Catabolite gene activator protein activation of lac transcription

Reznikoff, W S

doi:10.1128/jb.174.3.655-658.1992

Cited by 72 publications

(50 citation statements)

References 23 publications

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“…This heterologous activation shows that the plasticity observed in hutUp is not a unique feature of this promoter. Furthermore, we have shown that NAC can activate lacZp 3 and lacZp 2 , two promoters that are repressed by the CAP-cAMP complex (21,30). The activation of lacZp 3 and lacZp 2 shows that heterologous activation by NAC is not restricted to promoters activated by CAP-cAMP and that it is possible with other weak promoters.…”

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confidence: 89%

Activation of the Escherichia coli lacZ promoter by the Klebsiella aerogenes nitrogen assimilation control protein (NAC), a LysR family transcription factor

Pomposiello

Bender

1995

J Bacteriol

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A chimeric promoter with the nitrogen assimilation control protein binding site from hutUp of Klebsiella aerogenes fused to the lacZ core promoter from Escherichia coli was built and cloned in a lacZ reporter plasmid. This construct showed a 14-fold increase of ␤-galactosidase activity upon nitrogen limitation. Primer extension experiments showed that the nitrogen assimilation control protein activates lacZp 1 in a position-dependent manner.The enteric bacterium Klebsiella aerogenes responds to a decrease in the concentration of ammonia, the preferred nitrogen source, by modifying transcription rates at many operons. Some of these nitrogen-regulated operons allow the cell to assimilate ammonia in low concentration or to use alternative nitrogen sources. This global response to nitrogen starvation is regulated by the Ntr system, a regulatory network built around a two-component system (see references 16 and 27 for reviews). Essentially, nitrogen starvation results in the activation of NtrC, a DNA-binding protein that enhances transcription from 54 -dependent promoters. Although the Ntr system is necessary for the modulation of all nitrogen-regulated operons, transcriptional activity of a subset of these operons is also dependent on the nitrogen assimilation control protein (NAC) (4,5,15). NAC is a member of the LysR family of DNA binding transcriptional factors (23,24). The transcription of the nac gene is 54 dependent and under positive control by the Ntr system (8, 15). The apparent paradox posed by a second, positively regulated transcription factor in the Ntr cascade is explained by the functions NAC adds to the Ntr repertoire: NAC regulates 70 -dependent promoters and can either activate or repress transcription (see reference 2 for a review). Therefore, we see NAC as the coupling agent between 54 -dependent Ntr regulation and the 70 -dependent operons. NAC activates the expression of operons involved in the catabolism of alternative nitrogen sources: histidine utilization (hut), proline utilization (put), and urease (ure) (15). The bestcharacterized NAC-activated system is that of the hutUH operon (25). The transcriptional activation of the hut operon by NAC has been reproduced in vitro with purified components (11). These in vitro transcription experiments have shown that NAC is sufficient for the activation of 70 -directed transcription. In addition, the binding site for NAC has been determined by gel mobility retardation and footprinting assays (11). NAC protects a 26-bp fragment of the hutU promoter centered 64 bp upstream of the hutU transcriptional initiation site. A sequence comparison of the NAC-protected regions from the hutU, putP, and ureD promoters revealed a minimal NAC binding consensus: 5Ј-ATA-N 9 -TAT-3Ј.Interestingly, some NAC-regulated operons have functions other than the generation of reduced nitrogen, such as carbon and energy provision, and are regulated independently by other control signals. For example, the hutUH operon, whose products provide glutamate and energy, is activated by c...

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confidence: 89%

Activation of the Escherichia coli lacZ promoter by the Klebsiella aerogenes nitrogen assimilation control protein (NAC), a LysR family transcription factor

Pomposiello

Bender

1995

J Bacteriol

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“…One of the most striking aspects of the Lrp regulon is the number of different patterns of regulation that are caused by the interaction of Lrp and leucine. Like some other important global regulators, for example, Crp (14), Lrp activates the expression of some operons and represses the expression of others. For some of the operons activated by Lrp, that activation is negated by leucine (as is the case for the ilvIH operon), requires leucine, or is independent of leucine (3,11).…”

Section: Methodsmentioning

confidence: 99%

In vitro transcription from the Escherichia coli ilvIH promoter

Willins

Calvo

1992

J Bacteriol

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Lrp (leucine-responsive regulatory protein) activates the expression of the Escherichia coli ilvIH operon in vivo and mediates the repression of the operon by exogenous leucine. In Lrp (leucine-responsive regulatory protein), a newly recognized global regulatory protein in Escherichia coli, regulates the expression of at least 12 operons (3, 11). Here we address Lrp-mediated regulation of ilvIH, an E. coli operon that encodes an enzyme catalyzing the first step in branchedchain amino acid biosynthesis (2). This operon is unusual in that sequences required for optimal expression extend at least 250 bp upstream of the presumed start point of transcription (6). Lrp (formerly called IHB) binds ilvIH DNA in this region and activates transcription of the ilvIH operon (13,15). Like many of the operons regulated by Lrp, ilvIH is also regulated by leucine (3, 11). Exogenous leucine reduces transcription of the ilvIH operon 5-to 10-fold in vivo, an effect that is mediated by Lrp (6,13,15,19).

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“…1B). CRP binds two cAMP molecules and undergoes a conformational change in the DNA-binding domains that enables the protein to interact with high affinity and specificity with DNA promoter sequences (13)(14)(15)(16)(17). The molecular architecture of CRP is ideal for quantitative studies on the mechanisms of transduction of allosteric signals because: 1) cAMP binding reports on intersubunit communication and cooperative interactions, and 2) DNA binding reports on intrasubunit interactions and cAMP-induced conformational changes of the protein (13,18).…”

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confidence: 99%

Asymmetric configurations in a reengineered homodimer reveal multiple subunit communication pathways in protein allostery

Lanfranco

Gárate

Engdahl³

et al. 2017

Journal of Biological Chemistry

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Many allosteric proteins form homo-oligomeric complexes to regulate a biological function. In homo-oligomers, subunits establish communication pathways that are modulated by external stimuli like ligand binding. A challenge for dissecting the communication mechanisms in homo-oligomers is identifying intermediate liganded states, which are typically transiently populated. However, their identities provide the most mechanistic information on how ligand-induced signals propagate from bound to empty subunits. Here, we dissected the directionality and magnitude of subunit communication in a reengineered single-chain version of the homodimeric transcription factor cAMP receptor protein. By combining wild-type and mutant subunits in various asymmetric configurations, we revealed a linear relationship between the magnitude of cooperative effects and the number of mutant subunits. We found that a single mutation is sufficient to change the global allosteric behavior of the dimer even when one subunit was wild type. Dimers harboring two mutations with opposite cooperative effects had different allosteric properties depending on the arrangement of the mutations. When the two mutations were placed in the same subunit, the resulting cooperativity was neutral. In contrast, when placed in different subunits, the observed cooperativity was dominated by the mutation with strongest effects over cAMP affinity relative to wild type. These results highlight the distinct roles of intrasubunit interactions and intersubunit communication in allostery. Finally, dimers bound to either one or two cAMP molecules had similar DNA affinities, indicating that both asymmetric and symmetric liganded states activate DNA interactions. These studies have revealed the multiple communication pathways that homo-oligomers employ to transduce signals.Allosteric proteins are the basic building blocks in the transmission of biological signals, allowing communication between and within cells and from the extracellular environment to the cytosol (1). Because many allosteric proteins form homo-oligomeric complexes to modulate a ligand-induced biological response (2, 3), intersubunit communication must play a crucial role in the transduction of an allosteric signal (4, 5). Identifying the molecular mechanisms of intersubunit communication has important implications, from understanding how biological systems detect and transduce signals (6, 7) to reengineering of signaling proteins (8 -10) and to developing allosteric therapeutic modulators with enhanced affinities and specificities (11,12). Despite its importance, dissecting the mechanisms of transduction of allosteric signals from one protein subunit to another has proven difficult because it requires monitoring intermediate liganded states that are poorly populated, especially if the protein displays positive ligand binding cooperativity. Therefore, one of the remaining unresolved issues in allostery is dissecting the directionality of pathways of signal transmissions across protein subunits.A widely used s...

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Catabolite gene activator protein activation of lac transcription

Cited by 72 publications

References 23 publications

Activation of the Escherichia coli lacZ promoter by the Klebsiella aerogenes nitrogen assimilation control protein (NAC), a LysR family transcription factor

Activation of the Escherichia coli lacZ promoter by the Klebsiella aerogenes nitrogen assimilation control protein (NAC), a LysR family transcription factor

In vitro transcription from the Escherichia coli ilvIH promoter

Asymmetric configurations in a reengineered homodimer reveal multiple subunit communication pathways in protein allostery

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