The 5'-upstream cis-acting sequences of a cyanobacterial psbA gene: analysis of their roles in basal, light-dependent and circadian transcription.
Transcription of the psbA2 gene in the unicellular photosynthetic cyanobacterium Microcystis aeruginosa K-81 is modulated by light and follows a circadian rhythm. In this study, we further characterized psbA transcription using a series of 5'-upstream deletions and mutant promoters which were tested in both photosynthetic and non-photosynthetic bacteria. Specific psbA2 transcripts were obtained from a minimal promoter sequence (-38/+14) with Escherichia coli RNA polymerases (RNAPs) both in vivo and in vitro, indicating the presence of a common regulatory mechanism for basal transcription. A DNase I footprinting assay showed that the E. coli RNAP, which is structurally similar to that of cyanobacteria, specifically binds to a large segment (from -115 to +23) of the sequence upstream of psbA2. In cyanobacteria, the -10 sequence (TAGTAT), but not the -35 motif (TTTACA), is essential for basal transcription by homologous and heterologous RNAPs that contain the major sigma factor. Each of the conserved thymidine nucleotides at positions -12 and -7 (underlined above) was essential, and both an insertion and a deletion in the spacer region of the promoter caused reductions in transcription. RNAP was able to bind to a mutant promoter lacking the -10 sequence, though this did not actually lead to transcription. Interestingly, a high level of arrhythmic circadian transcription was observed in mutants lacking the -35 region. In contrast, a mutation in the AU-box mutation, which controls the stability of the psbA2 mRNA, did not affect the circadian pattern of transcription. These findings demonstrate that light-dependent psbA2 expression is controlled at the transcriptional and post-transcriptional levels, whereas the circadian pattern of expression is regulated at the transcriptional level.
Shibato
J
J
Laboratory of Molecular Genetics, School of Agriculture, Ibaraki University, Ami-machi, Inashiki-gun, Ibaraki 300-0332, Japan.
Agrawal
G K
GK
Kato
H
H
Asayama
M
M
Shirai
M
M
D001483:Base Sequence; D002940:Circadian Rhythm / Q000235:genetics; D000458:Cyanobacteria / Q000235:genetics*; D004926:Escherichia coli; D015964:Gene Expression Regulation, Bacterial; D008027:Light; D008969:Molecular Sequence Data; D045322:Photosynthetic Reaction Center Complex Proteins / Q000235:genetics*; D045332:Photosystem II Protein Complex; D011401:Promoter Regions, Genetic; D017384:Sequence Deletion; D020506:Untranslated Regions; D015854:Up-Regulation
D045322:Photosynthetic Reaction Center Complex Proteins; D045332:Photosystem II Protein Complex; D020506:Untranslated Regions; C422369:photosystem II, psbA subunit