The Escherichia coli Ada protein can interact with two distinct determinants in the sigma70 subunit of RNA polymerase according to promoter architecture: identification of the target of Ada activation at the alkA promoter

Abstract

The methylated form of the Ada protein (meAda) activates transcription from the Escherichia coli ada, aidB, and alkA promoters with different mechanisms. In this study we identify amino acid substitutions in region 4 of the RNA polymerase subunit sigma70 that affect Ada-activated transcription at alkA. Substitution to alanine of residues K593, K597, and R603 in sigma70 region 4 results in decreased Ada-dependent binding of RNA polymerase to the alkA promoter in vitro and impairs alkA transcription both in vivo and in vitro, suggesting that these residues define a determinant for meAda-sigma70 interaction. In a previous study (P. Landini, J. A. Bown, M. R. Volkert, and S. J. W. Busby, J. Biol. Chem. 273:13307-13312, 1998), we showed that a set of negatively charged amino acids in sigma70 region 4 is involved in meAda-sigma70 interaction at the ada and aidB promoters. However, the alanine substitutions of positively charged residues K593, K597, and R603 do not affect meAda-dependent transcription at ada and aidB. Unlike the sigma70 amino acids involved in the interaction with meAda at the ada and aidB promoters, K593, K597, and R603 are not conserved in sigmaS, an alternative sigma subunit of RNA polymerase mainly expressed during the stationary phase of growth. While meAda is able to promote transcription by the sigmaS form of RNA polymerase (EsigmaS) at ada and aidB, it fails to do so at alkA. We propose that meAda can activate transcription at different promoters by contacting distinct determinants in sigma70 region 4 in a manner dependent on the location of the Ada binding site.

FIG. 1

In vivo transcription from the alkA promoter. The effects of the substitutions in ς⁷⁰ region 4 on alkA expression are shown as percentages of alkA expression with wild-type ς⁷⁰. Values (± standard deviations) are averages of four independent experiments. The average value for the wild type was 342 Miller units.

FIG. 2

In vitro transcription with reconstituted RNA polymerases (50 nM). Lanes 1 to 3, wild-type Eς⁷⁰; lanes 4 to 6, Eς⁷⁰_KA593; lanes 7 to 9, Eς⁷⁰_KA597; lanes 10 to 12, Eς⁷⁰_RA603. Lanes 1, 4, 7, and 10, no Ada protein; lanes 2, 5, 8, and 11, 0.2 μM unmethylated Ada; lanes 3, 6, 9, and 12, 0.2 μM methylated Ada. The positions of the main lacUV5 and alkA transcripts are indicated.

FIG. 3

Ratio of alkA/lacUV5 transcripts. Values are percentages of transcription by wild-type Eς⁷⁰ in the presence of ^meAda and are averages of three independent experiments. Dark grey bars, transcription levels in the absence of Ada; light grey bars, transcription levels in the presence of unmethylated Ada; black bars, transcription levels in the presence of methylated Ada. Standard deviations were less than 15%.

FIG. 4

Gel retardation assays performed with reconstituted RNA polymerase. Odd-numbered lanes, no ^meAda; even-numbered lanes, 0.2 μM ^meAda. Lanes 1 and 2, no RNA polymerase; lanes 3 and 4, wild-type Eς⁷⁰; lanes 5 and 6, Eς⁷⁰_KA593; lanes 7 and 8, Eς⁷⁰_KA597; lanes 9 and 10, Eς⁷⁰_RA603. F, unbound alkA promoter DNA; C, RNA polymerase-alkA complex.

FIG. 5

In vitro transcription with Eς⁷⁰ (lanes 1 to 3) and Eς^S (lanes 4 to 6) forms of RNA polymerase. Lanes 1 and 4, no Ada protein; lanes 2 and 5, 0.2 μM Ada; lanes 3 and 6, 0.2 μM ^meAda. The positions of the main lacUV5 and alkA transcripts are indicated.

FIG. 6

Gel retardation assays performed with Eς⁷⁰ and Eς^S forms of RNA polymerase. Odd-numbered lanes, no ^meAda; even-numbered lanes, 0.2 μM ^meAda. Lanes 1 and 2, no RNA polymerase; lanes 3 and 4, Eς⁷⁰; lanes 5 and 6, Eς^S. F, unbound alkA promoter DNA; C₇₀ and C_S, Eς⁷⁰-alkA and Eς^S-alkA complexes, respectively.