Domain structure, oligomeric state, and mutational analysis of PpsR, the Rhodobacter sphaeroides repressor of photosystem gene expression.
The transcription factor PpsR from the facultative photoheterotroph Rhodobacter sphaeroides is involved in repression of photosystem gene expression under aerobic growth conditions. We have isolated a number of spontaneous mutations as well as constructed directed mutations and deletions in ppsR. Repressor activities and the oligomeric state of the wild-type and mutant proteins were assayed. Our results suggest that the wild-type PpsR exists in cell extracts as a tetramer. Analysis of the PpsR mutants confirmed that the carboxy-terminal region of PpsR (residues 400 to 464) is involved in DNA binding. The central region of the protein (residues 150 to 400) was found to contain two PAS domains (residues 161 to 259 and 279 to 367). PAS domains are ubiquitous protein modules involved in sensory transduction as well as in protein-protein interactions. All spontaneously isolated mutations, which significantly impaired repressor activity and which mapped outside the DNA binding region, were positioned in the PAS domains. None of these, however, affected the overall oligomeric state. This implies that the conformation of the PAS domains within the tetramer is critical for repressor activity. Upstream of the first PAS domain resides a putative glutamine-rich hinge (residues 127 to 136) that connects the first PAS domain to the amino-terminal region (residues 1 to 135). The role of the amino terminus of PpsR is not obvious; however, extended deletions within this region abolish repressor activity, thus suggesting that the amino terminus is essential for structural integrity of the protein. We present a model of the domain architecture of the PpsR protein according to which PpsR is comprised of three regions: the carboxy terminus responsible for DNA binding, the central region primarily involved in protein oligomerization and possibly signal sensing, and the amino terminus of unknown function. This model may prove useful for determining the mode of PpsR action.
Gomelsky
M
M
Department of Microbiology and Molecular Genetics, University of Texas Medical School, Houston, Texas 77030, USA.
Horne
I M
IM
Lee
H J
HJ
Pemberton
J M
JM
McEwan
A G
AG
Kaplan
S
S
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001665:Binding Sites; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D008969:Molecular Sequence Data; D010788:Photosynthesis / Q000235:genetics*; D045322:Photosynthetic Reaction Center Complex Proteins / Q000235:genetics*; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D012097:Repressor Proteins / Q000737:chemistry* / Q000378:metabolism; D012242:Rhodobacter sphaeroides / Q000235:genetics*; D017386:Sequence Homology, Amino Acid
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D045322:Photosynthetic Reaction Center Complex Proteins; D012097:Repressor Proteins