Figure 4.

Mutational analysis of the conserved S-box in the psbH 5' UTR. (A) Mutagenesis of the psbH 5' UTR. The sequence of the psbB 5' UTR is shown at the top for comparison, and the sequence of the psbH 5' UTR below it, with the translation start codons in bold. The conserved ‘S-box’ is highlighted in bold and underlined. The sequences of the sRNAs are shown in lowercase above the respective sequences. The black arrows show the positions of the major 5'-ends (see Figure 2 and Supplementary Figure S3). The transformation vector (p41A) carries a selectable aadA spectinomycin resistance cassette inserted downstream of psbH (Materials and Methods). The Chlamydomonas host strain (ΔH) carries a substitution of the psbH gene and its 5' UTR to ensure that transformants carrying the aadA marker also bear the desired mutation. (B) Immunoblot analysis of PSII components. Total protein extracts were analyzed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and immunoblotting with the antibodies indicated on the left. From left to right, the samples are the WT, the mbb1-222E mutant (mbb1), the ΔH transformation host (ΔH), the psbB m26-31 mutant (m26-31), the transformation host rescued with a WT psbH vector (p41A) and the three mutants shown in (A). (C) RNA blot hybridization analysis of the psbB/T/H transcripts. Total RNA was analyzed by agarose gel electrophoresis and RNA blot hybridization using the psbH and psbB probes as indicated on the left. A probe for psaA was used as a control. (D) Mapping of the psbH transcripts. The schematic map shows the positions of the 5'-end at −54/−53 with a black arrow and of the 3' termini with white arrows [according to (30)]. The upstream probe (5') and coding sequence probe (cod) are depicted below the map as open bars. Total RNA from the WT and a representative mutant (ApaS) was analyzed by agarose gel electrophoresis and RNA blot hybridization using the probes indicated at the bottom of each panel.