Diverse directional changes of cGMP relative to cAMP in E. coli.

The relationships of the changes of cAMP and cGMP concentrations in $\text{E. coli}$ varied as a function of experimental conditions. (1) Cells starved for carbon source for a short time period had high cAMP and low cGMP concentrations. Addition of carbon source (succinate, glucose or α-methyl glucoside) led to a decrease in cAMP and an increase in cGMP (bi-directional change). (2) Washed cells starved for glucose for long time periods had low cAMP levels which did not change on glucose addition. Addition of succinate or glucose to such cells led to a transient increase in cGMP levels (uncoupled change). The cGMP concentration peaked at 15 minutes or 1 hour after glucose or succinate addition, respectively. (3) Sham shift-up experiments (addition of α-methyl glucoside to cultures growing in succinate) in $\text{E. coli}$ 1100 and CA 8000 showed decreases in cGMP levels in both strains; however, cAMP levels increased in the former (bi-directional change) and decreased in the latter (unidirectional change).     

Characterization of and lipopolysaccharide binding to the E. coli LptC protein dimer

Lipopolysaccharide (LPS, endotoxin) is the major component of the outer leaflet of the outer membrane of Gram‐negative bacteria such as Escherichia coli and Salmonella typhimurium. LPS is a large lipid containing several acyl chains as its hydrophobic base and numerous sugars as its hydrophilic core and O‐antigen domains, and is an essential element of the organisms' natural defenses in adverse environmental conditions. LptC is one of seven members of the lipopolysaccharide transport (Lpt) protein family that functions to transport LPS from the inner membrane (IM) to the outer leaflet of the outer membrane of the bacterium. LptC is anchored to the IM and associated with the IM LptFGB₂ complex. It is hypothesized that LPS binds to LptC at the IM, transfers to LptA to cross the periplasm, and is inserted by LptDE into the outer leaflet of the outer membrane. The studies described here comprehensively characterize and quantitate the binding of LPS to LptC. Site‐directed spin labeling electron paramagnetic resonance spectroscopy was utilized to characterize the LptC dimer in solution and monitor spin label mobility changes at 10 sites across the protein upon addition of exogenous LPS. The results indicate that soluble LptC forms concentration‐independent N‐terminal dimers in solution, LptA binding does not change the conformation of the LptC dimer nor appreciably disrupt the LptC dimer in vitro, and LPS binding affects the entire LptC protein, with the center and C‐terminal regions showing a greater affinity for LPS than the N‐terminal domain, which has similar dissociation constants to LptA.     

Comparison of cAMP receptor protein (CRP) and a cAMP-independent form of CRP by Raman spectroscopy and DNA binding.

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)     

Molecular cloning and nucleotide sequencing of the gene for E. coli cAMP receptor protein.

The crp gene of E. coli, which codes for cAMP receptor protein (CRP), has been cloned in the plasmid pBR322 on the basis of a genetic complementation. One of the recombinant plasmids, pHA1, was shown to direct the synthesis of CRP in a cell-free system. The location of the crp gene was determined by constructing subclones carrying various portions of pHA1. The nucleotide sequence of the crp gene has been determined. The coding region consists of 627 base pairs (bp), which specify a protein of 209 amino acids. The predicted amino acid sequence from the DNA sequence is consistent with the amino acid sequence partially known and the amino acid composition of CRP. After the coding region, there is a G-C rich inverted repeat sequence followed by a run of Ts, which could be a terminator of the crp gene. A possible promoter sequence was found about 180 bp upstream from the initiation codon and was shown to act as a promoter in vitro and in vivo. There are two dyad symmetry regions in a 167 bp leader sequence.     

Interaction of CRP L124 with cAMP affects CRP cAMP binding constants,cAMP binding cooperativity,and CRP allostery

A cyclic nucleotide-binding pocket of the CRP dimer is composed of amino acid residues contributed by both subunits. Leucine (L) 124 of one subunit packs against the adenine ring of cAMP bound to the opposing subunit. We have undertaken a study designed to evaluate the role of L124 in CRP allostery. Wild-type (WT) apo-CRP is a 47 kDa protease-resistant dimer composed of identical subunits that exhibits a biphasic isotherm in cAMP titration studies. The WT CRP-cAMP complex is a protease-sensitive dimer degraded by protease to a dimer core that ranges between 26.5 and 30.5 kDa. Substitution of L124 with isoleucine (I), valine (V), cysteine (C), or alanine (A) generated a series of CRP variants that exhibited unique differences in apo-CRP resistance to protease, the mass of the core fragments generated in protease digestion reactions, cAMP-mediated allostery, and CRP-cAMP complex functionality. Differences in the affinity of the position 124 CRP variants for cAMP were observed. The binding constants that drive the formation of the WT and L124I CRP-cAMP complexes deviated by not more than a factor of 1.5. In contrast, the L124V, L124A, and L124C forms of CRP exhibited both a decreased K(cAMP1)(app) and an increased K(cAMP2)(app) to produce 2.4-, 55-, and 204-fold reductions, respectively, in the difference between these two parameters compared to that observed for WT CRP. The data indicate that the van der Waals volume and/or the hyrophobicity of the L124 side chain are important determinants of CRP cAMP binding properties and affect, either directly or indirectly, cAMP-mediated conformation changes in CRP.     

Specific binding of the cAMP receptor protein of Escherichia coli to the lactose operon promoter

The nitrocellulose filter binding assay has been used to study effects of pH, temperature, ionic strength and magnesium ions on the specific binding of the cyclic adenosine 3',5'-monophosphate (cAMP) receptor protein (CAP) to the promoter of the lactose (lac) operon of Escherichia coli. The pH has a significant effect on binding with the greatest amount of specific binding appearing at pHs near 7 with a gradual decrease in binding as the pH is increased to 8. Specific binding was observed at temperatures of 22 degrees C and 37 degrees C but not at 4 degrees C. The specific binding was also found to be a function of the concentration of magnesium acetate and potassium chloride, being dependent on the specific cation present, the total ionic strength, and the concentration of the CAP protein. All binding decreases as the ionic strength, increases, but this decrease occurs at a lower ionic strength in magnesium acetate than in potassium chloride. In a double label experiment the filter assay demonstrates that the cAMP-CAP complex preferentially binds to the wild-type lac promoter in the presence of a lac promoter mutated at the CAP binding site. Based on these results and comparisons with other experiments reported in the literature, buffer conditions that approximate the physiological state of a cell appear to be best for studying the interaction between CAP and the lactose promoter in vitro.     

Kinetic and structural studies of the allosteric conformational changes induced by binding of cAMP to the cAMP receptor protein from Escherichia coli

The cAMP receptor protein, allosterically activated by cAMP, regulates the expression of more than 100 genes in Escherichia coli. CRP is a homodimer of two-domain subunits. It has been suggested that binding of cAMP to CRP leads to a long-distance signal transduction from the N-terminal cAMP binding domain to the C-terminal domain of the protein responsible for interaction with specific sequences of DNA. In this study, the stopped-flow and time-resolved fluorescence lifetime measurements were used to observe the kinetics of the distance changes between the N-terminal and C-terminal domain of CRP induced by binding of cAMP to high-affinity binding sites. In these measurements, we used the constructed CRP heterodimer, which possesses a single Trp85 residue localized at the N-terminal domain of one CRP subunit, and fluorescently labeled by 1,5-I-AEDANS Cys178 localized at the C-terminal domain of the same subunit or at the opposite one. The F?rster resonance energy transfer method has been used to study the distance changes, induced by binding of cAMP, between Trp85 (fluorescence donor) and Cys178-AEDANS (fluorescence acceptor) in the CRP structure. The obtained results show that the allosteric transitions of CRP at micromolar cAMP concentrations follow the sequential binding model, in which binding of cAMP to high-affinity sites causes a 4 A movement of the C-terminal domain toward N-terminal domains of the protein, with kinetics faster than 2 ms, and CRP adopts the "closed" conformation. This fast process is followed by the slower reorientation of both CRP subunits.     

UV resistance of E. coli K-12 deficient in cAMP/CRP regulation.

Deletion of genes for adenylate cyclase (delta cya) or cAMP receptor protein (delta crp) in E. coli K-12 confers a phenotype that includes resistance to UV radiation (254 nm). Such mutations lead to UV resistance of uvr+, uvrA, lexA and recA strains which could partly be abolished by the addition of cAMP to delta cya but not to delta crp strain culture medium. This effect was not related to either inducibility of major DNA repair genes or growth rate of the bacteria. Enhanced survival was also observed for UV-irradiated lambda bacteriophage indicating that a repair mechanism of UV lesions was involved in this phenomenon.     

Fluorescence quenching and kinetic studies of conformational changes induced by DNA and cAMP binding to cAMP receptor protein from Escherichia coli

Cyclic AMP receptor protein (CRP) regulates the expression of more then 100 genes in Escherichia coli. It is known that the allosteric activation of CRP by cAMP involves a long-distance signal transmission from the N-terminal cAMP-binding domain to the C-terminal domain of CRP responsible for the interactions with specific sequences of DNA. In this report we have used a CRP mutant containing a single Trp13 located in the N-terminal domain of the protein. We applied the iodide and acrylamide fluorescence quenching method in order to study how different DNA sequences and cAMP binding induce the conformational changes in the CRP molecule. The results presented provide evidence for the occurrence of a long-distance conformational signal transduction within the protein from the C-terminal DNA-binding domain to the N-terminal domain of CRP. This conformational signal transmission depends on the promoter sequence. We also used the stopped-flow and Forster resonance energy transfer between labeled Cys178 of CRP and fluorescently labeled DNA sequences to study the kinetics of DNA-CRP interactions. The results thus obtained lead to the conclusion that CRP can exist in several conformational states and that their distribution is affected by binding of both the cAMP and of specific DNA sequences.     

Specific and cooperative binding of E. coli single-stranded DNA binding protein to mRNA.

Fluorometric titration of E. coli single-stranded DNA binding protein with various RNAs showed that the protein specifically and cooperatively binds to its own mRNA. The binding inhibited in vitro expression of ssb and bla but not nusA. This inhibition takes place at a physiological concentration of SSB. The function of the protein in gene regulation is discussed.     

Site-directed mutants of the cAMP receptor protein--DNA binding of five mutant proteins

Oligonucleotide-directed mutagenesis was employed to generate mutants of the cAMP receptor protein (CRP) of Escherichia coli. The mutant proteins were purified to homogeneity and tested for stability and DNA binding. It is shown that mutations at the position of Arg180 abolish specific DNA binding, whereas those at the position Arg185 have very little effect. Both positions have previously been implicated as crucial for the specific interaction between CRP and DNA. The Ser128----Ala mutant shows a slight reduction in DNA binding affinity relative to wild-type. All mutants investigated show similar stability profiles to wild-type CRP with respect to thermolysin proteolysis as a function of temperature.     

Characterization of E. coli manganese superoxide dismutase binding to RNA and DNA

Bacterial manganese superoxide dismutase (MnSOD) has been shown to localize to the chromosomal portion of the cell and impart protection from ionizing radiation to DNA. The binding affinity of bacterial MnSOD to non-sequence specific double stranded oligomeric DNA has been quantitated previously by nitrocellulose filter binding and gel shift assays. In the current study we have examined the equilibrium binding of Escherichia coli MnSOD to poly(U), poly(A), poly(C), poly(dU) and double-stranded (ds) DNA. Equilibrium association constant, K_obs, was measured by monitoring intrinsic tryptophan fluorescence quenching. Based on the extent of quenching, K_obs was determined as a function of monovalent salt (MX) concentration and type, as well as temperature, from which ΔG°_obs and ΔH°_obs were determined. It was found that the polynucleotides bind to MnSOD in the following affinity hierarchy, poly(dU) > poly(U) > dsDNA > poly(A) > poly(C). The differences in the hierarchy were not large in magnitude as the poly(dU) bound with less than a 100-fold higher affinity than poly(C) at any given [MX]. For each polynucleotide, K_obs decreases only slightly with increasing [K⁺], surprising for a relatively non-specific nucleic acid protein. Thus, our finding that MnSOD can bind to RNA leads to the possibility that MnSOD may confer protection to RNA, as well. This is, as of yet, untested. Typically one would expect strong electrostatic interactions to dominate a non-specific binding event like that, but our results show an unexpectedly strong non-electrostatic contribution to the binding.     

The role of loops 3 and 4 in the interdomains and intersubunits communication of E. coli cAMP receptor protein

Cyclic AMP serves as an intracellular messenger in cells and regulates a variety of biological functions by transmitting information through proteins. These proteins of different functions all consist of a cAMP-binding motif, and the structure of this motif is highly conserved with an exception of the loop 3 and 4. In current study, cAMP receptor protein was employed as a model system to investigate the function of the two loops. The results indicated that the loop 3 involves in the intersubunits communication of CRP, whereas the loop 4 involves in cAMP binding and interdomains communication.     

Comparison of the binding sites for the Escherichia coli cAMP receptor protein at the lactose and galactose promoters

Polyacrylamide gel electrophoresis has been used to visualise and quantitate complexes between the Escherichia coli cyclic AMP receptor protein (CRP) and DNA fragments containing the promoter region of either the E. coli galactose or lactose operons. We show that, although CRP binding to the gal fragment is weaker than binding to the lac fragment, in each case, stable complexes are formed between one dimer of CRP and one molecule of DNA. We have examined the effects of a series of deletions and point mutations in the gal promoter region on CRP binding. From the position of deletions and mutations which prevent the formation of stable complexes, we deduce the location and extent of the sequence at the CRP binding site. We show that it covers approximately the same length of sequence as the binding site at the lac promoter. Unlike the lac site, the gal site contains no palindromic sequence. We discuss the importance of symmetry in the sequence at CRP binding sites and the validity of CRP binding consensus sequences which have been proposed.