An Asp-Arg-Tyr triad occurs in a majority of rhodopsin-like G protein-coupled receptors. The fully conserved Arg is critical for G protein activation, but the function of the flanking residues is not well understood. We expressed in COS-7 cells m1 muscarinic receptors that were mutated at Asp122 and Tyr124. Most mutations at either position strongly attenuated or prevented the expression of binding sites for the antagonist [3H]N-methylscopolamine. However, sites that were expressed displayed unaltered affinity for the antagonist. Receptor protein, visualized with a carboxyl-terminally directed antibody, was reduced but never completely abolished. The effects of these mutations were partially reversed by the deletion of 129 amino acids from the third intracellular loop of the receptor. In several cases, comparison of immunocytochemistry with binding measurements suggested the presence of substantial amounts of inactive, presumably misfolded, receptor protein. Some of the variants that bound [3H]N-methylscopolamine underwent small changes in their affinities for acetylcholine. All retained nearly normal abilities to mediate an acetylcholine-induced phosphoinositide response. We propose that Asp122 and Tyr124 make intramolecular contacts whose integrity is important for efficient receptor folding but that they do not participate directly in signaling. The role of these residues is completely distinct from that of Arg123, whose mutation abolishes signaling without diminishing receptor expression.

Plasmids carrying different portions of the polycistronic spoIIA locus, and unable to replicate autonomously in Bacillus subtilis, were able to transform a Spo+ B. subtilis strain, BR151, for the plasmid-determined chloramphenicol resistance by Campbell-like insertion into the region of homology on the chromosome. Two such plasmids, pPP35 and pPP36, yielded Spo- transformants, indicating that the cloned regions of these plasmids were entirely within the chromosomal spoIIA transcriptional unit. The cloned regions overlapped the end of a known spoIIA cistron, so that the transcriptional unit was larger than this cistron, and was polycistronic. This is the first demonstration of such a polycistronic sporulation transcriptional unit. The DNA sequence of the region has now been determined (given in an accompanying paper) and suggests a transcriptional unit with three open reading frames. Two other plasmids yielded Spo+ transformants of BR151, and these define the outer limits of the transcriptional unit. The adjacent sporulation locus identified by the spoV A89 mutation was not part of the same transcriptional unit.

Transmembrane domain 6 of the muscarinic acetylcholine (ACh) receptors is important in ligand binding and in the conformational transitions of the receptor but the roles of individual residues are poorly understood. We have carried out a systematic alanine-scanning mutagenesis study on residues Tyr381 to Val387 within the binding domain of the M(1) muscarinic ACh receptor. The seven mutations were then analyzed to define the effects on receptor expression, agonist and antagonist binding, and signaling efficacy. Tyr381Ala produced a 40-fold reduction in ACh affinity and a 50-fold reduction in ACh-signaling efficacy. Leu386Ala had similar but smaller effects. Asn382Ala caused the largest inhibition of antagonist binding. The roles of the hydroxyl group and benzene ring of Tyr381 were probed further by comparative analysis of the Tyr381Phe and Tyr381Ala mutants using three series of ligands: ACh analogs, azanorbornane- and quinuclidine-based ligands, and atropine analogs. These data suggested that the hydroxyl group of Tyr381 is primarily involved in forming hydrogen bond interactions with the oxygen atoms present in the side chain of ACh. We propose that this interaction is established in the ground state and preserved in the activated state of the receptor. In contrast, the Tyr381 benzene ring may form a cation-pi interaction with the positively charged head group of ACh that contributes to the activated state of the receptor but not the ground state. However, the hydroxyl group and benzene ring of Tyr381 both participate in interactions with azanorbornane- and quinuclidine-based ligands and atropine analogs in the ground state as well as the activated state of the receptor.

The control of expression of the BacUlus subtilis spollA locus was analyzed by titrating gene expression against gene copy number. A plasmid integrated into the B. subtilis chromosome and carrying the spoIlA control region fused to Escherichia coli lacZ was forced to form tandem repeats by the selection of clones that grow on high levels of chloramphenicol, the antibiotic against which the plasmid determines resistance. DNA from the clones was digested with BglIl, which did not cut in the reiterated region, and the size of the fragment was determined by orthogonal-field-alternation gel electrophoresis to determine the copy number. Most clones had fairly homogeneous copy numbers. Gene expression was monitored by P-galactosidase activity. dosages and level of expression proportional to increase of copy number above a threshold. We consider that this approach will be generally applicable to the task of unraveling the controls in complex networks of gene expression.The spoIIA locus used for this study is a polycistronic operon (8,24). It appears to have a regulatory role in sporulation. The putative protein product of the third gene of spollA has strong amino acid sequence homology with several RNA polymerase sigma factors (6, 8). MATERIALS AND METHODSBacterial strains. The B. subtilis 168 strain used was MB24, metC3 trpC2 rif-2. The E. coli strain was DH1 (12).Plasmids. All plasmids were maintained in E. coli DH1 unless otherwise stated. The lac fusion vector pDB1 was kindly provided by A. L. Sonenshein. It has the same promoter-cloning site as pCED6 (5) but is unable to replicate in B. subtilis and cannot transform B. subtilis; it expresses resistance to chloramphenicol rather than to kanamycin. The positive selection vector pJAB1 was described by Sargent and Bennett (25) and kindly provided by M. G. Sargent. Plasmid pPP22 contains the 5' end of the spoIIA transcriptional unit and includes the first two genes of the operon (Fig. 1). pPP22 consists of the 5.9-kilobase-pair (kbp) HindIII fragment from pHM2 (16) self-ligated and maintained in E. coli, where it replicates autonomously. The construction of pPP81 and pPP136 is described in Results.DNA preparation. Plasmid DNA was prepared from E. coli by the method of Guerry et al. (10) or, for small-scale preparations, by the method of Ish-Horowicz and Burke (13).B. subtilis chromosomal DNA was generally prepared as described previously (23). However, a method based on the agarose bead procedure of Cook (4) was used when it was necessary to prepare high-molecular-weight DNA susceptible to restriction nucleases. Bacterial cultures (30 ml) were harvested by centrifugation, and the pellets were suspended thoroughly in 1 ml of 10 mM Tris hydrochloride (pH 7.5)-i mM Na2 EDTA and placed in a 50-ml round-bottom flask.