An AU-box motif upstream of the SD sequence of light-dependent psbA transcripts confers mRNA instability in darkness in cyanobacteria.
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.
Agrawal
G K
GK
Laboratory of Molecular Genetics, School of Agriculture, Ibaraki University, Ami 3-21-1, Inashiki, Ibaraki 300-0332, Japan.
Kato
H
H
Asayama
M
M
Shirai
M
M
D020121:5' Untranslated Regions; D001483:Base Sequence; D017124:Conserved Sequence; D000458:Cyanobacteria / Q000235:genetics* / Q000378:metabolism; D003624:Darkness; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D008027:Light; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009154:Mutation; D045322:Photosynthetic Reaction Center Complex Proteins / Q000096:biosynthesis / Q000235:genetics*; D045332:Photosystem II Protein Complex; D020871:RNA Stability; D012329:RNA, Bacterial / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid; D012270:Ribosomes / Q000378:metabolism; D016415:Sequence Alignment; D014158:Transcription, Genetic
D020121:5' Untranslated Regions; D045322:Photosynthetic Reaction Center Complex Proteins; D045332:Photosystem II Protein Complex; D012329:RNA, Bacterial; D012333:RNA, Messenger