E. coli RNA polymerase, deleted in the C-terminal part of its alpha-subunit, interacts differently with the cAMP-CRP complex at the lacP1 and at the galP1 promoter.
A deletion of the C-terminal part of the alpha-subunit of RNA polymerase is known to affect differently promoters activated by CRP depending on the location of the CRP binding site at the promoter. When the CRP binding site is located at -61.5, as at lacP1 (a type I promoter), activation is strongly impaired while it is not significantly affected at galP1 where CRP binds 41.5 bp upstream of the start of the message (type II promoter). We have investigated the differences in the architecture of the corresponding open complexes by comparing the positioning of holoenzymes reconstituted respectively with native or with truncated alpha-subunits (containing the first 235 or 256 residues of a) at two 'up' promoter mutants of the lacP1 and galP1 promoters (respectively lacUV5 and gal9A16C). First, the affinity of wild-type RNA polymerase for both promoters is increased by the presence of CRP and cAMP. By contrast, holoenzymes reconstituted with truncated alpha-subunits, show cooperative binding at the galP1 promoter only. Second, footprinting data confirm these observations and indicate that the truncated holoenzymes are unable to recognize regions of the promoter upstream from position -40. The absence of contacts between the truncated enzymes and CRP at the lacP1 promoter can explain the deficiency in activation. At the galP1 promoter, where the CRP site is closer to the initiation site, protein-protein contacts can still occur with the truncated polymerases, showing that the C-terminal part of the alpha-subunit is not involved in activation.
Institut Pasteur, Unité de Physicochimie des Macromolécules Biologiques (URA 1149 du CNRS), Paris, France.
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Kolb
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Igarashi
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Ishihama
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Lavigne
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Buckle
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Buc
D002352:Carrier Proteins / Q000378:metabolism*; D000242:Cyclic AMP / Q000378:metabolism; D002373:Cyclic AMP Receptor Protein; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D010618:Phenanthrolines; D011401:Promoter Regions, Genetic; D011953:Receptors, Cyclic AMP / Q000378:metabolism*
D002352:Carrier Proteins; D002373:Cyclic AMP Receptor Protein; D010618:Phenanthrolines; D011953:Receptors, Cyclic AMP; D000242:Cyclic AMP; D012321:DNA-Directed RNA Polymerases; C025205:1,10-phenanthroline