Purification, characterization, and gene expression of all sigma factors of RNA polymerase in a cyanobacterium.
The expression of RNA polymerase (RNAP) sigma factor genes and proteins was characterized as a first step toward understanding their functions in a unicellular cyanobacterium Synechocystis sp. PCC 6803, which can perform photosynthesis. All nine sigma factors (group 1, SigA; group 2, SigB to SigE; and group 3, SigF to SigI) and each RNAP core subunit (RpoA, RpoB, RpoC1 and RpoC2) were overproduced and purified from Escherichia coli cells, then polyclonal antibodies were prepared. Western blot and primer extension analyses revealed that the intracellular levels of group 1 and 2 sigma factors ranged from 0.9 fmol to 9.3 fmol per microgram of the total protein under conditions of steady-state growth, and that growth phase-dependent or constitutive transcripts were observed. Interestingly, no group 3 sigma factor proteins were detected under normal physiological conditions whereas their transcripts were robust, implying a possible regulation of translational attenuation and/or protein instability. Phylogenetic analysis also revealed that group 3 sigma factor homologues of cyanobacteria are conserved with evolutionary or functionary divergence among them. In vitro and in vivo results indicated significant evidence of high-light responsive SigD expression and its promoter recognition of the photosynthesis gene, psbA. On the other hand, autoregulated sigB transcription, a dramatically increased SigB expression upon the exposure of cells to heat-shock, and specific promoter recognition by SigB with redundancy of other sigma factors on the heat-shock hspA promoter were observed. These findings clearly indicated that SigB is a heat-shock responsive sigma factor. The unique promoter architecture and expression of the relevant sigma factor gene are also discussed herein.
Imamura
Sousuke
S
Laboratory of Molecular Genetics, College of Agriculture, Ibaraki University, Ami, Inashiki, 300-0393, Ibaraki, Japan.
Yoshihara
Satoshi
S
Nakano
Serina
S
Shiozaki
Noriko
N
Yamada
Akiko
A
Tanaka
Kan
K
Takahashi
Hideo
H
Asayama
Munehiko
M
Shirai
Makoto
M
D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D000458:Cyanobacteria / Q000201:enzymology*; D017931:DNA Primers; D012321:DNA-Directed RNA Polymerases / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D018390:Gene Targeting; D005818:Genetic Engineering; D006360:Heat-Shock Proteins / Q000096:biosynthesis; D018869:Heat-Shock Response; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D010802:Phylogeny; D011401:Promoter Regions, Genetic; D021122:Protein Subunits; D011817:Rabbits; D012689:Sequence Homology, Nucleic Acid; D012808:Sigma Factor / Q000235:genetics* / Q000302:isolation & purification*; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D017931:DNA Primers; D006360:Heat-Shock Proteins; D021122:Protein Subunits; D012808:Sigma Factor; D012321:DNA-Directed RNA Polymerases