Transcriptional terminator is a positive regulatory element in the expression of the Escherichia coli crp gene (original) (raw)
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Transcriptional effects of CRP* expression in Escherichia coli
Journal of Biological Engineering, 2009
Background: Escherichia coli exhibits diauxic growth in sugar mixtures due to CRP-mediated catabolite repression and inducer exclusion related to phosphotransferase system enzyme activity. Replacement of the native crp gene with a catabolite repression mutant (referred to as crp*) enables co-utilization of glucose and other sugars in E. coli. While previous studies have examined the effects of expressing CRP* mutants on the expression of specific catabolic genes, little is known about the global transcriptional effects of CRP* expression. In this study, we compare the transcriptome of E. coli W3110 (expressing wild-type CRP) to that of mutant strain PC05 (expressing CRP*) in the presence and absence of glucose.
Plasmid, 1998
Plasmids derived from bacteriophage are known as plasmids. These plasmids contain the ori region and replication genes O and P. Typical plasmids also contain the cro gene, the product of which is a repressor of the p R promoter when present at relatively high concentrations. These genes stably maintain the plasmid in Escherichia coli at copy numbers of 20 to 50 per cell. According to a generally accepted model, stable maintenance of plasmids is possible due to the Cro repressor autoregulatory loop (the cro gene is under control of p R). Here we demonstrate that plasmids devoid of the Cro autoregulatory loop can also be stably maintained in E. coli strains. We present data for two such plasmids: pTC1 in which the p R-cro region has been replaced by the p tetA promoter and the tetR gene (coding for the TetR repressor), and a standard plasmid with inactivated cro gene (cro-null plasmid). Thus, the presence of the Cro repressor autoregulatory loop does not appear to be essential to the maintenance of plasmids in vivo.
Cell Biochemistry and Function, 2008
The cyclic AMP receptor protein (CRP) of Escherichia coli regulates the activity of more than 150 genes. Allosteric changes in CRP structure accompanied by cAMP binding, initiate transcription through protein binding to specific DNA sequences. Initially, researchers proposed a two-site cAMP-binding model for CRP-dependent transcription activation since biophysical methods showed two transitions during titration experiments. Three conformational states were considered; apo-CRP, CRP:(cAMP) 1 and CRP:(cAMP) 2 , and CRP:(cAMP) 1 was proposed as the active form in this initial model. X-ray data indicated an anti conformation and in contrast NMR experiments suggested a syn conformation for bound cAMPs. For years this paradigm about ligand conformation has been ambiguous. When CRP was crystallized with four bound cAMP in the last decade, two cAMPs were assigned to syn and the other two to anti conformations. Again three conformational states were suggested; apo-CRP, CRP:(cAMP) 2 , and CRP:(cAMP) 4 . This new structure changed the view of CRP allosteric activation from a two-site model to a four-site model in the literature and the new model has been supported by biochemical and genetic data so far. According to the accepted model, binding of the first two cAMP molecules displays positive cooperativity, however, binding of the last two cAMP molecules shows negative cooperativity. This resolved the conflict between dynamic and static experimental observations. However, this new model cannot explain the initiation mechanism as previously proposed because functionally active CRP has only one cAMP equivalent. Gene regulation and transcription factors are involved in regulating both prokaryotic and eukaryotic metabolism. Although gene regulation and expression are much more complex in eukaryotes, CRP-mediated transcription initiation is a model of general interest to life sciences and medicine. Therefore, the aim of this review is to summarize recent works and developments on the cAMP-dependent CRP activation mechanism in E. coli.
Transcriptome Analysis of Crp-Dependent Catabolite Control of Gene Expression in Escherichia coli
Journal of Bacteriology, 2004
We report here the transcriptome analyses of highly expressed genes that are subject to catabolite repression or activation mediated by the cyclic AMP receptor protein (Crp). The results reveal that many operons encoding enzymes of central carbon metabolic pathways (e.g., Krebs cycle enzymes), as well as transporters and enzymes that initiate carbon metabolism, are subject to direct Crp-mediated catabolite repression. By contrast, few enzyme-encoding genes (direct regulation) but many ribosomal protein- and tRNA-encoding genes (indirect regulation) are subject to Crp-dependent glucose activation. Additionally, Crp mediates strong indirect catabolite repression of many cytoplasmic stress response proteins, including the major chaperone proteins, five ATP-dependent protease complexes, and several cold and heat shock proteins. These results were confirmed by (i) phenotypic analyses, (ii) real-time PCR studies, (iii) reporter gene fusion assays, and (iv) previously published reports abo...
Nucleic Acids Research, 1996
At class II CRP-dependent promoters the DNA site for CRP overlaps the DNA site for RNA polymerase, covering the -35 region. Transcription activation at class II CRP-dependent promoters requires a contact between an activating region in the upstream subunit of the bound CRP dimer and a contact site in the C-terminal domain of the α-subunit of RNA polymerase. Transcription activation is suppressed by amino acid substitutions in the activating region, but activation can be restored by second site substitutions at K52 or E96. These substitutions identify two separate regions on the surface of CRP that appear to be able to interact with RNA polymerase specifically at class II promoters. Using the method of 'oriented heterodimers' we show that these alternative activating regions are functional in the downstream subunit of the bound CRP dimer.
Nucleic Acids Research, 1991
We have investigated a number of mutations that alter the ability of the the E.coli transcription factors CRP and FNR to activate transcription. In CRP, some mutations at position 159 (H159L, H1591 and A159) prevent transcription activation at a number of naturally-occurring and semi-synthetic CRP-dependent promoters. We suggest that some feature of the surface-exposed turn around residue 159 is recognised by RNA polymerase during transcription activation at these promoters. Mutations at position 52 increase CRP activity and reverse the effects of H159L and A159, most likely by creating a new contact with RNA polymerase. However this new contact only gives increased expression when the CRP binding site is located 41 1/2 base pairs upstream of the transcription start site and fails to reverse the effects of H159L and Al 59 at promoters where the CRP site is located further upstream. To explain our results we propose that the two surface-exposed turns around residues 52 and 159 contain elements that are potential RNA polymerase docking sites: in the CRP dimer these two active patches are located on adjacent faces of different subunits. FNR, a related transcription activator, contains amino acid sequences homologous to the CRP sequence around position 52. Mutations in this zone (from residues 81 -88 in FNR) reduce expression from an FNR-dependent promoter without stopping FNR binding to its target. This defines a patch on FNR, which is homologous to the CRP surface-exposed loop around position 52, which is involved in transcription activation, most likely by contacting RNA polymerase.
Downregulation of the Escherichia coli guaB Promoter by Upstream-Bound Cyclic AMP Receptor Protein
Journal of Bacteriology, 2009
The Escherichia coli guaB promoter (P guaB ) is responsible for directing transcription of the guaB and guaA genes, which specify the biosynthesis of the nucleotide GMP. P guaB is subject to growth rate-dependent control (GRDC) and possesses an UP element that is required for this regulation. In addition, P guaB contains a discriminator, three binding sites for the nucleoid-associated protein FIS, and putative binding sites for the regulatory proteins DnaA, PurR, and cyclic AMP receptor protein (CRP). Here we show that the CRP-cyclic AMP (cAMP) complex binds to a site located over 100 bp upstream of the guaB transcription start site, where it serves to downregulate P guaB . The CRP-mediated repression of P guaB activity increases in media that support lower growth rates. Inactivation of the crp or cyaA gene or ablation/translocation of the CRP site relieves repression by CRP and results in a loss of GRDC of P guaB . Thus, GRDC of P guaB involves a progressive increase in CRP-mediated repression of the promoter as the growth rate decreases. Our results also suggest that the CRP-cAMP complex does not direct GRDC at P guaB and that at least one other regulatory factor is required for conferring GRDC on this promoter. However, PurR and DnaA are not required for this regulatory mechanism.