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Interactions between activating region 3 of the Escherichia coli cyclic AMP receptor protein and region 4 of the RNA polymerase sigma(70) subunit: application of suppression genetics.

Rhodius V.A., Busby S.J.

The Escherichia coli cyclic AMP receptor protein, CRP, induces transcription at Class II CRP-dependent promoters by making three different activatory contacts with different surfaces of holo RNA polymerase. One contact surface of CRP, known as Activating Region 3 (AR3), is functional in the downstream subunit of the CRP dimer and is predicted to interact with region 4 of the RNAP sigma(70) subunit. We have previously shown that a mutant CRP derivative that activates transcription primarily via AR3, CRP HL159 KE101 KN52, requires the positively charged residues K593, K597 and R599 in sigma(70) for activation. Here, we have used the positive control substitution, EK58, to disrupt AR3-dependent activation by CRP HL159 KE101 KN52. We then screened random mutant libraries and an alanine scan library of sigma(70) for candidates that restore activation by CRP HL159 KE101 KN52 EK58. We found that changes at R596 and R599 in sigma(70) can restore activation by CRP HL159 KE101 KN52 EK58. This suggests that the side-chains of both R596 and R599 in sigma(70) clash with K58 in CRP. Maximal activation by CRP HL159 KE101 KN52 EK58 is achieved with the substitutions RE596 or RD596 in sigma(70). We propose that there are specific charge-charge interactions between E596 or D596 in sigma(70) and K58 in AR3. Thus, no increase in activation is observed in the presence of another positive control substitution, EG58 (CRP HL159 KE101 KN52 EG58). Similarly, both sigma(70) RE596 and sigma(70) RD596 can restore activation by CRP EK58 but not CRP EG58, and they both decrease activation by wild-type CRP. We suggest that E596 and D596 in sigma(70) can positively interact with K58 in AR3, thereby enhancing activation, but negatively interact with E58, thereby decreasing activation. The substitution, KA52 in AR3 increases Class II CRP-dependent activation by removing an inhibitory lysine residue. However, this increase is not observed in the presence of either sigma(70) RE596 or sigma(70) RD596. We conclude that the inhibitory side-chain, K52 in AR3, clashes with R596 in sigma(70). Finally, we show that the sigma(70) RE596 and RD596 substitutions affect CRP-dependent activation from Class II, but not Class I, promoters.

J. Mol. Biol. 299:311-324(2000) [PubMed] [Europe PMC]

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