Abstract
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.
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Acknowledgments
We thank D.L. Herrin (University of Texas-Austin) for kindly providing a peptide antibody against CrRpoD, and T. Bollenbach for assistance in developing the refolding technique for CrRpoD. Work at the Boyce Thompson Institute was supported by NSF International award 0089253, and NSF award MCB 0091020. Work at the Humboldt University was supported by the Deutsche Forschungsgemeinschaft (SFB 429 and We 1535/6-4). A.-V.B. was supported by a travel grant from the DAAD to A.W. (Weihe-315/PPP).
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Communicated by F.-A. Wollman
Accession number: the DNA sequence reported in this paper is available from EMBL under accession number AJ627180.
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Bohne, AV., Irihimovitch, V., Weihe, A. et al. Chlamydomonas reinhardtii encodes a single sigma70-like factor which likely functions in chloroplast transcription. Curr Genet 49, 333–340 (2006). https://doi.org/10.1007/s00294-006-0060-7
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DOI: https://doi.org/10.1007/s00294-006-0060-7