The two alpha subunits of Escherichia coli RNA polymerase are asymmetrically arranged and contact different halves of the DNA upstream element.
RNA polymerase core enzyme of Escherichia coli is composed of two alpha subunits and one each of the beta and beta' subunits. The C-terminal domain of the RNA polymerase alpha subunit plays a key role in molecular communications with class I transcription factors and upstream (UP) elements of promoter DNA, using the same protein surface. To identify possible differences in the functional roles of the two alpha subunits, we have developed a reconstitution method for hybrid RNA polymerases containing two distinct alpha subunit derivatives in a defined orientation ("oriented alpha-heterodimer"). The binding sites of two alpha C-terminal domains on the UP element DNA were determined by hydroxyl radical-based DNA cleavage mediated by (p-bromoacetamidobenzyl)-EDTA x Fe, which was bound at Cys-269 on the UP recognition surface of one or both alpha subunits. The results clearly indicated that the two alpha subunits bind in tandem to two helix turns of the rrnBP1 UP element, and that the beta'-associated alpha subunit is bound to the promoter-distal region.
Murakami
K
K
Department of Molecular Genetics, National Institute of Genetics, Mishima, Shizuoka, Japan.
Kimura
M
M
Owens
J T
JT
Meares
C F
CF
Ishihama
A
A
D001665:Binding Sites; D015153:Blotting, Western; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry* / Q000378:metabolism*; D004492:Edetic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D017665:Hydroxyl Radical; D007502:Iron Chelating Agents / Q000378:metabolism; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D009942:Organometallic Compounds / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins; D012045:Regulatory Sequences, Nucleic Acid; D014158:Transcription, Genetic
D004269:DNA, Bacterial; D007502:Iron Chelating Agents; D009942:Organometallic Compounds; D011993:Recombinant Fusion Proteins; D017665:Hydroxyl Radical; C042007:1-(4-bromoacetamidobenzyl)EDTA; D004492:Edetic Acid; D012321:DNA-Directed RNA Polymerases