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. 2001 May 1;29(9):1835–1843. doi: 10.1093/nar/29.9.1835

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

Ganesh K Agrawal 1, Hideki Kato 1, Munehiko Asayama 1, Makoto Shirai 1,a
PMCID: PMC37257  PMID: 11328866

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.

INTRODUCTION

It is generally accepted that the ancestors of cyanobacteria gave rise to plant plastids by endosymbiotic events, conferring the photosynthetic ability found in present-day algae and plants. Members of the genus Microcystis (Synechocystis) are cyanobacteria (blue-green algae) which can perform oxygenic photosynthesis involving two photosystems, PS I and PS II, as in higher plants. One such member, the unicellular, colony-forming Microcystis aeruginosa K-81 (hereafter referred to as K-81) strain was isolated (1) and characterized with regard to its biological and genetic properties (2,3). The K-81 strain possesses multiple psbA genes in its chromosome. Previously, we cloned one gene family member, psbA2 (4), whose deduced amino acid sequence exhibited extensive homology to a D1 homolog (a core protein in PS II) corresponding to the type II form as defined in Synechococcus sp. strain PCC 7942 (hereafter called PCC 7942) psbAII and psbAIII. Although we observed specific transcription from the Escherichia coli consensus-type promoter of cyanobacterium K-81 psbA2 in both K-81 and E.coli, transcript accumulation was light-dependent in K-81 whereas it was constitutive in E.coli grown in either the light or the dark (L/D) (5). Previously, specific transcription from the psbA2 promoter was also detected in vitro using RNA polymerases containing either E.coli principal σ factor σ70, or K-81 principal σ factor σA1 found in K-81 cells grown under L/D cycles (5–7). This pattern of transcription required a minimal sequence from –38 to +14 containing the core promoter (–38 to +6), along with cis- up- (Cis-U: –80 to –39) and/or down- (Cis-D: +7 to +46) regulatory element(s) (5). Light-dependent and circadian rhythm-associated expression of psbA2 transcripts have been observed in both homologous K-81 and heterologous PCC 7942 cyanobacterial strains. Furthermore, psbA2 expression in a recombinant strain of PCC 7942, AG400, in which the region from –404 to +111 of psbA2 is transcriptionally fused to lacZ, exhibited a clear circadian-type rhythmic expression, while very little or no rhythmicity was observed in the second strain, AG429 (containing the reporter gene fusion from –38 to +14), suggesting that the region(s) around the promoter was required for the rhythmic expression (8). These results suggest that psbA2 is transcribed from a minimal promoter sequence under both light and dark conditions via a holoenzyme containing the principal σ factor in K-81. Therefore, sequences in the 5′-UTR appear to function as a negative regulatory cis-element required for light-dependent or circadian-associated oscillations in the level of psbA2 transcript accumulated in cyanobacteria.

The psbA genes of cyanobacteria are found as a multi-gene family in the chromosome. Its members are differentially transcribed in response to changes in light intensity. For example, transcripts of the psbAI gene, which encodes the form I-type D1 protein in PCC 7942, are predominantly expressed at low light intensity, whereas transcript levels of psbAII and psbAIII genes, encoding the form II-type D1 protein, rapidly increase upon a shift to high light intensity along with a correlative decrease in the psbAI transcript (9). Gene disruption analyses showed that only one of the three kinds of genes is necessary and produces sufficient D1 protein to support photosynthesis in actively growing cyanobacteria (10–13). In the alga Chlamydomonas and in higher plants, there is only one kind of psbA gene, corresponding to the cyanobacterial form II-type (Fig. 1), and it is encoded in the chloroplast genome. Light-responsive psbA gene expression in Chlamydomonas and higher plants is regulated in part at both post-transcriptional and translational levels via elements in the 5′-UTR (14–17). A combination of mRNA stability, processing and translational regulation facilitates the expression of chloroplast psbA genes in Chlamydomonas and higher plants, and the light-activated translation mediated by the 5′-UTR appears to be mechanistically a hybrid between prokaryotic and eukaryotic regulatory systems (18,19). A portion (+1 to +45) of the 5′-UTR of psbAII in strain PCC 7942 is conserved in the 5′-UTR sequence (+1 to +52) of the psbA2 gene of K-81, and confers high light induction and regulates mRNA stability (20,21).

Figure 1.

Figure 1

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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.

In this article, we aligned the 5′-upstream DNA sequences among psbA genes, and identified a conserved AU- (AT-) box motif encompassing a region just upstream of the ribosome-binding (SD) sequence. The 5′-upstream region from –38 to +36 containing the AU- (AT-) motif is essential for light-dependent expression. Mutational analyses indicated that the motif is important as a post-transcriptional negative regulatory element involved in light-responsive changes in transcript accumulation.

MATERIALS AND METHODS

Medium and culture conditions

The recombinant PCC 7942 strain was grown in BG11 liquid medium (10) containing 40 µg/ml of spectinomycin sulfate at 30°C under continuous white light illumination (1900 lux; Cont. L) until the mid-log phase (OD750 = 0.4 to 0.5) followed by either L/D cycles (1900 lux, L/D cycles of 12/12 h) or continuous white light illumination (1900 lux).

Site-directed mutagenesis

An EcoRI–HindIII fragment was isolated from pHNL7-up (22) carrying the psbA 5′-upstream region (–404/+111) and inserted into the EcoRI–HindIII sites of the M13mp19 vector (23) to create pGK4. Site-directed mutagenesis for constructs of the mutant AT- (AU-) box was performed as described previously (24) with single-stranded template DNA from pGK4 and the following oligonucleotides: GKA40 (for pAG451), 5′-TTGACAATTAAATACATATAAATACAGCAGAAATTATCCAATC-3′; GKA41 (for pAG452), 5′-GACAATTAAATAAATAGCAGAAATTATCC-3′; GKA44 (for pAG455), 5′-GAACCTTGACAATACATAAATTAGCAGAAATTATCCAATC-3′; GKA45 (for pAG456), 5′-GAACCTTGACAATTGTATTTATAGCAGAAATTATCCAATC-3′; GKA46 (for pAG457), 5′-GAACCTTGACAATCAAACAAATAGCAGAAATTATCCAATC-3′; GKA47 (for pAG458), 5′-GAACCTTGACAATCGGGCGGGTAGCAGAAATTATCCAATC-3′; GKA51 (for pAG461), 5′-CAAACAAAGAACCTTGACGCAGAAATTATCCAATCATGAC-3′.

Construction of plasmids and PCC 7942 mutants

For promoter-deletion plasmids, fragments of the 5′-upstream region of psbA2 were amplified by PCR with the template DNA of pHNL7-up and a set of primers as follows (Fig. 2): GKA7 (forward: 5′-TTGCCCCGGGCCCTTTACATAACTAT-3′) + GKA10 (reverse: 5′-GCGGAAGATCTATAATTTCTGCTATG-3′); GKA7 (forward) + GKA9 (reverse: 5′-GCGGAAGATCTTGACATGAATTTAAT-3′); GKA7 (forward) + GKA32 (reverse: 5′-CGGAAGATCTCTATGTATTTAATTGT-3′); GKA7 (forward) + GKA34 (reverse: 5′-CGGAAGATCTTCTTTGTTTGACATGA-3′); GKA38 (forward: 5′-GGCCCCCGGGGATCTCATAGAAACGA-3′) + GKA2 (reverse: 5′-GCGAAGATCTTCCACTGGCAGAACTG-3′). Since forward and reverse primers contain restriction enzymes for SmaI or BglII, the PCR fragments were digested with BglII and SmaI, then inserted into the SmaI–BglII sites of pAM990 (25) to yield pAG407, 410, 427, 429 and 500, respectively. To avoid products of PsbA2::LacZ translated from the psbA2 SD sequence and only synthesize the LacZ products (β-galactosidase, without PsbA2::LacZ) translated from the lacZ SD sequence, each psbA2 insert was out-frame fused to the lacZ reporter gene on the transcriptional fusion vector, pAM990 (Fig. 2). A 0.7 kb EcoRI (blunt-end by Klenow)–BglII fragment, which contains the 5′-upstream region of rpoD1 (6), was isolated from pKXCΔ1016B and inserted into the SmaI–BglII sites of pAM990, to create pD1Δ1016B (26). On the other hand, for a linker adaptation of the AT- (AU-) box mutant fragments, a series of resultant pAG plasmids (see above) as template DNA was subjected to PCR with a set of GKA38 and GKA2 primers. Each amplified 0.5 kb fragment (–404/+113) was digested with BglII and SmaI, inserted into the SmaI–BglII sites of pAM990 to make pAG451, 452, 455, 456, 457, 458 and 461, respectively. Sequences of the inserts on the pAM990 derivatives were verified. These plasmids or the original pAM990 vector were introduced into the chromosome of Synechococcus sp. strain PCC 7942 by natural transformation (27) and the recombinant cells, referred to as strains AG407∼500 (psbA2–lacZ), AM990 (lacZ only) and 1016 (rpoD1–lacZ), which are spectinomycin resistant, were obtained.

Figure 2.

Figure 2

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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.

RNA isolation and primer extension

Total RNA was isolated from 30 ml of cell culture by a hot phenol method as described previously (4). Primer extension analyses were also carried out with a lacZ-R1 (5′-AGTTGGGTAACGCCAGGGTTTTCCCA-3′) or a D2II (4) primer for the K-81 psbA2–lacZ transcripts, as described previously (4,5,8), except that the temperature was 50°C for the lacZ-R1 primer annealing. Primer 1 (5′-AAGGGTTAGTACATCGTGGG-3′) was also used for the K-81 rpoD1–lacZ transcript (6). Products of the extension were dissolved in 7 µl of stop solution, denatured at 95°C for 5 min and then 3 µl of aliquot was resolved on a 6% polyacrylamide gel containing 8 M urea.

β-Galactosidase assay

The recombinant PCC 7942 cells were cultivated under continuous white light illumination until mid-log phase (see above), then a portion of the culture was subjected to the assay described previously (26).

RESULTS

The 5′-upstream sequences and AU- (AT-) boxes of psbA genes

Recent studies have revealed the unique DNA structure of the 5′-upstream region of psbA2 and its role in light-dependent and circadian rhythm expression in M.aeruginosa K-81 (4,5,8). To characterize in more detail the structure of possible regulatory elements located upstream of the psbA gene, we aligned the promoter sequences and surrounding regions corresponding to the 5′-UTR from four cyanobacteria, Chlamydomonas, and two higher plant psbA genes (Fig. 1). Promoter sequences (tT–aca, Ta–––T) of the E.coli consensus type, and the ribosome-binding (SD) sequence are extensively conserved, as reported previously (5). Interestingly, AT-rich sequences, TAAATAAA, located immediately 5′ to the SD sequences on the non-transcribed strand are just as well conserved. We therefore designated this sequence the AT-box or the AU-box, for the sequence found in DNA or RNA, respectively. This extended AT-box containing TAAA was also found in the 5′-region of some psbA genes (Fig. 1). Although the cyanobacterial psbA 5′-UTRs are ∼30–40 nt shorter than those of Chlamydomonas or plants, they still contain several adenine or uracil tracts.

Identification of effective cis-elements on 5′-UTR

We previously established a recombinant system for analysis of the light-dependent expression of the psbA2 gene of cyanobacterium K-81 in PCC 7942, a heterologous, genetically amenable cyanobacterium (8). A series of deletion fragments of the psbA2 promoter region was constructed and introduced into the genome of PCC 7942 as single copy promoter–lacZ reporter gene fusions to facilitate identification of the cis-elements required for wild-type psbA expression in vivo (Fig. 2). The 5′-end mapping of psbA2–lacZ transcripts was performed using total RNA prepared from cells of recombinant strains grown in continuous light. The relative psbA2–lacZ mRNA levels were measured from signal intensities on a gel with the value from strain AG500 carrying the largest upstream sequence (–404/+113) designated as standard and set at 100% (Fig. 2). No activity was observed in strain AM990 carrying no promoter or in strain AG410 containing only the core promoter sequence (–38/+6). This result is compatible with previous in vitro data (5). The transcript level increased in strain AG407 (–38/+46) compared to that in AG500. Compared to the value from strain AG407, there was a slight decrease in transcript levels in AG427 (–38/+36), but further deletion (–38/+14) led to a marked increase. Relative β-galactosidase activities did not contradict the mRNA expression level results (Fig. 2). The region containing the SD sequence contributed to a high expression level of β-galactosidase in AG407. These results show that the region from +36 to +46 carrying the SD sequence is a positive cis-element and the region +14/+36 containing the AT-box is a cis-negative element, for psbA2 transcript accumulation (Fig. 1).

We further examined the effect of darkness and re-exposure to light on the expression of psbA2–lacZ fusion transcripts in various recombinant cyanobacterial strains (Fig. 3, lanes 2 and 3). For the initial part of the experiment, we measured transcript levels under continuous light (Fig. 3, lane 1), and obtained the same relative values as in the previous experiment shown in Figure 1, confirming the reproducibility of the differences between the constructs/strains. For any given fusion gene, the level measured under continuous light was designated as the basal level of expression. In AG500 (–404/+113), a clear pattern of light-dependent expression was observed, with no transcript detectable in the dark. The basal level was approximately two times higher in AG407 (–38/+46) than in AG500, and the transcript was weakly expressed in the dark. In AG427 (–38/+36), a robust pattern of light-dependent expression was again exhibited, showing that the –38/+36 region was sufficient for the light-dependent expression of psbA2. In contrast, the basal levels were increased in AG429 (–38/+14) and the transcripts were almost constitutively expressed in both dark and light conditions (Fig. 3, lanes 2 and 3), whereas no signal was detected in AG410 (–38/+6). From these results, we conclude that the –38/+14 region is an essential (minimal) cis-element for basal psbA2 transcription, and the +14/+46 region is required for light-dependent expression of the transcript. The +36/+46 region containing the SD sequence might enhance transcript stability, while the +14/+36 region (light-dependent cis-element) carrying the AT-box contributes to the clear pattern of light-dependent expression. In other words, the AT-box and SD sequence might be negative and positive cis-elements, respectively, for light-dependent expression of psbA2 transcript (Fig. 1). In contrast, in control strain 1016, harboring a reporter gene for K-81 rpoD1, transcripts accumulated in a constitutive manner regardless of light (Fig. 3, bottom), as shown previously (6). In this study, the expression of the K-81 rpoD1 transcript was also constant in the recombinant PCC 7942 cells grown in light and darkness. The reason for the weak pattern of rpoD1 transcript will be discussed later.

Figure 3.

Figure 3

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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.

Characterizations of the AT-box mutants

To further investigate the influence of the AT-box on light-dependent expression, a series of AT-box mutants were constructed and transformed into the heterologous cyanobacterium (Fig. 4), and the relevant transcripts examined under the same conditions (light and dark) as in Figure 3 (Fig. 5). The AT-box was mutagenized as follows: a tandem dimer was inserted in the same phase of the double-helix DNA strand (strain AG451); the cyanobacterial AT-box was replaced with the plant consensus AT-box sequence (TAAATAAA, Fig. 1) (strain AG452); the AT-box was inserted in the reverse direction on the non-transcribed strand; i.e. on the mRNA strand (strain AG455); the AT-box was placed in the reverse direction on the transcribed strand (strain AG456); thymidines (T) in the plant AT-box consensus sequence were converted to cytidines (C) (strain AG457); Ts were converted to Cs and adenines (A) were converted to guanidines (G) (strain AG458); the AT-box was deleted (strain AG461). AG500 showed an absolute, light-specific pattern of expression. The basal level decreased in AG451 and 452, indicating that an increase in the number of AT-boxes or replacement of the cyanobacterial sequence with the plant consensus sequence had an enhanced negative effect, supporting our conclusions from the results in Figures 2 and 3. In contrast, transcript levels were higher in AG457, 458 and 461 than in AG500 (Fig. 5, lane 1). In particular, in AG458 and 461, transcripts were expressed in the dark (Fig. 5, lane 2). Although light-induced expression was observed in AG457 (Fig. 5, lane 3), the amount was lower than that in AG500. From these results, we conclude that Ts (or Us) and A tracts in the AT-box are important for the negative effect on expression of transcript even in light. In AG455, transcripts accumulated in the dark, as in strains AG458 and AG461, but not in AG500, indicating that the direction of the AT-box is also important to the function of this element in regulating light-specific expression. In AG456, the transcripts were detected at the same level as in AG500 (Fig. 5, lane 1) but a low level of light-induced expression was observed as in AG457, reinforcing that the sequence direction of the AT-box affects not only mRNA instability in darkness but also transcript accumulation in light. The low expression level of transcripts in AG451, 456 and 457 (Fig. 5, lane 3) will be discussed later. Of note, we also obtained a similar pattern of β-galactosidase activities corresponding to psbA2–lacZ transcripts from the recombinant cells grown under the same culture conditions (Fig. 4).

Figure 4.

Figure 4

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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.

Figure 5

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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.

A negative effect of AT-box and mRNA instability in darkness

To confirm the negative effect of the AT- (AU-) box, we examined reporter gene transcript levels in various recombinant strains over a shorter time course so that the early stages of expression under different light intensities could be studied (Fig. 6). A clear pattern of loss and increase was detected within 90 min of transfer to darkness and upon re-exposure to light, respectively, in control strain AG407 (Fig. 6A and B). In contrast, the expression of reporter gene transcripts in AG458 and AG461 was almost constant and responded slowly compared to that in AG407. In addition, transcript levels were higher overall in AG458 and AG461 than AG407. This is strong confirmatory evidence that the AT- (AU-) box is a negative cis-element for light-responsive expression. Having more clearly defined the time course for the accumulation and loss of transcript under light and dark conditions, respectively, we next investigated the effects of antibiotics on these expression patterns (Fig. 6C and D). As in the experiment shown in Figure 6A, when no antibiotics were supplemented, a clear pattern of graded loss was observed in AG407 upon exposure to dark (Fig. 6C, top). To examine psbA2–lacZ mRNA stability, we added rifampicin (Rif), a bacteriocidal antibiotic that inhibits RNA synthesis by binding to and inhibiting the β-subunit of RNA polymerase. A marked and quick loss in transcript was observed within 10 min of the addition of Rif (Fig. 6C, middle). The amount of transcript expressed in darkness was about five times less than that in light, on addition of Rif (data not shown), indicating that the transcripts are degraded faster in the dark. These results further show that the psbA2 transcripts are unstable in the dark. In addition, that the loss of transcript in darkness is slow and graded in AG407 without Rif suggests that constitutive transcription from the wild-type psbA2 promoter occurs in the dark (Fig. 6C, top), further supporting the view that negative regulation of mRNA instability in darkness occurs at the post-transcriptional and/or translational step. Since chloramphenicol (Cm) inhibits protein synthesis by interacting with the 50S ribosomal subunit and inhibiting the peptidyltransferase reaction, large quantities of total cellular proteins synthesized before the addition of Cm might be sufficient to regulate the light-dependent expression in the dark (Fig. 6C, bottom). Interestingly, on addition of Cm, the amount of transcript increased, indicating that the mRNA might be stable in the presence of Cm even in darkness. These results imply that mRNA instability in darkness involves some negative factor(s) and the addition of Cm resulted in inhibition of the mRNA degradation.

Figure 6.

Figure 6

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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.

DISCUSSION

This study revealed that the sequence –38/+36 is the essential cis-element in vivo for light-dependent expression of K-81 psbA2 transcript. Evidence was also obtained that the region –38/+14 containing the promoter is the minimal element for the basal transcription, and the additional regions +14/+36 carrying the AT-box and +36/+46 possessing the SD sequence are the negative and positive elements, respectively, for light-dependent expression (Fig. 1). A scheme for the initiation of K-81 psbA2 transcription and translation is presented in Figure 7. It has been suggested that transcription was driven by RNA polymerase (EσA1) containing the principal σ factor, σA1, in both light and dark in the K-81 cells (5–7). However, in this study, psbA2–lacZ transcript levels decreased in the dark in a strain with the promoter region –38/+36 (AG427) whereas expression was detected under the same conditions in strain AG429 containing the promoter region –38/+14. This result indicates that the +14/+36 region containing the AT- (AU-) box motif is required for the clear pattern of light-dependent expression and this motif might be essential for mRNA decay (= a negative effect, Fig. 7) in darkness (Fig. 3). Results of mutation and deletion analyses in the AT- (AU-) box also supported a negative effect on transcript accumulation (Figs 4–6). In view of the findings in this article and of previous results, we propose a model involving negative regulation as a mechanism for light-dependent expression of psbA2 as follows: (i) the psbA2 gene is transcribed by EσA1 in both light and dark conditions through the minimal cis-element –38/+14 for the basal transcription; (ii) these transcripts (or the transcription) are negatively modified by factor(s) associated with the AU- (AT-) box; (iii) ribosome binding to the SD sequence increases mRNA stability and might lead to interaction with the factor(s). The action by the putative factor(s), e.g. ribonuclease (Fig. 7), is apparently unique in that it causes mRNA instability particularly in the dark. The increase in the amount of transcript in the presence of Cm also supports the involvement of factor(s) associated with the AU- (AT-) box (Fig. 6). Since the AU-box at 5′-UTR locates just upstream from the ribosome-binding site, the binding of the ribosome might protect the binding of the putative negative factor(s) by sequestration of the AU-box, resulting in an increase in mRNA stability (= positive effect, Figs 1 and 7). Deletion of the ribosome-binding site resulted in a reduction in the level of transcript, a result which supports this concept (Figs 2 and 3, compare AG407 to AG427). Toeprint analysis of soluble and membrane polysome-associated psbA mRNA has also revealed that ribosomes can bind to the initiator region carrying the conserved AU-box (15).

Figure 7.

Figure 7

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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).

It was recently reported that possible endonucleases or RNA-binding proteins recognize and process mRNAs at 5′-UTRs in eukaryotic genes (17,18). The ribosomal protein S1 (43 kDa), isolated from the stroma and membranes of the spinach chloroplast, binds to the region from +38 to +78 and endonucleolytic cleavage sites at +38/+39 have been identified in vitro on the 5′-UTR, which has a possible stem–loop structure [region +7 to +61 around the (30 bp) stretch shown in Fig. 1] (18,28). The tobacco psbA 5′-UTR is also sufficient to confer light-dependent translational regulation on a reporter gene (14), and a protein complex has been identified that recognizes a critical AU-rich region in the 5′-UTR (16). On the other hand, light-activated translation of the psbA mRNA in Chlamydomonas involves an RNA-binding complex, consisting of four primary proteins (RB60 = PDI, RB55, RB47 = PABP and RB38) which bind the region at 5′-UTR. In this case, following transcription, the mRNA is processed at a stem–loop structure (Fig. 1) by a small ribosomal subunit and/or additional proteins in a light-independent manner; such a message-specific complex can then activate a complete translational initiation at the SD sequence in a light-dependent manner (17,19). To date, although some proteins which are encoded by nuclear genes and which bind to the psbA 5′-UTR have been isolated and characterized for light-induced expression of transcripts in eukaryotes, no homologs have been found in cyanobacteria.

Since the cyanobacterial psbA 5′-UTR is ∼30–40 nt shorter than those of the eukaryotic psbA mRNAs (Fig. 1), no typical stem–loop element upstream of the SD sequence has been identified on the K-81 psbA2 mRNA. In the cyanobacterium PCC 7942, the 5′-UTR of psbA might regulate mRNA stability by modulating translation, or by recruiting RNA-binding proteins that affect mRNA turnover (21). In that report the authors suggested the 5′-ends of the transcripts to be the binding site of factors involved directly in mRNA decay, such as endonucleases, and pointed out that the binding of ribosomes to the 5′-end, and coupling between transcription and translation, influence mRNA stability. Furthermore, a stabilizer cis-element (CRS) was identified within the open reading frame that was a site for ribosome pausing. The accumulation of ribosomes caused by the pause site resulted in an increase in the psbA mRNA levels. It remains unclear whether factors directly associate with the conserved AU-box sequence and cleave the K-81 psbA2 mRNA at 5′-UTR or whether there is interaction between the factors and ribosomes at the SD sequence in cyanobacteria. However, a clue as to the possible cleavage sites within the AU-box sequence may be obtained from mRNA sampled from dark-grown cells. To further test this notion, protein extracts will be prepared from cells grown in the dark or light, mixed with mRNAs synthesized in vitro, then tested to see whether the mRNA stability is lower in the extract isolated from cells grown in darkness than in light. mRNA instability will be recovered by the AU-box mutants but potentiated by the SD sequence mutants. In addition, other factors might cause psbA2 mRNA instability by non-specific or global negative effects because a slight loss of transcript was observed in AG429 (–38/+14, the minimal element for the basal transcription) in darkness (Fig. 3, lane 2). Furthermore, transcriptional, post-transcriptional or translational regulation through trans-acting factors associated at the coding and 3′-UTR regions has been reported for other prokayotic, chloroplast and mammalian transcripts (29–32).

We cannot exclude the possibility that the light-dependent expression of psbA2 in K-81 is also regulated at transcription (Fig. 7). For example, a potential repressor would specifically bind the AT-box and inhibit the synthesis of psbA2 mRNA in darkness. In fact, changing the AT-box sequences from the preset direction or number on the double-helix DNA influenced the light/dark pattern of transcript expression (Figs 4 and 5, AG451, AG455 and AG456). In addition, the low recovery of light-induced expression observed in AG451, 456 and 457 (Fig. 5, lanes 2 and 3) also implies positive regulation by trans-acting factor(s) (= inducers, Fig. 7) in light. It has been reported that one or more soluble proteins from PCC 7942 grown in high light conditions specifically bind to the DNA segment upstream of psbAII that corresponds to the 5′-UTR (20). When 12 bp were deleted from the region associated with the protein, protein binding was impaired and high light induction of both transcriptional and translational psbAII–lacZ reporters was significantly reduced (20). These findings demonstrate that there is a clear transcriptional component to high light-inducible psbAII expression in PCC 7942, as in light-induced expression by the inducers shown in Figure 7. We have analyzed the pattern of rpoD1 expression as a control. The rpoD1 transcript levels derived from Promoter 1 do not change under different light intensities as shown in Figure 3. The 5′-UTR and the region around Promoter 1 similarly have no typical AT- (AU-) box just upstream of the SD sequence (6). Promoter 1 also possesses an E.coli consensus-type sequence like K-81 psbA2, and replacement of the K-81 psbA2 promoter with rpoD1 Promoter 1 in the upstream fragment of psbA2 also produced a pattern of light-dependent expression (data not shown), strongly indicating that the AT- (AU-) box plays a significant role in the 5′-UTR rather than promoter function.

Why do light-responsive psbA transcripts appear under light/dark conditions? Is there a common regulatory mechanism for the light-responsive transcripts among photosynthetic organisms? Since we have identified the AT-box motif, which confers mRNA instability under darkness, in the 5′-UTRs of other photosynthetic genes in cyanobacteria and plants, this mechanism of regulation coupling transcription, translation initiation and/or mRNA degradation may be more widely distributed than previously thought. Mutational analyses of relevant sequences have provided further evidence of an important role in the regulation of transcript levels in the dark and light. Cloning and characterization of such trans-acting factor(s) should be explored in cyanobacteria.

Acknowledgments

ACKNOWLEDGEMENTS

We thank Dr J.Y.Suzuki for critical reading of the manuscript. This work was supported by a grant for Scientific Research on Priority Areas from the Ministry of Education, Science, and Culture of Japan.

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