Catabolite Activator Protein Binding Research Articles

Dynamic Allostery of the Catabolite Activator Protein Revealed by Interatomic Forces.

The Catabolite Activator Protein (CAP) is a showcase example for entropic allostery. For full activation and DNA binding, the homodimeric protein requires the binding of two cyclic AMP (cAMP) molecules in an anti-cooperative manner, the source of which appears to be largely of entropic nature according to previous experimental studies. We here study at atomic detail the allosteric regulation of CAP with Molecular dynamics (MD) simulations. We recover the experimentally observed entropic penalty for the second cAMP binding event with our recently developed force covariance entropy estimator and reveal allosteric communication pathways with Force Distribution Analyses (FDA). Our observations show that CAP binding results in characteristic changes in the interaction pathways connecting the two cAMP allosteric binding sites with each other, as well as with the DNA binding domains. We identified crucial relays in the mostly symmetric allosteric activation network, and suggest point mutants to test this mechanism. Our study suggests inter-residue forces, as opposed to coordinates, as a highly sensitive measure for structural adaptations that, even though minute, can very effectively propagate allosteric signals.

3D reconstruction and comparison of shapes of DNA minicircles observed by cryo-electron microscopy

We use cryo-electron microscopy to compare 3D shapes of 158 bp long DNA minicircles that differ only in the sequence within an 18 bp block containing either a TATA box or a catabolite activator protein binding site. We present a sorting algorithm that correlates the reconstructed shapes and groups them into distinct categories. We conclude that the presence of the TATA box sequence, which is believed to be easily bent, does not significantly affect the observed shapes.

Genome-wide analysis of deoxyadenosine methyltransferase-mediated control of gene expression in Escherichia coli.

Deoxyadenosine methyltransferase (Dam) methylates the deoxyadenine residues in 5'-GATC-3' sequences and is important in many cellular processes in Escherichia coli. We performed a computational analysis of the entire E. coli genome and confirmed that GATC sequences are distributed unevenly in regulatory regions, which suggests that Dam might regulate gene transcription. To test this, a high-density DNA microarray of 4097 E. coli genes was constructed and used to assess the gene expression profiles of the wild type and the dam-16::kam mutant strain grown under four different conditions. We also used two-dimensional electrophoretic analysis of the proteome to assess the protein profiles. The expression of a large number of genes was affected by the dam deficiency. Genes involved in aerobic respiration, stress and SOS responses, amino acid metabolism and nucleotide metabolism were expressed at higher levels in the mutant cells, especially in aerobic conditions. In contrast, transcription of genes participating in anaerobic respiration, flagella biosynthesis, chemotaxis and motility was decreased in the dam mutant strain under both aerobic and low aerobic conditions. Thus, Dam-controlled genes are involved in adjusting the metabolic and respiratory pathways and bacterial motility to suit particular environmental conditions. The promoters of most of these Dam-controlled genes were also found to contain GATC sequences that overlap with recognition sites for two global regulators, fumarate nitrate reduction (Fnr) and catabolite activator protein (CRP). We propose that Dam-mediated methylation plays an important role in the global regulation of genes, particularly those with Fnr and CRP binding sites.

Hidden Markov models from molecular dynamics simulations on DNA.

An enhanced bioinformatics tool incorporating the participation of molecular structure as well as sequence in protein DNA recognition is proposed and tested. Boltzmann probability models of sequence-dependent DNA structure from all-atom molecular dynamics simulations were obtained and incorporated into hidden Markov models (HMMs) that can recognize molecular structural signals as well as sequence in protein-DNA binding sites on a genome. The binding of catabolite activator protein (CAP) to cognate DNA sequences was used as a prototype case for implementation and testing of the method. The results indicate that even HMMs based on probabilistic roll/tilt dinucleotide models of sequence-dependent DNA structure have some capability to discriminate between known CAP binding and nonbinding sites and to predict putative CAP binding sites in unknowns. Restricting HMMs to sequence only in regions of strong consensus in which the protein makes base specific contacts with the cognate DNA further improved the discriminatory capabilities of the HMMs. Comparison of results with controls based on sequence only indicates that extending the definition of consensus from sequence to structure improves the transferability of the HMMs, and provides further supportive evidence of a role for dynamical molecular structure as well as sequence in genomic regulatory mechanisms.

The role of DNA-protein salt bridges in molecular recognition: a model study.

A theoretical study is presented of the influence of salt bridges between cationic side chains and DNA phosphates on DNA conformation and flexibility. The DNA sequence studied is that of the catabolite activator protein binding oligomer from the crystallized complex. The effect of salt bridges is modeled by neutralization of net phosphate charges for the groups involved in such interactions in the crystallized complex. Energy-optimized conformations are obtained by molecular mechanics using the JUMNA program. Base sequence dependence is studied by moving the phosphate neutralization pattern along the sequence and also by point mutations. Normal mode analysis is used to evaluate DNA flexibility. The results obtained show that the free oligomer is already precurved in the direction favored by the protein, and the effect of phosphate neutralization is principally to increase this curvature. This effect is, however, strongly sequence dependent. In addition, it is shown that oligomer flexibility cannot be explained by a simple superposition of the properties of successive dinucleotide steps, strong long-range coupling effects are observed. In all the cases examined, phosphate neutralization, however, leads to a reduction in oligomer flexibility.

CAP, the −45 region, and RNA polymerase: three partners in transcription initiation at lacP1 in Escherichia coli

The lac operon of Escherichia coli is positively regulated by the catabolite activator protein (CAP) bound upstream of the −45 region (CAP binding is centered at −61.5; the −45 region extends from −50 to −38). Certain mutations within the −45 region generate sequences that resemble UP elements in base composition and mimic the stimulation by the rrnBP1 UP element, yielding up to 15-fold stimulation in vivo. These −45 region “UP mutants” are compromised in their CAP stimulation. CAP and UP elements do not act in a fully additive manner in vivo at the lac operon. Transcription assays with the wild-type lac promoter and an UP mutant of lac indicate that CAP and UP DNA also fail to act in a completely additive manner in vitro. RNA polymerase can stabilize CAP binding to promoter DNA with a −45 region UP element against a heparin challenge. This shows that CAP and the UP DNA do not compete for the α-CTD as a mechanism for their lack of additivity. CAP and UP elements both demonstrate decreased stimulation of transcription as RNA polymerase concentration is increased from 0.05 to 10 nM in in vitro transcription experiments. In addition CAP also stimulates transcription in a manner that does not decrease as RNA polymerase is varied over this concentration range. This invariable stimulation is by two- to threefold and occurs both in vivo and in vitro. It is not dependent upon the α-CTD of RNA polymerase and is maintained in the presence of the AR1 CAP mutant HL159. This two- to threefold invariable CAP stimulation appears to depend on the −45 region sequence as our −45 region mutants demonstrate different responses to HL159 CAP stimulation in vivo.

The question of long-range allosteric transitions in DNA.

The question of long-range allosteric transitions of DNA secondary structure and their possible involvement in transcriptional activation is discussed in the light of new results. A variety of recent evidence strongly supports a fluctuating long-range description of DNA secondary structure. Balanced equilibria between two or more different secondary structures, and the occurrence of very large domain sizes, have been documented in several instances. Long-range allosteric effects stemming from changes in sequence or secondary structure over a small region of the DNA have been observed to extend over distances up to hundreds of base pairs in some cases. The discovery that coherent bending strain beyond a threshold level in small (N < or = 250 base pairs (bp)] circular DNAs significantly alters the DNA secondary structure has important implications, especially for transcriptional activators that either bend the DNA directly or are involved in the formation of DNA loops of sufficiently small size (N < or = 250 bp). Whether the RNA polymerase is activated primarily via protein: protein contacts, as is widely believed, or instead via a bend-induced allosteric transition of the DNA in such a small loop, is now an open question. Binding of the transcriptional activator Sp1 to linear DNA induces a remarkably long-range change in its secondary structure, and catabolite activator protein binding to a supercoiled DNA behaves similarly, though possibly for different reasons. Compelling evidence for a bend-induced long-range structural transmission effect of the transcriptional activator integration host factor on RNA polymerase activity was recently reported. These results may augur a new paradigm in which allosteric transitions of duplex DNA, as well as of the proteins, are involved in the regulation of transcription.

Uneven Distribution of GATC Motifs in the Escherichia coliChromosome, its Plasmids and its Phages

This work reconsiders the GATC motif distribution in a 1.6 Mb segment of the Escherichia coligenome, compared to its distribution in phages and plasmids. At first sight the distribution of GATC words looks random. But when a realistic model of the chromosome (made of average genes having the same codon usage as in the real chromosome), is used as a theoretical reference, strong biases are observed. GATC pairs such as GATCNNGATC are under-represented while there is a strong positive selection for motifs separated by 10, 19, 70 and 1100 bp. The last class is the only one present in E. coliparasites. It can be ascribed to the triggering sequences of the long-patch mismatch repair system. The 6 bp class overlaps with the consensus of CAP (catabolite activator protein) and FNR (fumarate/nitrate regulator) binding sites, thus accounting for counter-selection. The other classes, which could be targets for a nucleic acid binding protein, are almost always present inside protein coding sequences, and are members of clusters of GATC motifs. Analysis of the genes containing these motifs suggests that they correspond to a regulatory process monitoring the shift from anaerobic to aerobic growth conditions. In particular this regulation, closing down transcription of a large number of genes involved in intermediary metabolism would be well suited for the cold and oxygen shift from the mammal's gut to the standard environmental conditions. In this process the methylation status of GATC clusters would be very important for tuning transcription, and a DNA binding protein, probably a member of the cold-shock proteins family would be needed for alleviating the effects mediated by slackening of the pace of methylation during the shift.

Cloning and initial characterization of the Bordetella pertussis fur gene.

In several Gram-negative pathogens the fur (ferric uptake regulator) gene product controls the expression of many genes involved in iron uptake and virulence. To facilitate the study of iron-regulated gene expression in Bordetella pertussis, we cloned the fur gene from this organism. The B. pertussis fur gene product was 54% identical to the Escherichia coli Fur and complemented two E. coli fur mutants. As with the E. coli fur gene, sequences upstream of the B. pertussis fur were homologous to the consensus Fur-binding site and to the consensus catabolite activator protein binding site.

Protein-induced bending and DNA cyclization.

We have applied T4 ligase-mediated DNA cyclization kinetics to protein-induced bending in DNA. The presence and direction of a static bend can be inferred from J factors for cyclization of 150- to 160-base-pair minicircles, which include a catabolite activator protein binding site phased against a sequence-directed bend. We demonstrate a quasi-thermodynamic linkage between cyclization and protein binding; we find that properly phased DNAs bind catabolite activator protein approximately 200-fold more tightly as circles than as linear molecules. The results unambiguously distinguish DNA bends from isotropically flexible sites and can explain cooperative binding by proteins that need not contact each other.

Downstream deletion analysis of the lac promoter.

We have generated a series of deletions in the downstream region of the lac promoter. The promoter activities of these mutations were compared by measuring the levels of beta-galactosidase gene expression in vivo. Our results show that deletion of downstream lac promoter sequences changes the promoter strength only two- to threefold. The effects of these deletions on transcription initiation site location were studied through primer extension assay of in vivo mRNAs. We found that the transcription start sites are primarily chosen as an approximate distance from the -10 region of the lac promoter; however, starts are sometimes manifested at a GAATT(C) sequence, which is identical to the wild-type preferred start site. lac promoter P2 and a newly identified promoter, P3, are transcribed in vivo at low levels. Catabolite activator protein complexed with cyclic AMP represses P2 and P3 expression in vivo. The secondary catabolite activator protein binding site plays at most a modest role in catabolite repression in vivo.

Cloning and characterization of the N-acetylglucosamine operon of Escherichia coli

Three enzymes are required for N-acetylglucosamine (NAG) utilization in Escherichia coli: enzyme IInag (gene nagE), N-acetylglucosamine-6-phosphate deacetylase (gene nagA), and glucosamine-6-phosphate isomerase (gene nagB). The three genes are located near 16 min on the E. coli chromosome. A strain of E. coli, KPN9, incapable of utilizing N-acetylglucosamine, was used to screen a genomic library of E. coli for a complementing recombinant colicin E1 plasmid that allowed for growth on N-acetylglucosamine. Plasmid pLC5-21 was found to contain all three known nag genes on a 5.7-kilobase (5.7-kb) fragment of DNA. The products of these nag genes were identified by complementation of E. coli strains with mutations in nagA, nagB, and nagE. The gene products from the 5.7-kb fragment were identified by [35S]methionine-labelled maxicells and autoradiography of sodium dodecyl sulphate-polyacrylamide electrophoresis gels. The gene products had the following relative masses (Mrs: nagE, 62,000; nagA, 45,000; nagB, 29,000. In addition, another product of Mr 44,000 was detected. The genes have been sequenced to reveal an additional open reading frame (nagC), a putative catabolite activator protein binding site that may control nagB and nagE, putative rho-independent terminator sites for nagB and nagE, and sequence homologies for RNA polymerase binding sites preceding each of the open reading frames, except for nagA. The calculated molecular weight (MWs) of the gene products derived from the sequence are as follows: nagA, 40,954; nagB, 29,657; nagC, 44,664; nagE, 68,356. No role is known for nagC, although a number of regulatory roles appear to be plausible. No obvious transcriptional termination site distal to nagC was found and another open reading frame begins after nagC. This gene, nagD, was isolated separately from pLC5-21, and the sequence revealed a protein with a calculated MW of 27,181. The nagD gene is followed by repetitive extragenic palindromic sequences. The nag genes appear to be organized in an operon: nagD nagC nagA nagB nagE.

Activation of the lac promoter and its variants : Synergistic effects of catabolite activator protein and supercoiling in vitro

Escherichia coli lac promoter variants are shown to be subject to large synergistic transcriptional activation by catabolite activator protein (CRP) and DNA supercoiling in vitro. Activation was studied for the lac wild-type promoter, a promoter with a variant spacing ( lac Δl) and two promoters with variant −10 regions ( lac p s , lac UV5). The variant promoters respond to the simultaneous presence of CRP and supercoiling by exhibiting large multiplicative activation at the low to moderate superhelicities that are most pertinent in vivo. Although all four promoters can be activated by CRP, those made stronger by changing downstream promoter elements are less CRP-activated even though each contains an identical CRP binding site. When each of the variant promoters is made stronger by introducing DNA supercoils, the apparent CRP activation initially remains constant but eventually declines at higher superhelicities. Thus, strengthening the lac promoter through either DNA sequence changes or the introduction of high-level DNA supercoiling can lead to diminished potential for activation by CRP. These results are interpreted in terms of a role for CRP in providing extra stabilizing contacts for RNA polymerase binding that are necessary only when other stabilizing features of promoter structure are lacking.

Nucleotide sequencing and expression of the fadL gene involved in long-chain fatty acid transport in Escherichia coli.

The fadL gene of Escherichia coli codes for an outer membrane protein involved in long-chain fatty acid transport. Its product was purified from outer membrane proteins. We determined the nucleotide sequence of a 2.8-kb chromosomal DNA segment that contains the fadL gene. The fadL gene consists of a 1149-nucleotide coding region and contains a highly hydrophobic polypeptide of 383 amino acids with a calculated molecular weight of 42,380. We have used S1-mapping analysis to identify the transcription initiation site. A region exhibiting extensive dyad symmetry and perfect homology to the catabolite activator protein binding site was detected.

Interactions of the catabolite activator protein (CAP) at the galactose and lactose promoters of Escherichia coli probed by hydroxyl radical footprinting. The second CAP molecule which binds at gal and the one CAP at lac may act to stimulate transcription in the same way.

The catabolite activator protein (CAP) binding sites of the Escherichia coli galactose and lactose operons were probed by hydroxyl radical footprinting. This method reveals each base that is protected by the bound protein. The patterns of protection seen for the primary CAP sites at gal and lac were virtually identical. In the presence of RNA polymerase the footprint of the second CAP molecule at gal was found to be very similar to those at the other two sites. This upstream site in gal align's perfectly with the lac CAP site with respect to the start of P1 transcription. Replacing most of the gal second CAP site DNA with heterologous sequences did not abolish binding although it became noticeably weaker. In vitro transcription studies of this hybrid gal promoter DNA further demonstrated the reduced affinity of the second CAP. These results are consistent with molecular models proposed for specific CAP binding and suggest that the second CAP at gal may be responsible for overall stimulation of transcription at this operon. Thus, in spite of differences in stoichiometry, the mechanisms of activation by CAP at gal and lac may be quite similar.

Molecular Cloning of Streptococcus bovis Lactose Catabolic Genes

SUMMARY: The hydrolysis of lactose in Streptococcus bovis was shown to be catalysed by β-D-galactosidase. To isolate lactose catabolic genes, a gene library of S. bovis DNA was constructed in bacteriophage λ47.1 and recombinants expressing β-D-galactosidase activity were detected using 5-bromo-4-chloro-indoyl-β-D-galactoside (X-Gal). The gene was cloned on a 7·8 kb DNA restriction fragment into pBR322 to generate the recombinant plasmid pHG1, which also encoded S. bovis lactose permease, thiogalactoside transacetylase and lactose repressor protein. The position and orientation of the four genes were determined by subcloning and transposon mutagenesis. The β-D-galactosidase, lactose permease and thiogalactoside transacetylase genes constitute an operon controlled by the repressor protein. β-D-Galactosides induced expression of the S. bovis lac genes in Escherichia coli while glucose had no effect. The nucleotide sequence of the presumptive regulatory region of the S. bovis lac operon was determined and compared with the corresponding E. coli sequence. The operators and the 5’ end of the lacZ genes showed strong identity. The catabolite activator protein binding sequence, present in the E. coli promoter, was absent from the corresponding S. bovis region.

A model for catabolite activator protein binding to supercoiled DNA.

Catabolite activator protein (CAP) is a dimeric molecule (M(r) = 2 x 22,500) involved in transcription initiation of several catabolite-sensitive genes of Escherichia coli. The present communication proposes a model for the interaction of CAP with DNA. The model is based upon known geometrical features of the CAP molecule [McKay, D. B. & Steitz, T. A. (1981) Nature (London) 290, 744-749], which allow interaction between dyad-related alpha-helices of the dimeric protein and major grooves in adjacently aligned sections of right-handed B-DNA. These geometrical features suggest that in vivo CAP binding to closed-circular DNA involves CAP bridging adjacent loops of a DNA solenoidal coil. This interaction pattern is shown to be consistent with the geometrical and stoichiometric properties of nonspecifically bound CAP complexes observed by Chang et al. [Chang, J. J., Dubochet, J., Baudras, A., Blazy, B. & Takahashi, M. (1981) J. Mol. Biol. 150, 435-439]. CAP-induced coil formation is related to in vivo CAP potentiation of RNA polymerase activity in underwound closedcircular DNA. Specifically, it is proposed that CAP binding to the right-interwound form of supercoiled DNA effects a local redistribution of DNA twist-strain energy, thus resulting in the formation of a left-handed solenoidal loop. The production of this localized solenoidal loop, which reflects compensatory alterations in DNA twist and writhe, may provide a conformationally unique site for RNA polymerase binding where the DNA is partially unwound. The proposed interaction pattern is consistent with both recent DNA unwinding experiments and various nuclease protection data. Moreover, features of the model suggest that the repetitive and symmetric character of many promoter sequences may provide the structural basis for a switching mechanism operative in the differential control of gene transcription.