The 5′-upstream cis-acting sequences of a cyanobacterial psbA gene: analysis of their roles in basal, light-dependent and circadian transcription

Abstract

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.

SigC, the Group 2 Sigma Factor of RNA Polymerase, Contributes to the Late-stage Gene Expression and Nitrogen Promoter Recognition in the CyanobacteriumSynechocystissp. Strain PCC 6803

“…Specific recognition by RNAP holoenzyme reconstituted with an E. coli core and cyanobacterial sigma factors has been reported. 26,36,38,[42][43][44] When an E. coli RNAP holoenzyme with PCC6803 NtcA was used, a strong signal was observed from the À35 site, which is more than 2 bases upstream from the À33 site, identified in vivo as the position of P1, using linear template DNAs (Fig. 5A, lane 2).…”

Two Distinct Curved DNAs Upstream of the Light-responsivepsbAGene in a Cyanobacterium

“…2,3) Ourˆndings in this study might be unique, in that a single promoter region has two distinct types of DNA curvatures, one a sequencedirected CIT and the other a protein-induced RIB. This also presents a novel feature of RNA polymerase induced-DNA bend the center of which is located downstream (＋10) from the transcription start point (＋1).…”

“…1) It has been characterized for basal, light-responsive, and circadian-rhythmic transcription of the psbA2 gene. [2][3][4][5] We have found and characterized an intrinsic (static or sequence-directed, the same meaning) DNA curvature, CIT (Changeable bending-center sites of an Intrinsic curvature under Temperature conditions) located in the 5?-upstream region (-241 to -80) of psbA2. 6) We have reported the universality of the curved DNA structure in psbA genes of another cyanobacterium, red alga, and in plants, and also showed the roles of the curvature in basal transcription in cyanobacteria.…”

“…3,5) In this study, we tested whether RNA polymerase binding to the psbA2 promoter caused proteininduced DNA bending or not. A 126-bp DNA fragment ampliˆed by PCR with the oligonucleotides GKA6 and GKA10, 3) containing the psbA2 promoter region from -80 to ＋46, was cloned into an SmaIBgl II site of the plasmid pCY7 12) to create pGK3, which can supply a series of 526-bp permuted fragments (the length of each insert is the same, Fig. 1a) for Circular Permutation Analysis (CPA).…”

“…The recombinant PCC 7942 cells were selected on BG11 liquid medium 16) containing spectinomycin sulfate (40 mg W ml). Activities of b-galactosidase for the basal transcription of K-81 psbA2 3,10) were measured using cells grown under continuous white-light illumination (35 mE m -2 s -1 ). A reduction in the activity and no activity of b-galactosidase was found for AG430 (＋CIT) versus AG407 (-CIT), and AG429 (＋RIB) versus AG410 (-RIB), respectively.…”