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Circadian regulation of chloroplast transcription in Chlamydomonas is accompanied by little or no fluctuation in RPOD levels or core RNAP activity

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Abstract

In Chlamydomonas growing under 24 h light–dark cycles, chloroplast transcription is under circadian clock control, and peaks early in the morning. The peak (but not trough) requires ongoing cytoplasmic translation, as it is sensitive to cycloheximide (CH). The chloroplast transcriptional apparatus in Chlamydomonas is simpler than in land plants, with only one type of RNA polymerase (RNAP, bacterial) and apparently only one sigma factor (RPOD). Core RNAP can be assayed in vitro with a non-sigma factor dependent template, and is sensitive to rifampicin. We developed a membrane-based assay for RNAP activity, and used it to determine that core activity is only weakly affected by pre-treating cells with CH. Moreover, core chloroplast RNAP activity was steady during a 24 h light–dark cycle. Levels of the sigma factor (RPOD) were examined using western blots, and found to fluctuate less than 25 % during light–dark cycles. These data indicate that circadian regulation of chloroplast transcription is distinct from regulation by sulfur availability, which involves significant changes in RPOD levels. The implications of this data for hypotheses that purport to explain the circadian control mechanism are discussed.

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Acknowledgments

This research was supported by grants from the Texas Advanced Research Program (003658-0144-2007) and the Robert A. Welch Foundation (F-1164) to DLH, and by an Undergraduate Research Fellowship to BV.

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Author notes

  1. Kristina M. Mahan

    Present address: University of California at Davis, Davis, CA, 95616, USA

  2. Brad E. Venghaus

    Present address: University of Texas Health Science Center at San Antonio, San Antonio, TX, 78229, USA

  3. Matthew L. Carter

    Present address: Agilent Technologies, Cedar Creek, TX, 78612, USA

Authors and Affiliations

  1. Section of Molecular Cell & Developmental Biology, Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX, 78712, USA

    Ryo Kawazoe, Kristina M. Mahan, Brad E. Venghaus, Matthew L. Carter & David L. Herrin

  2. 205 W. 24th St, STOP A6700, Austin, TX, 78712-1240, USA

    David L. Herrin

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  1. Ryo Kawazoe
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  4. Matthew L. Carter
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  5. David L. Herrin
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Corresponding author

Correspondence to David L. Herrin.

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Fig. S1

Determining the response and capacity of the DEAE-membrane for the core RNAP reaction. a The core RNAP assay used 32P-UTP (to estimate RNA synthesis) and a highly active RNAP extract (PEG-prec) under standard conditions, except for increasing the reaction vol by 500 l. The terminated reactions were aliquoted (as indicated) into separate tubes, adjusted to 400 l with 1× stop solution (0.5 M NaH2PO4 pH 7.2, 50 mM EDTA) and applied to the DEAE-membrane. Each horizontal row of dots is from the same reaction mixture, and a set of unincubated (U) reactions (100 μl) were included to estimate background. b Plot of the membrane-bound signal versus the reaction mixture vol (per well). The data points are averages of the three separate reactions, which did not vary more than 7%. They were normalized relative to the 200-μl reaction, which was considered to be 100%. (DOC 25 kb)

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Kawazoe, R., Mahan, K.M., Venghaus, B.E. et al. Circadian regulation of chloroplast transcription in Chlamydomonas is accompanied by little or no fluctuation in RPOD levels or core RNAP activity. Mol Biol Rep 39, 10565–10571 (2012). https://doi.org/10.1007/s11033-012-1942-z

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  • DOI: https://doi.org/10.1007/s11033-012-1942-z

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