitro. This chimeric holoenzyme specifically bound the spinach psbA and Chlamydomonas rrn16 promoters in gel mobility shift assays and exhibited specific transcription initiation from the same two promoters, providing evidence for the role of CrRpoD as a functional transcription factor.
Bohne
Alexandra-V
AV
Institut für Biologie, Humboldt-Universität, Chausseestr. 117, 10115, Berlin, Germany.
Irihimovitch
Vered
V
Weihe
Andreas
A
Stern
David B
DB
D020418:Algal Proteins / Q000235:genetics* / Q000502:physiology*; D000595:Amino Acid Sequence; D000818:Animals; D016825:Chlamydomonas reinhardtii / Q000235:genetics*; D002736:Chloroplasts / Q000235:genetics*; D003001:Cloning, Molecular; D012321:DNA-Directed RNA Polymerases / Q000235:genetics* / Q000502:physiology*; D020224:Expressed Sequence Tags; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012808:Sigma Factor / Q000235:genetics* / Q000502:physiology*; D013879:Thioredoxins / Q000235:genetics; D014158:Transcription, Genetic
D020418:Algal Proteins; D011993:Recombinant Fusion Proteins; D012808:Sigma Factor; D013879:Thioredoxins; C081775:RNA polymerase sigma 70; D012321:DNA-Directed RNA Polymerases