An AU-box motif upstream of the SD sequence of light-dependent psbA transcripts confers mRNA instability in darkness in cyanobacteria

Abstract

The psbA2 gene of a unicellular cyanobacterium, Microcystis aeruginosa K-81, encodes a D1 protein homolog in the reaction center of photosynthetic Photosystem II. The expression of the psbA2 transcript has been shown to be light-dependent as assessed under light and dark (12/12 h) cycling conditions. We aligned the 5'-untranslated leader regions (UTRs) of psbAs from different photosynthetic organisms and identified a conserved sequence, UAAAUAAA or the 'AU-box', just upstream of the SD sequences. To clarify the role of 5'-upstream cis-elements containing the AU-box for light-dependent expression of psbA2, a series of deletion and point mutations in the region were introduced into the genome of heterologous cyanobacterium Synechococcus sp. strain PCC 7942, and psbA2 expression was examined. A clear pattern of light-dependent expression was observed in recombinant cyanobacteria carrying the K-81 psbA2 -38/+36 region (which includes the minimal promoter element and a light-dependent cis-element with the AU-box), +1 indicating the transcription start site. A constitutive pattern of expression, in which the transcripts remained almost stable under dark conditions, was obtained in cells harboring the -38/+14 region (the minimal element), indicating that the +14/+36 region with the AU-box is important for the observed light-dependent expression. Point mutations analyses within the AU-box also revealed that changes in number, direction and identity (as assayed by adenine/uridine nucleotide substitutions) influenced the light-dependent pattern of expression. The level of psbA2 transcripts increased markedly in CG- or deletion-box mutants in the dark, strongly indicating that the AU- (AT-) box acts as a negative cis-element. Furthermore, characterization of transcript accumulation in cells treated with rifampicin suggests that psbA2 5'-mRNA is unstable in the dark, supporting the view that the light-dependent expression is controlled at the post-transcriptional level. We discuss various mechanisms that may lead to altered mRNA stability such as the binding of factor(s) or ribosomes to the 5'-UTR and possible roles of the AU-box motif and the SD sequence.

Figure 1

A sequence alignment of the psbA 5′-upstream region. The promoter (box), transcription start site (+1, arrow), AT-box (shading), extended AT-box (light shading), ribosome-binding site (SD, bold and underlined) and initiation codon (ATG, bold and italics) are shown. For each sequence, accession numbers are given in parentheses, as follows: K-81 psbA2 (D84228), PCC 7942 psbAII (X04617), PCC 6803 psbA2 (X13547), PCC 7120 psbAII (U21332), Chlamydomonas psbA (X01424), Spinach psbA (J01442) and Tobacco psbA (Z00044). Positions for possible stem–loop structures (paired arrows) and processing sites (arrowheads) are indicated. Multiple ribosome-binding sites (RBS1, RBS2, RBS3) of Tobacco are also shown.

Figure 2

Deletion constructs. Cloned K-81 psbA2 5′-upstream region with the promoter, AT-box and SD positions indicated. Recombinant Synechococcus sp. strain PCC 7942, designated strain AG, contains the psbA2–lacZ fusion constructs whose psbA2 fragments were amplified by PCR with a set of GKA primers indicated in the first column. Specific primers (D2II and lacZ-R1) were used for primer extension in this study. The results of primer extension with lacZ-R1 (ZR1) and relative signal intensities (% mRNA) corresponding to the psbA2 transcripts under continuous white light (same condition as in lane 1 of Fig. 3) are indicated. The relative β-galactosidase activities are also shown.

Figure 3

The psbA2 transcripts in the deletion mutants grown under light/dark conditions. Total RNA (5 µg) was isolated from the AG407 to AG500 (psbA2–lacZ) or 1016 (rpoD1–lacZ) recombinants grown under white light at 4 h and darkness at 4 h in the L/D (12/12 h) cycle, then subjected to primer extension with the lacZ-R1 primer for psbA2–lacZ transcript or Primer 1 for rpoD1–lacZ transcript which was transcribed from rpoD1 Promoter 1 (6). Relative signal intensities corresponding to the transcripts synthesized by primer extension are shown on the right. Other details are described in the text.

Figure 4

AT- (AU-) box mutants. Relevant characteristics of constructs and sequences mutagenized at the AT-box are shown. Total RNA (5 µg) was isolated from the AG451–AG461 recombinants, which are derived from AG500 [psbA2(–404/+113)–lacZ], grown under continuous white light (corresponding to the conditions in lane 1 of Fig. 5), then subjected to primer extension with the lacZ-R1 primer. Relative transcripts were measured from the transcription start point (+1) of psbA2. The relative β-galactosidase activities are also shown.

Figure 5

The psbA2 transcripts in the AT-box mutants grown under light/dark conditions. Total RNA (5 µg) was isolated from the AG451–AG500 (psbA2–lacZ) recombinants grown under light and dark conditions, then subjected to primer extension with the lacZ-R1 primer. Relative signal intensities corresponding to the psbA2 transcripts are shown on the right. Other details are as in Figure 3.

Figure 6

mRNA stability in AT-box mutants and effects of antibiotics on the transcripts. (A) The AG transformants (psbA2–lacZ) were cultivated under white light until mid-log phase, then exposed to darkness (12 h) and white light (12 h). Total RNA (5 µg) was prepared from the strains, and subjected to primer extension with the lacZ-R1 (for AG407, AM990) or D2II (for AG458, AG461) primer (see Fig. 2). (B) Relative psbA2 transcripts in (A) were schematically shown as circles (AG407), triangles (AG458) and squares (AG461), respectively. (C) AG407 cells were cultivated as in (A) and the BG11 cell culture was supplemented with antibiotic (Rif, 200 µg/ml or Cm, 250 µg/ml) to at the end of the light condition (0 min). (D) Relative psbA2 transcripts in (C) were also shown as circles (without antibiotics), triangles (+ Rif) and squares (+ Cm), respectively.

Figure 7

Initiation of cyanobacterial psbA2 transcription and translation with AT- and AU-boxes. A summarized scheme for psbA2 expression in strain K-81 is presented and discussed in the text. +, positive effect; –, negative effect (dark > light).