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؉ -and NEM-sensitive residues were found at the cytoplasmic end of TMH5 and suggest a possible connection of this region to the NEM-and Ag ؉ -sensitive region of TMH4 described previously. From the now complete pattern of TMH residue reactivity, we conclude that aqueous access from the periplasmic side of F 0 to cAsp-61 at the center of the membrane is likely to be mediated by residues of TMHs 2, 3, 4, and 5 at the center of a four-helix bundle. Further, aqueous access between cAsp-61 and the cytoplasmic surface is likely to be mediated by residues in TMH4 and TMH5 at the exterior of the four-helix bundle that are in contact with the c-ring.

The global anaerobic regulator FNR is a DNA binding protein that activates transcription of genes required for anaerobic metabolism in Escherichia coli through interactions with RNA polymerase (RNAP). Alaninescanning mutagenesis of FNR amino acid residues 181 to 193 of FNR was utilized to determine which amino acid side chains are required for transcription of both class II and class I promoters. In vivo assays of FNR function demonstrated that a core of residues (F181, R184, S187, and R189) was required for efficient activation of class II promoters, while at a class I promoter, FF(؊61.5), only S187 and R189 were critical for FNR activation. Site-directed mutagenesis of positions 184, 187, and 189 revealed that the positive charge contributes to the function of the side chain at positions 184 and 189 while the serine hydroxyl is critical for the function of position 187. Subsequent analysis of the carboxy-terminal domain of the ␣ subunit (␣CTD) of RNAP, using an alanine library in single copy, revealed that in addition to previously characterized side chains (D305, R317, and L318), E286 and E288 contributed to FNR activation of both class II and class I promoters, suggesting that ␣CTD region 285 to 288 also participates in activation by FNR. In conclusion, this study demonstrates that multiple side chains within region 181 to 192 are required for FNR activation and the surface of ␣CTD required for FNR activation is more extensive than previously observed.A common mechanism of activation by transcription factors employs a series of macromolecular interactions, from binding DNA to multiple contacts with RNA polymerase (RNAP), which allows target promoters to overcome intrinsic defects in transcription initiation (21). This study focused on the Escherichia coli global anaerobic regulator FNR and the protein requirements essential for transcription activation. In particular, this study further investigated the residues of FNR that have been proposed to interact with the ␣ subunit of RNAP.FNR, which regulates transcription in response to O 2 deprivation, belongs to a family of transcriptional regulators related to the cyclic AMP receptor protein, CRP (13). In its active form, FNR is a homodimer (16) which protects an ϳ22-bp sequence in DNase I footprinting experiments (9, 14). The vast majority of FNR-activated promoters contain an FNR binding site centered approximately 41.5 bp upstream of the transcriptional start site and are termed class II promoters (Fig. 1A) (6). At this position, FNR is able to make multiple contacts with RNAP through three domains: activating region 1 (AR1), AR2, and AR3 (3,5,14,15,31,33,34). FNR-AR3 is active in the downstream subunit (3) and is likely to contact the 70 subunit of RNAP (23). FNR-AR2, which plays a minor role in activation, is also active in the downstream subunit (5), but its interaction partner is proposed to be the amino-terminal domain of the ␣ subunit of RNAP (5). FNR-AR1 is active in the upstream subunit (3, 33) and is proposed to interact with the carboxy-terminal dom...

Subunit c in the membrane-traversing F 0 sector of Escherichia coli ATP synthase is known to fold with two transmembrane helices and form an oligomeric ring of 10 or more subunits in the membrane. Models for the E. coli ring structure have been proposed based upon NMR solution structures and intersubunit cross-linking of Cys residues in the membrane. The E. coli models differ from the recent x-ray diffraction structure of the isolated Ilyobacter tartaricus c-ring. Furthermore, key cross-linking results supporting the E. coli model prove to be incompatible with the I. tartaricus structure. To test the applicability of the I. tartaricus model to the E. coli c-ring, we compared the cross-linking of a pair of doubly Cys substituted c-subunits, each of which was compatible with one model but not the other. The key finding of this study is that both A21C/M65C and A21C/I66C doubly substituted c-subunits form high yield oligomeric structures, c 2 , c 3 . . . c 10 , via intersubunit disulfide bond formation. The results indicate that helical swiveling, with resultant interconversion of the two conformers predicted by the E. coli and I. tartaricus models, must be occurring over the time course of the cross-linking experiment. In the additional experiments reported here, we tried to ascertain the preferred conformation in the membrane to help define the most likely structural model. We conclude that both structures must be able to form in the membrane, but that the helical swiveling that promotes their interconversion may not be necessary during rotary function.The F 1 F 0 -ATP synthases of oxidative phosphorylation utilize the energy of a transmembrane electrochemical gradient of H ϩ or Na ϩ to mechanically drive the synthesis of ATP via two coupled rotary motors in the F 1 and F 0 sectors of the enzyme (1-3). In the intact enzyme, ATP synthesis or hydrolysis takes place in the F 1 sector at the surface of the membrane, with synthesis being coupled to H ϩ or Na ϩ transport through the transmembrane F 0 sector. Homologous enzymes are found in mitochondria, chloroplasts, and many bacteria (4). In Escherichia coli and other eubacteria, F 1 consists of five subunits in an ␣ 3 ␤ 3 ␥ 1 ␦ 1 ⑀ 1 stoichiometry (4). F 0 is composed of three subunits in a likely ratio of a 1 b 2 c 10 in E. coli and Bacillus PS3 or a 1 b 2 c 11 in the Na ϩ -translocating Ilyobacter tartaricus ATP synthase (3, 5-7). A 3.9-Å resolution crystal structure of a yeast mitochondrial F 1 -c 10 depicts 10 c-subunits arranged in a ring-like structure (8). Other bacterial c-rings may have as many as 15 c-subunits (9). Subunit c spans the membrane as a hairpin of two transmembrane helices (TMHs), 2 with the first TMH on the inside and the second TMH on the periphery of the c-ring (7, 10, 11). Helical hairpin structures resembling those predicted for the membrane have been solved by NMR using chloroform/methanol solvent mixtures (12-14). A high resolution x-ray structure of the I. tartaricus c 11 -ring, which differs significantly from the NMR structures, has rev...

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