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Here it is shown, with the use of protein-protein photocrosslinking, that the carboxyl-terminal region of the alpha subunit of RNA polymerase (RNAP) is in direct physical proximity to the activating region of the catabolite gene activator protein (CAP) in the ternary complex of the lac promoter, RNAP, and CAP. These results strongly support the proposal that transcription activation by CAP involves protein-protein contact between the carboxyl-terminal region of the alpha subunit and the activating region of CAP.

Escherichia coli cAMP receptor protein (CRP) regulates the expression of a large number of catabolite-sensitive genes. The mechanism of CRP regulation most likely involves communication between subunits and domains. A specific message, such as the activation of CRP, may be manifested as a change in the interactions between these structural entities. Hence, the elucidation of the regulatory mechanism would require a quantitative evaluation of the energetics involved in these interactions. Thus, a study was initiated to define the conditions for reversible denaturation of CRP and to quantitatively assess the energetics involved in the intra- and intersubunit interactions in CRP. The denaturation of CRP was induced by guanidine hydrochloride. The equilibrium unfolding reaction of CRP was monitored by three spectroscopic techniques, namely, fluorescence intensity, fluorescence anisotropy, and circular dichroism. The spectroscopic data implied that CRP unfolds in a single cooperative transition. Sedimentation equilibrium data showed that CRP is dissociated into its monomeric state in high concentrations of denaturant. Unfolding of CRP is completely reversible, as indicated by fluorescence and circular dichroism measurements, and sedimentation data indicated that a dimeric structure of CRP was recovered. The functional and other structural properties of renatured and native CRP have also been examined. Quantitatively identical results were obtained. Results from additional studies as a function of protein concentration and from computer simulation demonstrated that the denaturation of CRP induced by guanidine hydrochloride proceeds according to the following pathway: (CRP2)Native<-->2(CRP)Native<-->2(CRP)Denatured. The delta G values for dissociation (delta Gd) and unfolding (delta G(u)) in the absence of guanidine hydrochloride were determined by linear extrapolation, yielding values of 12.0 +/- 0.6 and 7.2 +/- 0.1 kcal/mol, respectively. To examine the effect of the DNA binding domain on the stability of the cAMP binding domain, two proteolytically resistant cAMP binding cores were prepared from CRP in the presence of cAMP by subtilisin and chymotrypsin digestion, yielding S-CRP and CH-CRP, respectively. Results from an equilibrium denaturation study indicated that the denaturation of both CH-CRP and S-CRP is also completely reversible. Both S-CRP and CH-CRP exist as stable dimers with similar delta Gd values of 10.1 +/- 0.4 and 9.5 +/- 0.4 kcal/mol, respectively. Results from this study in conjunction with crystallographic data [McKay, D. B., Weber, I. T., & Stietz, T. A. (1982) J. Biol. Chem. 257, 9518-9524] indicate that the DNA binding domain and the C-helix are not the only structural elements that are responsible for subunit dimerization.(ABSTRACT TRUNCATED AT 400 WORDS)

We have isolated 21 mutants of catabolite gene activator protein (CAP) defective in transcription activation at the lac promoter but not defective in DNA binding. The amino acid substitutions in the mutants map to a single region of CAP: amino acids 156-162. As assessed in vitro, the substituted CAP variants are nearly completely unable to activate transcription at the lac promoter but bind to DNA with the same affinity and bend DNA to the same extent as wild-type CAP. Our results establish that amino acids 156-162 are critical for transcription activation at the lac promoter but not for DNA binding and DNA bending. In the structure of CAP, amino acids 156-162 are part of a surface loop. We propose that this surface loop makes a direct protein-protein contact with RNA polymerase at the lac promoter.

Catabolite gene activator protein (CAP)-dependent promoters can be grouped into three classes, based on the requirement for transcription activation and the position of the DNA site for CAP. Class I CAP-dependent promoters require only CAP for transcription activation and have the DNA site for CAP located upstream of the DNA site for RNA polymerase. Amino acids 156 to 162 of the promoter-proximal subunit of CAP are essential for transcription activation at Class I CAP-dependent promoters, but are not essential for DNA binding, and are not essential for DNA bending. In the structure of the CAP-DNA complex, these amino acids are located in a surface loop and form a cluster on the surface of the CAP-DNA complex. Amino acids 261, 265, and 270 of the alpha subunit of RNA polymerase are essential for response to transcription activation by CAP at Class I CAP-dependent promoters. Several lines of evidence indicate that transcription activation at Class I CAP-dependent promoters requires a direct protein-protein contact between amino acids 156 to 162 of the promoter-proximal subunit of CAP and a molecule of RNA polymerase bound adjacent to CAP on the same face of the DNA helix. It is a strong possibility that this direct protein-protein contact involves amino acids 261 and 265 of the alpha subunit of RNA polymerase.

We have developed a gel electrophoretic approach for visualizing and cloning protein binding sites from complete genomes. This system consists of a simple two-dimensional band shift, in which protein-DNA complexes are retarded in the first dimension, performed at low temperature, and disrupted in the second dimension, performed at high temperature. We present here results obtained with the integration host factor (IHF) and cAMP receptor protein (CRP) proteins of Escherichia coli, and discuss some of the important methodological aspects of the technique.

Single amino acid substitutions for residue Glu171 in helix E of the catabolite gene activator protein (CAP) of Escherichia coli have been reported to abolish activation of transcription without impairing binding to the CAP site of the lac promoter. The negative charge of Glu171 was proposed to transmit the activating signal from CAP to RNA polymerase. However, this idea has been challenged by later work. We set up a system to re-examine this issue. We analysed the ability of mutant CAP-E171L and CAP-E171K proteins to bind a near-consensus CAP site in vivo and found it to be diminished fourfold relative to wild type in each case. Activation of lac transcription by these mutant proteins remains the same as with wild-type CAP. Thus our results confirm that Glu171 in helix E of CAP is not involved directly in the activation of transcription. Yet CAP-E171K does not activate transcription as well as wild-type CAP under all circumstances. Possible reasons for this absence of activation are discussed.

The structure of GH5, the globular domain of the linker histone H5, has been solved to 2.5 A resolution by multiwavelength anomalous diffraction on crystals of the selenomethionyl protein. The structure shows a striking similarity to the DNA-binding domain of the catabolite gene activator protein CAP, thereby providing a possible model for the binding of GH5 to DNA.

Protein-protein interactions between cAMP receptor protein (CRP) and RNA polymerase (RNAP) have been proposed to be essential in RNAP activation by CRP in type I promoters. These two proteins were shown to interact in solution in the absence of promoter DNA (Heyduk et al., 1993). In this report we describe the preparation of fluorescent derivatives of CRP (fluorescent probes at position 13 and 85); and of the alpha-subunit of RNAP (at position 321). The specific incorporation of fluorescence probes was achieved by expressing protein in a bacteria strain, auxotrophic for tryptophan, in media containing 5-hydroxytryptophan (5-OH-Trp). The absorbance spectrum of a protein containing 5-OH-Trp is shifted towards longer wavelengths as compared to the native protein. This allows selective monitoring of the fluorescence signal of 5-OH-Trp derivative of a protein even in the presence of high concentration of tryptophan containing protein(s). The CRP derivative is shown to retain 100% of the native protein cAMP binding and specific DNA binding activity. Using a fluorescence polarization assay, it is also shown that 5-OH-Trp derivative of CRP interacts with RNAP as well as the native protein. The RNAP reconstituted with 5-OH-Trp derivative of the alpha-subunit retained the enzymatic activity. Fluorescence quenching studies show that Trp 321 of alpha-subunit is located in the region of the protein which is exposed to a solvent.(ABSTRACT TRUNCATED AT 250 WORDS)

We have developed a simple procedure to incorporate an EDTA-metal complex at a rationally selected site within a full-length protein. Our procedure has two steps: In step 1, we use site-directed mutagenesis to introduce a unique solvent-accessible cysteine residue at the site of interest. In step 2, we derivatize the resulting protein with S-(2-pyridylthio)cysteaminyl-EDTA-metal, a novel aromatic disulfide derivative of EDTA-metal. We have used this procedure to incorporate an EDTA-iron complex at amino acid 2 of the helix-turn-helix motif of each of two helix-turn-helix motif sequence-specific DNA binding proteins, catabolite gene activator protein (CAP) and Cro, and we have analyzed EDTA-iron-mediated DNA affinity cleavage by the resulting protein derivatives. The CAP derivative cleaves DNA at base pair 2 of the DNA half-site in the protein-DNA complex, and the Cro derivative cleaves DNA at base pairs -3 to 5 of the DNA half-site in the protein-DNA complex. We infer that amino acid 2 of the helix-turn-helix motif of CAP is close to base pair 2 of the DNA half-site in the CAP-DNA complex in solution and that amino acid 2 of the helix-turn-helix motif of Cro is close to base pairs -3 to 5 of the DNA half-site in the Cro-DNA complex in solution.(ABSTRACT TRUNCATED AT 250 WORDS)

We have developed a straightforward biochemical method to determine the orientation of the DNA binding motif of a sequence-specific DNA binding protein relative to the DNA site in the protein-DNA complex. The method involves incorporation of a photoactivatable crosslinking agent at a single site within the DNA binding motif of the sequence-specific DNA binding protein, formation of the derivatized protein-DNA complex, UV-irradiation of the derivatized protein-DNA complex, and determination of the nucleotide(s) at which crosslinking occurs. We have applied the method to catabolite gene activator protein (CAP). We have constructed and analyzed two derivatives of CAP: one having a phenyl azide photoactivatable crosslinking agent at amino acid 2 of the helix-turn-helix motif of CAP, and one having a phenyl azide photoactivatable crosslinking agent at amino acid 10 of the helix-turn-helix motif of CAP. The results indicate that amino acid 2 of the helix-turn-helix motif is close to the top-strand nucleotides of base pairs 3 and 4 of the DNA half site in the CAP-DNA complex, and that amino acid 10 of the helix-turn-helix motif is close to the bottom-strand nucleotide of base pair 10 of the DNA half site in the CAP-DNA complex. The results define unambiguously the orientation of the helix-turn-helix motif relative to the DNA half site in the CAP-DNA complex. Comparison of the results to the crystallographic structure of the CAP-DNA complex [Schultz, S., Shields, S. & Steitz, T. (1991) Science 253, 1001-1007] indicates that the method provides accurate, high-resolution proximity and orientation information.

The Xanthomonas campestris catabolite gene activator protein-like protein (CLP) can substitute for the Escherichia coli catabolite gene activator protein (CAP) in transcription activation at the lac promoter (V. de Crecy-Lagard, P. Glaser, P. Lejeune, O. Sismeiro, C. Barber, M. Daniels, and A. Danchin, J. Bacteriol. 172:5877-5883, 1990). We show that CLP has the same DNA binding specificity as CAP at positions 5, 6, and 7 of the DNA half site. In addition, we show that the amino acids at positions 1 and 2 of the recognition helix of CLP are identical to the amino acids at positions 1 and 2 of the recognition helix of CAP:i.e., Arg at position 1 and Glu at position 2.

Singlet oxygen (1O2), generated by exciting an eosin-Tris complex with a high intensity beam of radiation at 532 nm, was used to chemically modify bases in fragments of DNA containing the lac UV5 promoter in the presence of the DNA binding proteins, RNA polymerase and CRP (cAMP receptor protein). Subsequent treatment with piperidine selectively cleaved the DNA at specific modified bases in the sequence. Using this technique we show first that the reactivity of DNA bound by CRP differs in the presence and absence of RNA polymerase. Hence the local conformation of CRP-bound DNA must change during the transition to the open complex. However, no reactivity is observed at the sites of the 40 degrees kinks described in the cocrystal structure (Steitz, 1990). Secondly we show that there is unique CRP-dependent reactivity at a specific site (position -46 on the upper strand) in the open complex. Finally, in the open complex, 1O2 also reacts with sites 90 bp upstream from the transcription start point. This reactivity is qualitatively CRP-independent. We infer that 1O2 reacts at sites where the promoter DNA is significantly distorted, and suggest that the pattern observed reflects the functional orientation of an active transcriptional complex in which the DNA is bent to form an extended loop.

The structure of the cyclic GMP-binding domain of the cyclic GMP-gated ion channel from bovine retinal rod photoreceptors has been modeled by analogy to the crystal structure of the homologous cyclic AMP-binding domain of catabolite gene activator protein (CAP). The modeled cyclic GMP-binding domain has a three-residue deletion and a five-residue insertion between beta strands compared to CAP. The major interactions of the ion channel with cyclic GMP are similar to those observed for cyclic AMP bound to CAP and predicted for cGMP bound to the cGMP-dependent protein kinase: Gly 543 and Glu 544 make hydrogen-bond interactions with the ribose 2'-OH, Arg 559 forms an ion pair with the charged phosphate oxygen, and Thr 560 forms hydrogen-bond interactions with an exocyclic phosphate oxygen and with the 2-amino group of cGMP. Three additional potential interactions were predicted from the model structure. Ile 545 O and Ser 546 OH form hydrogen-bond interactions with an exocyclic phosphate oxygen, and Phe 533 may interact with the aromatic ring of cGMP. This model is in agreement with both the analogue binding experiments and the mutational analysis of Thr 560.

The arginine-dependent repressor-activator from Bacillus subtilis, AhrC, has been overexpressed in Escherichia coli and purified to homogeneity. AhrC, expressed in E. coli, is able to repress a Bacillus promoter (argCp), which lies upstream of the argC gene. The purified protein is a hexamer with a subunit molecular mass of 16.7 kDa. Its ability to recognize DNA has been examined in vitro using argCp in both DNase I and hydroxyl radical protection assays. AhrC binds at two distinct sites within the argCp fragment. One site, argCo1, with the highest affinity for protein, is located within the 5' promoter sequences, whilst the other, argCo2, is within the coding region of argC. The data are consistent with the binding of a single hexamer of AhrC to argCo1 via four of its subunits, possibly allowing the remaining two subunits to bind at argCo2 in vivo forming a repression loop similar to those observed for the E. coli Lac repressor.

The Escherichia coli catabolite gene activator protein (CAP) is a helix-turn-helix motif sequence-specific DNA binding protein. CAP contains a unique solvent-accessible cysteine residue at amino acid 10 of the helix-turn-helix motif. In published work, we have constructed a prototype semi-synthetic site-specific DNA cleavage agent from CAP by use of cysteine-specific chemical modification to incorporate a nucleolytic chelator-metal complex at amino acid 10 of the helix-turn-helix motif [Ebright, R., Ebright, Y., Pendergrast, P.S. and Gunasekera, A., Proc. Natl. Acad. Sci. USA 87, 2882-2886 (1990)]. Construction of second-generation semi-synthetic site-specific DNA cleavage agents from CAP requires the construction of derivatives of CAP having unique solvent-accessible cysteine residues at sites within CAP other than amino acid 10 of the helix-turn-helix motif. In the present work, we have constructed and characterized two derivatives of CAP having no solvent-accessible cysteine residues: [Ser178]CAP and [Leu178]CAP. In addition, in the present work, we have constructed and characterized one derivative of CAP having a unique solvent-accessible cysteine residue at amino acid 2 of the helix-turn-helix motif: [Cys170;Ser178]CAP.

A frequent cellular response to organismal stress is the increase in ligand binding by beta-adrenergic receptors. The extracellular signal is amplified by intracellular increases in cyclic AMP and the ensuing activation of cyclic AMP-dependent protein kinase (cAPK). The molecular mechanisms involve the binding of cyclic AMP to regulatory (R) subunits of cAPK, thus freeing the catalytic subunit for protein phosphorylation. This study was carried out to determine the cellular compartmentalization of the cyclic AMP-receptor proteins in heart ventricular tissue obtained from rats flown on the Cosmos 1887 mission. Photoaffinity labeling of soluble and particulate cell fractions with an [32P]-8-azido analog of cyclic AMP was followed by electrophoretic separation of the proteins and by autoradiographic identification of the labeled isoforms of cAPK R subunits. The results showed that RII in the particulate subcellular fraction was significantly decreased in heart cells from rats in the flight group when compared to controls. Protein banding patterns in both the cytoplasmic fraction and in a fraction enriched in chromatin-bound proteins showed some variability in tissues of individual animals, but exhibited no changes that could be directly attributed to flight conditions. No significant change was apparent in the distribution of RI or RII cyclic AMP binding in the soluble fractions. These findings indicate that the cardiac cell integrity or its protein content is not compromised under flight conditions. There is, however, what appears to be an adaptive molecular response which can be detected using microanalytical methods, indicating that a major hormone regulated mechanism may be affected during some phase of travel in space.

The 3 angstrom resolution crystal structure of the Escherichia coli catabolite gene activator protein (CAP) complexed with a 30-base pair DNA sequence shows that the DNA is bent by 90 degrees. This bend results almost entirely from two 40 degrees kinks that occur between TG/CA base pairs at positions 5 and 6 on each side of the dyad axis of the complex. DNA sequence discrimination by CAP derives both from sequence-dependent distortion of the DNA helix and from direct hydrogen-bonding interactions between three protein side chains and the exposed edges of three base pairs in the major groove of the DNA. The structure of this transcription factor--DNA complex provides insights into possible mechanisms of transcription activation.

CRP is resistant to attack by carboxypeptidase Y at 37 degrees C, whereas cAMP-CRP is digested yielding a core terminating at Thr-202 and lacking the seven carboxyl-terminal amino acid residues. A similar core (CRPCY) is formed when CRP is incubated with carboxypeptidase Y at 47 degrees C in the absence of cAMP. CRPCY has a more open conformation than CRP at 37 degrees C. While unliganded CRP is resistant to trypsin, CRPCY is sensitive to tryptic attack. Dithionitrobenzoic acid-mediated intersubunit disulfide crosslinking of CRP requires cAMP, CRPCY subunits are crosslinked in the absence of cAMP. The carboxyl-terminal region of unliganded CRP is conformationally restricted at 37 degrees C. The CRPCY retains cAMP binding activity. The CRPCY which terminates at Thr-202, no longer binds lac P+ DNA nor stimulates abortive initiation by RNA polymerase from the lac P+ promoter. The results indicate that the C-terminal region of CRP participates in the conformational stability of the closed form of CRP and indirectly in DNA binding by the open cAMP-CRP conformer.

The secondary structures of the cAMP receptor protein (CRP), a complex of CRP and cAMP, and a cAMP-independent receptor protein mutant (CRP*141 gln) were examined by using Raman spectroscopy. Spectra were obtained from CRP and CRP*141 gln dissolved in 0.3 M NaCl and 30 mM sodium phosphate at protein concentrations of 30-40 mg/mL. CRP and CRP.cAMP1 were compared at lower protein concentrations (10-12 mg/mL) in a solvent of 0.35 M NaCl and 20 mM sodium phosphate. Raman analysis indicates that CRP structural changes induced by one bound cAMP or by the Gly to Gln mutation at residue 141 are small. Spectra of the three CRP samples are essentially identical from 400 to 1900 cm-1. This result differs from the Raman spectroscopy study of CRP and CRP.cAMP2 cocrystals [DeGrazia et al. (1990) Biochemistry 29, 3557]. The latter work showed spectral differences between CRP and CRP.cAMP2 consistent with alterations in the protein conformation. These studies indicate that CRP and CRP.cAMP1 in solution are similar in structure and differ from CRP.cAMP2 cocrystals. Protease digestion and a DNA binding assay were also employed to characterize the wild-type and mutant proteins. CRP*141 gln exhibited the same conformational characteristics of previously reported cAMP-independent mutant proteins. It was sensitive to proteolytic attack in the absence of cAMP, or upon addition of cGMP. In the absence of cAMP, both wild-type and mutant CRPs bound noncooperatively to a 62 bp lac promoter DNA. The equilibrium constants were approximately 10(6) M-1 in 0.1 M Na+. CRP*141 gln had a 2-4-fold higher affinity for the 62 bp DNA than CRP.(ABSTRACT TRUNCATED AT 250 WORDS)

Endoglycoceramidase (EGCase) cleaves the linkage between oligosaccharides and ceramides of various glycosphingolipids (Ito, M., and Yamagata, T. (1986) J. Biol. Chem. 261, 14278-14282). Recently, by extensive purification, it was separated from cell-lytic factor (hemolysin) and found to consist of three molecular species each with its own specificity (EGCases I, II, and III) (Ito, M., and Yamagata, T. (1989) J. Biol. Chem. 264, 9510-9519). A detergent was required for EGCases to express full activity, possibly due to their hydrophobic nature, and thus EGCases cannot be used for research on live cells. This paper presents findings on activator proteins in the culture supernatant of Rhodococcus sp. M-777 regarding the stimulation of EGCase activity in the absence of detergents. The activator protein, exhaustively purified and designated as activator II in this study, showed a single protein band on sodium dodecyl sulfate-, native-, and isoelectrofocussing-polyacrylamide slab gel electrophoresis after being stained with Coomassie Brilliant Blue. Its molecular weight and pI were 69,200 and 4.0, respectively. The activator protein enhanced the hydrolysis of glycosphingolipids in vitro and on the cell-surface by EGCase II in the absence of detergents in a concentration-dependent manner. Interestingly, activator II stimulated the activity of EGCase II much more than that of EGCase I on using asialo-GM1 as the substrate. This activator protein was found nonspecific to substrates susceptible to hydrolysis with EGCase II. Besides activator II, strain M-777 produced a second minor molecular species of activator protein designated as activator I which appeared specific for stimulating the activity of EGCase I in contrast to activator II. Following the addition of activator II, EGCase II hydrolyzed cell-surface glycosphingolipids quite efficiently at neutral pH at which hydrolysis hardly occurred at all in its absence. When using activator II in place of Triton X-100 for stimulating EGCase II activity, it was also noted to cause no damage to intact cells. It is thus possible by activator proteins to elucidate the biological functions of endogenous glycosphingolipids in situ by EGCases.

Two similar, but not identical, models have been proposed for the amino acid-base pair contacts in the CAP-DNA complex ('Model I,' Weber, I. and Steitz, T., Proc. Natl. Acad. Sci. USA, 81, 3973-3977, 1984; 'Model II,' Ebright, et al., Proc. Natl. Acad. Sci. USA, 81, 7274-7278, 1984). One difference between the two models involves Glu181 of CAP. Model I predicts that Glu181 of CAP makes two specificity determining contacts: one H-bond with the cytosine N4 atom of G:C at base pair 7 of the DNA half site, and one H-bond with the adenine N6 atom of T:A at base pair 6 of the DNA half site. In contrast, Model II predicts that Glu181 makes only one specificity determining contact: one H-bond with the cytosine N4 atom of G:C at base pair 7 of the DNA half site. In the present work, we show that replacement of T:A at base pair 6 of the DNA half site by T:N6-methyl-adenine has no, or almost no, effect on the binding of CAP. We conclude, contrary to Model I, that Glu181 of CAP makes no contact with the adenine N6 atom of base pair 6 of the DNA half site.