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The thermodynamics of the binding of cyclic adenosine monophosphate (cAMP) and its non-functional analog, cyclic guanosine monophosphate (cGMP), to cyclic AMP receptor protein (CRP) and its T127L mutant were investigated by isothermal titration calorimetry (ITC) in 0.2 and 0.5 M KCl phosphate buffer (pH 7.0) at 24 and 39 degrees C. Although, the binding of the first cAMP molecule to CRP is exothermic with an enthalpy change (delta Hb) of -6 kJ mol-1, a heat capacity change (delta Cp) of -0.300 kJ mol-1 K-1, and an entropy increase (delta Sb) of 72 J mol-1 K-1, the overall binding of cAMP to CRP is endothermic and positively cooperative: binding of the first cAMP molecule increases the affinity for the second one by more than an order of magnitude at 24 degrees C. The binding of the second cAMP molecule is accompanied by large changes of 48.1 kJ mol-1 in delta Hb, of -1.4 kJ mol-1 K-1 in delta Cp, and of 255 J mol-1 K-1 in delta Sb at 24 degrees C and 0.5 M KCl phosphate buffer. In contrast, the overall binding of cGMP to CRP is exothermic and non-cooperative with delta Hb, delta Cp, and delta Sb values close to the those values for binding of the first cAMP molecule to CRP. The point mutation, T127L, switches off the cooperativity between the cAMP ligated binding sites without affecting the binding constant of cAMP and changes the specificity of the protein so that transcription is now activated only upon cGMP binding. All the binding reactions to CRP and the mutant are mainly entropically driven at 24 degrees C.

Mutant forms (CRP*) of the Escherichia coli cAMP receptor protein (CRP) that activate CRP-dependent promoters in the absence of the normal allosteric effector (cAMP) have been described. A previous report (Harman, J. G., McKenney, K., and Peterkofsky, A. (1986) J. Biol. Chem. 261, 16332-16339) detailed the properties of three CRP* mutant proteins. One protein, 220 CRP, has amino acid substitutions at positions 127 and 170 and low CRP* activity in vivo. A second protein, 222 CRP, has the amino acid substitutions present in 220 CRP and a third substitution (arginine for leucine) at position 195. 222 CRP has high CRP* activity in vivo and high apparent affinity for lacP DNA relative to the 220 CRP in vitro. In this report, we evaluate the effect of a single amino acid substitution at position 195 (leucine to arginine) on CRP activity both in vivo and in vitro. Cells (cya delta crp delta/pJH8crpR195) containing R195 CRP were found to exhibit a CRP* phenotype, expressing a variety of CRP-dependent genes in the absence of added cAMP. R195 CRP exhibited both CRP* activity in vitro and increased apparent affinity for cAMP relative to wild-type CRP. CRP titration experiments performed using an in vitro lac transcription system suggest that the isolated substitution of arginine at position 195 does not confer on CRP the high lacP affinity that distinguishes the 220 and 222 forms of CRP. These findings lead us to the conclusion that the effects of multiple mutations in CRP can be both cumulative and interactive.

Mutations induced by (+/-)trans-benzo-[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BaP-diol epoxide-1) were selected by using a recombinant plasmid vector system designed for the study of transcription termination signals. The plasmid contains a transcription terminator positioned between a promotor signal and the Escherichia coli galactokinase structural gene (galK). By selections for the expression of galK (i.e., galK- to galK+), mutations are obtained in the terminator region that allow transcription from the promotor to read the galK gene. These mutations were characterized by direct DNA sequence of the terminator region. The DNA sequence changes caused by BaP-diol epoxide-1 were demonstrated for three different mutants. Two were found to be single-base-pair insertions of T.A into a cluster of consecutive T.A base pairs and the other change was a single-base-pair deletion of G.C from a cluster of consecutive G.C base pairs.

Roy et al. [Roy, A., Haziza, C. & Danchin, A. (1983) EMBO J. 2, 791-797] established that translation of Escherichia coli adenylate cyclase initiates at a UUG codon, and they suggested this might decrease the efficiency of translation. We investigated the effect of varying the initiation codon on the expression of the adenylate cyclase (cya) gene. Using oligonucleotide-directed mutagenesis, we changed the UUG initiation codon to GUG and the more common initiator AUG and assayed for cya gene expression in a number of ways. First, the GUG initiation codon, in place of UUG, doubled cya expression when cya was expressed from the dual cya P1/P2 promoters. The corresponding AUG codon construct was nonviable. Second, when the cya gene was placed under the transcriptional control of the thermoinducible phage lambda PL promoter, the relative amounts of cya gene product were 1:2:6 for the UUG, GUG, and AUG initiation codons, respectively. Finally, the cya P2 promoter, Shine-Dalgarno sequence, and the DNA corresponding to the first 86 codons of cya were fused to DNA encoding the E. coli galactokinase gene beginning at the second codon. The relative amounts of the fusion polypeptides, which had galactokinase activity, were 1:2:3 for the UUG, GUG, and AUG initiation codons, respectively. These results demonstrate that the cya UUG initiation codon limits cya expression at the level of translation.

The yeast CUP1 gene product, copper metallothionein, acts to repress the basal transcription of its own structural gene. By creating a series of truncation and amino acid substitutions in CUP1, we show that the ability of the protein to autoregulate is directly correlated to its ability to bind and detoxify copper. These results support a model in which metallothionein controls the level of free intracellular copper available to interact with positive transcription factors. In addition, mutations in chemically equivalent cysteine residues were functionally dissimilar, suggesting that partial sites in the molecule are critical for the formation of the sulfur-metal cluster.

Small angle neutron scattering (SANS) measurements were performed on solutions of cAMP receptor protein (CRP) and on solutions of the T127L,S128A double mutant of CRP (CRP*) in D2O K3PO4 buffer containing 0.5 M KCl, in the absence and presence of 3',5' cyclic adenosine monophosphate (cAMP). Energy-minimized structures of the CRP were calculated by minimization of the x-ray crystallographic structure of CRP in either the exclusively "closed" form where the alpha-helices of the carboxyl-terminal domain are folded close to the amino-terminal domain and in the exclusively "open" form where the alpha-helices of the carboxyl-terminal domain are folded away from the amino-terminal domain. Neutron scattering models show that the CRP SANS data follow closely the data curve predicted for unligated CRP in the open form, whereas the cAMP-ligated data are more in agreement with the data predicted for the minimized cAMP-ligated CRP structure in the closed form. Thus, it appears that CRP undergoes a conformational change from the open form to the closed form in solution upon ligation with cAMP. The SANS data from the CRP* and cAMP-ligated CRP* are coincidental, which implies that there is very little structural difference between the two species of CRP*. This is in agreement with in vivo results, which show that whereas CRP activates transcription in the cell only in the presence of cAMP, CRP* activates transcription in the absence of cAMP, implying that CRP* is already in the correct conformation for the activation of transcription.

Purified yeast copper-metallothionein lacks 8 amino-terminal residues that are predicted from the DNA sequence of its gene. The removed sequence is unusual for metallothionein in its high content of hydrophobic and aromatic residues and its similarity to mitochondrial leader sequences. To study the significance of this amino-terminal cleavage, several mutations were introduced into the metallothionein coding gene, CUP1. One mutant, which deletes amino acid residues 2-8, had a minor effect on the ability of the molecule to confer copper resistance to yeast but did not affect CUP1 gene regulation. A second mutation, which changes two amino acids adjacent to the cleavage site, blocked removal of the extension peptide but had no effect on copper detoxification or gene regulation. Immunofluorescence studies showed that both the wild-type and these two mutant proteins are predominantly cytoplasmic with no evidence for mitochondrial localization. The cleavage site mutation allowed isolation and structural characterization of a full length metallothionein polypeptide. The copper content and luminescent properties of this molecule were identical to those of the truncated wild-type protein indicating a homologous cluster structure. Moreover, the amino-terminal peptide was selectively removed by various endopeptidases and an exopeptidase suggesting that it does not participate in the tertiary fold. These results argue that the amino-terminal peptide is not required for either the structural integrity or biological function of yeast metallothionein.

Supercoiled DNA containing the replication origin of bacteriophage lambda can be replicated in vitro. This reaction requires purified lambda O and P replication proteins and a partially purified mixture of Escherichia coli proteins (Tsurimoto, T., and Matsubara, K. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 7639-7643; Wold, M. S., Mallory, J.B., Roberts, J. D., LeBowitz, J. H., and McMacken, R. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 6176-6180). The lambda origin region has four repeats of a 19-base pair sequence to which O protein binds. To the right of these sites on the lambda map is a 40-base pair region that is rich in adenine and thymine, followed by a 28-base pair palindromic sequence. To define more precisely the boundaries of the lambda origin, we cloned a 358-base pair piece of lambda DNA containing the origin region into M13mp8 in both orientations. In vitro replication of RF I DNAs prepared from cells infected with these two M13 ori lambda phage was dependent on lambda O and P proteins and a crude protein fraction from uninfected E. coli; with these conditions there was no replication of M13mp8 RF I DNA. We made deletions from the left and the right ends of the lambda origin DNA and determined the deletion end points by DNA sequencing. We have tested RF I DNAs prepared from cells infected with phage carrying ori lambda deletions for their ability to function as templates for O- and P-dependent replication in vitro. Our results show that lambda DNA between nucleotide positions 39072 and 39160 is required for efficient O- and P-dependent replication. This 89-base pair piece of DNA includes only two of the four 19-base pair O protein-binding sites (the two right-most) and the adjoining adenine- and thymine-rich region to the right of the O-binding sites.

cAMP receptor protein (CRP)-dependent operon expression in Escherichia coli requires the CRP X cAMP complex form of wild-type CRP. One class of crp mutants (crp*) activates CRP-dependent promoters in strains (cya) incapable of endogenous cAMP synthesis. Of fundamental interest is the difference in regulatory properties exhibited by crp* mutant strains, some of which exhibit glucose-mediated repression of beta-galactosidase synthesis, some of which do not. To gain a better understanding of the mechanisms of cAMP-independent promoter activation and repression we have: determined through cloning and DNA sequence analysis the primary structure of three CRP* forms of CRP; purified the mutant proteins; characterized the effect of these mutations on CRP secondary structure; and studied CRP*-activated lac promoter regulation in a purified in vitro transcription system. The results of this study provide strong evidence that mutations in crp alter the conformation of CRP and result in cAMP-independent activation of CRP-dependent promoters in vitro. In addition, a CRP allele-specific inhibition of CRP* activity by spermidine was observed in vitro that parallels crp* strain-specific sensitivity to glucose-mediated repression of CRP-dependent enzyme synthesis in vivo. This observation provides evidence that catabolite repression in cells lacking cAMP may be mediated through a mechanism that inhibits CRP* activity.

We fused segments of the Escherichia coli his regulatory region to galK in single-copy and multicopy vectors. These fusions demonstrated that (i) derepression of his by histidine starvation is due exclusively to attenuation; (ii) the his promoter is metabolically regulated; and (iii) both regulatory systems operate when the his regulatory region is present on a multicopy plasmid. Thus, there is no evidence for titration of his regulatory elements. Deletions of the his anti-attenuator region, carried on multicopy plasmids, cause low-level galK expression. This expression is not stimulated by histidine starvation, but is growth rate dependent. We replaced the his attenuator with the efficient lambda terminator, to. In the context of the his regulatory region, however, lambda to only partially terminates transcription.