Intracellular location of catabolite activator protein of Escherichia coli

Abstract

The catabolite activator protein was assayed in extracts from the minicellproducing Escherichia coli strain P678-54. The level of catabolite activator protein was found to be the same in both parent cells and purified minicells, regardless of whether the bacteria were grown on glucose (which leads to low intracellular cyclic adenosine monophosphate levels) or on glycerol-yeast extract or LB broth (which lead to high cyclic adenosine monophosphate concentrations in the cell). Thus, at any given time most catabolite activator protein molecules are found in the cytoplasm. The implications of this for the mechanism of catabolite activator protein action at catabolite-sensitive operons are discussed.The phenomenon of catabolite repression, in which the synthesis of certain inducible enzymes is inhibited when cells are grown in the presence of glucose, is mediated in Escherichia coli by cyclic AMP (cAMP) and by the catabolite activator protein (CAP), which is also known as the cAMP receptor protein or the catabolite gene activator protein (14,18). A variety of genetic and biochemical data imply that the cAMP-CAP complex is a positive effector of transcription, which stimulates the initiation of mRNA synthesis by RNA polymerase at "catabolitesensitive" operons (6,8,25). In vitro studies have shown that in the absence of cAMP, initiation of transcription at such operons is quite inefficient (6). This correlates with the in vivo data since cAMP levels are relatively low in glucosegrown cells (9,19). A working model for the mechanism of cAMP-CAP action involves its binding to DNA at the promoter region and somehow enhancing the opening of the double helix downstream so that RNA polymerase can melt in to form a stable initiation complex (7,18). CAP will interact with DNA in the absence of cAMP, although the binding affinity is heightened by the cyclic nucleotide (17,23). Specific binding to catabolite-sensitive promoters has been difficult to show in vitro, but has recently been demonstrated (15).To elucidate the molecular details of CAP action we have been quantifying the association of CAP with DNA. We showed that in the absence of cAMP the binding of CAP to nonspecific DNA (which contains no functional CAP sites) is cooperative; that is, the protein is found in clusters on the DNA molecules (23). Addition of cAMP eliminated the cooperativity while increasing the overall affinity of the protein for DNA. We measured association constants for the CAP-DNA interaction at various ionic strengths. The results were extrapolated to presumed physiological ionic conditions to give an estimate of the in vivo binding constant. This value was combined with data for the volume of the bacterial cell and amounts of DNA and of CAP in the cell in a theoretical analysis which predicts the fraction of CAP molecules which should be bound to the chromosome at any given time. The calculation implies that nonspecific binding of CAP to DNA might occur in vivo, perhaps even at low cAMP levels (23). This theoretical approach, when applied to the la...

Predictive Biophysical Neural Network Modeling of a Compendium ofin vivoTranscription Factor DNA Binding Profiles forEscherichia coli

“…One important observation is that clusters of weaker sites can lead to occupancy equivalent to that of a single strong site, consistent with reports in other organisms 32 . Finally, our results speak to the long-standing question of the cellular localization of TFs 37,42 . For five TFs, we estimate >79% of protein is bound to the genome as compared to being free in solution.…”

Comparison of protein binding to DNA in vivo and in vitro: defining an effective intracellular target.

“…Although a variety of in vitro asays confirm this view for many proteins (Revzin, 1990;Stickle et al, 1994), there have only been a few in vivo assessments of this phenomenon. Remarkably, one of them, the determination of protein distribution in minicells, indicates that intracellular conditions render negligible the non-specific binding of the cAMP receptor protein of E.coli (Cook and Revzin, 1980). The data presented here show that IHF function is very much influenced by non-specific binding in vivo and therefore predict a very different minicell distribution from that reported for CRP.…”

Transcriptional regulation by the numbers: models

“…This is represented mathematically as NS is the difference between the two. Note that for the purposes of this simple model we have assumed that the reservoir for the activator molecules is the genomic DNA, though there is strong evidence that in the case of the lac operon many of the activators (CRP) are actually in the cytoplasm [38]. By way of contrast, as will be seen in paper 2 [1], in our actual applications of thermodynamic models to real operons, the question of whether the reservoir is nonspecific DNA or the cytoplasm never arises.…”