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POSITIVE REGULATION – THE ARA OPERON -first example of positive regulation; Englesberg and colleagues -met with much opposition as it was thought that the wellcharacterized negative regulation of the lac system was representative of ALL gene regulation -ara operon encodes genes responsible for utilization of Larabinose -L-arabinose is converted to D-xylulose-5-phosphate, which can then be used by other pathways -3 structural genes; araB, araA, araD -araBAD are induced in presence of arabinose, much like lac genes are induced in presence of lactose -one regulatory gene transcribed in opposite direction; araC -AraC functions as an activator upon binding inducer arabinose by binding at araI and stimulating transcription -AraC also functions as a repressor in absence of inducer arabinose by binding to two operators (araO2 and araO1) and preventing transcription through DNA looping Identification of AraC -identified 15 mutants that are all non-inducible Ara-all failed to complement each other so must be in same gene – araC -all are recessive to araC+ (NB. Very different from lac!) -these mutants contained nonsense, missense, and deletion mutations in araC Lac operon -expression induced by lactose -lacI-= constitutive Lac+ -lacI-/lacI+ = Lac+; recessive Ara operon -expression induced by arabinose -araC- = non-inducible Ara-araC-/araC+ = Ara+; recessive -suggests araC- mutations knock out a POSITIVE function required for expression of araBAD Isolation of araCc alleles -constitutive mutants are very rare, suggesting that they do not inactivate the regulator AraC (reverse for LacI) -isolated using D-fucose -D-fucose binds AraC and prevents it from binding arabinose, thereby preventing induction of araBAD operon -wildtype E. coli can not grow on minimal arabinose plates containing fucose -mutants that constitutively express ara operon WILL grow -mutants all mapped to araC -araCc are dominant to araC- Identification of araI -critics believed that araC- mutations acted by interfering with a negative regulatory system -eg. they suggested that araC- mutations prevented accumulation of inducer in the presence of arabinose, which would alleviate repression -to try and identify a negative regulator, Englesberg et al. looked for suppressors of an araC deletion allele, araC∆719 -mutants were rare and all mapped to a site, araI, near, but not in, the araBAD genes -these mutations identify an AraC binding site necessary for inducible activation of araBAD expression Definitive Proof of Positive Regulation by AraC -not until several years later -an in vitro coupled transcription-translation system was developed -DNA encoding araB and control region + RNAP + ribosomes -require addition of AraC to get AraB synthesis -fucose blocks AraB synthesis -AraCc obtained from a fucose resistant strain is insensitive to fucose inhibition -requires RNAP binding site and araI -araIc mutant DNA does not require AraC for AraB synthesis AraC is also a Repressor! -if AraC is solely an activator, then AraCc should be dominant to wildtype AraC Genotype araC araCaraCc F’araC+/araCF’araCc/araC- Phenotype Inducible Ara+ Non-inducible AraConstitutive Ara+ Inducible Ara+ Constitutive Ara+ F’araC+/araCc Inducible, Ara+ + -what’s going on? -hypothesize that AraC can exist in 2 states, P1 and P2 -P1 – in absence of arabinose; functions as a repressor -P2 – in presence of arabinose; functions as an activator -AraCc mutant is locked in P2 form, but in a merodiploid F’ araC+/araCc, wildtype AraC represses expression in absence of arabinose, preventing activation by AraCc Identification of an Operator for the ara operon -deletions that extended beyond araC caused increased basal expression of the araBAD operon -propose that deletion removes an operator -now know that AraC binds to three sites in the araBAD regulatory region, araI, araO1, and araO2 -in absence of arabinose, AraC binds araO2 and araI and represses araBAD transcription -arabinose alters AraC such that repression is alleviated and transcription is initiated (requires a second activator, CAP) -in addition, AraC binds to araO1 in absence of arabinose to repress its own transcription CAP/CRP -Catabolite Activator Protein (CAP) or Catabolite Repression Protein (CRP) -transcription activator that controls expression of genes involved in carbon and energy source utilization -since glucose is the preferred carbon source of E. coli, CAP ensures that other carbon utilization pathways are not expressed in the presence of glucose à CATABOLITE REPRESSION -accordingly, CAP functions as a transcription activator of genes involved in metabolism of alternative carbon sources when glucose is NOT present (ie. lac, ara, gal, mal operons) -it does this by sensing the levels of cAMP (cyclic AMP) -when glucose is being metabolized, levels of cAMP (cyclic AMP) are decreased -conversely, when glucose is not being metabolized levels of cAMP are elevated -CAP is a symmetrical dimer of two identical subunits -when bound to cAMP (low glucose, high cAMP), CAP is active and binds to a specific palindrome found upstream of genes that are controlled by catabolite repression -consensus: 5’-AAATGTGATCT-AGATCACATTT-3’ -DNA binding mediated by a HTH present in each subunit and leads to DNA bending (crystal structure) Different CAP promoters -CAP-dependent promoters can be grouped into 3 classes based on location of the CAP binding site i) Class I -require only CAP for transcription activation -CAP binding site is upstream of RNAP binding site -binding site can be located at various distances from start site as long as it is on the same face of the helix as the RNAP binding site (-61.5, -72.5, -82.5, -92.5) -protoype is lacP1 promoter ii) Class II -require only CAP for transcription activation -CAP binding site overlaps RNAP binding site -replaces –35 binding determinants for RNAP -prototype is galP1 promoter (CAP binding site at –41.5) iii) Class III -require a regulon-specific activator in addition to CAP for transcription activation -CAP binding site usually located more than 90 bp from transcription start site -eg. araBAD promoter -much work suggests that the mechanisms of transcription activation vary at Class I, Class II, and Class III promoters Transcription Activation at Type I Promoters -determinants within CAP and RNAP that are essential for transcription activation specifically at Type I promoters have been identified 1) CAP determinants for transcription activation -mutants were sought which were specifically defective in transcription from Class I promoters, but not in DNA binding or DNA bending à POSITIVE CONTROL MUTANTS -random mutagenesis of gene encoding CAP, in vivo screen for loss of ability to activate transcription at Class I promoter, but retention of ability to bind DNA (still represses) -mutants are designated crppc (positive control) -all localize to a region that is surface exposed in the crystal structure à aa 156-162 (ACTIVATING REGION I, ARI) -purified mutant proteins all bind and bend DNA but fail to activate transcription of Class I promoters -therefore, an additional step after DNA binding necessary to activate transcription that requires AR1 2) RNAP determinants for transcription activation -deletion of or point mutations in the C-terminal domain of the α subunit preclude CAP-mediated activation at Class I promoters, although the RNAP molecules are still able to bind DNA and transcribe from an un-regulated promoter -anti-α monoclonal antibodies inhibit CAP-mediated activation at Class I promoters, but only partially inhibit CAP-independent transcription -aa 261, 265 and 270 essential for CAP-mediated transcription -mutations alter a tightly folded C-terminal domain that is joined to an N-terminal domain by a flexible linker -hypothesize this region plays a role in AR1 activation i) Mechanism of transcription activation -transcription activation at Class I CAP-dependent promoters requires a protein-protein contact between AR1 and the C-terminus of the a subunit of RNAP -since CAP functions as a symmetrical dimer, which AR1 region is required? Both? -test using ORIENTED HETERODIMER EXPERIMENT -CAP heterodimers are constructed that carry one subunit that has altered DNA binding specificity à V181 (but functional in transcription activation); and one subunit defective in AR1 à A158 (but having wild-type DNA binding specificity) -the subunits are oriented on the promoter by using CAP binding sites that are specific for the altered DNA binding mutant in one half of the palindrome à L29 or L8 -by determining transcription levels mediated by different combinations of dimers and binding sites can determine which AR1 region is important -this approach determined that only the promoter proximal AR1 region is important for transcription activation at Class I CAPdependent promoters Transcription Activation at Type II Promoters -transcription activation requires 2 distinct protein-protein interactions between different areas of CAP and RNAP that affect different stages of transcription initiation i) A second activating region in CAP -a screen was performed to determine if CAP contains a second AR required for activation at Class II CAP promoters -random mutagenesis of CAP à screen for mutants specifically defective in activation of Class II genes -isolated mutations in His-19, His-21, and Lys-101 -all are next to each other in crystal structure, all are positively charged à AR2 -positive charge of AR2 is required for transcription activation of Class II promoters -these mutations affect transcription activation at Class II, but not Class I CAP-dependent promoters -they have no effect on DNA binding or bending ii) Identification of residues in RNAP that interact with AR2 -mutations at amino acids 162-165 of rpoA (N-terminal domain of α subunit) yield RNAP that is defective in transcription activation at Class II promoters, but not Class I, or unregulated promoters -these amino acids are all negatively charged, suggesting they might interact with the positively charged AR2 -in complex of CAP, RNAP, and DNA, AR2 and aa 162-165 of α are located next to one another iii) Mechanism of transcription activation at Class II promoters -transcription activation at Class II promoters involves an interaction between AR2 and the NTD of the α subunit of RNAP -which AR2 in the dimer is required à ORIENTED HETERODIMERS -AR2 in the downstream CAP subunit of the dimer is required for transcription activation at Class II promoters -NB à AR1 is also required at Class II promoters -because Class II promoters don’t contain an optimal –35, formation of the closed complex is inhibited -binding between AR1 and the CTD of a overcomes this inhibition Class II promoters require 2 separate interactions between CAP and RNAP -AR1 enhances formation of closed complex, AR2 stimulates open complex formation Other Characterized Activator-RNAP Contacts -protein-protein contact between activators and RNAP is a common requirement for transcription activation α -transcription factors which bind upstream of the promoter require α CTD to activate transcription -RNAP carrying an α subunit with a deletion of the CTD no longer facilitates transcription from promoters of genes requiring some activator proteins -eg. PhoB, OmpR, OxyR β’ -ssDNA binding protein of bacteriophage N4 stimulates transcription of late phage genes by interacting with a conserved domain of β’ σ -lcI encodes dual function activator/repressor responsible for the lytic/lysogenic growth decision in λ -mutants in an N-terminal acidic patch are specifically defective in transcription activation, however still bind DNA normally -suppressor mutations in σ restore activation of transcription to these mutants DEBATE OVER THE MECHANISM -2 possible mechanisms of transcription activation through proteinprotein contact with RNAP: 1) recruitment of RNAP/stimulation of closed complex formation 2) stimulation of conformational changes in RNAP that facilitate transcription initiation (AR2 of CAP) -several experiments suggest that ANY protein-protein contact between a DNA binding protein located upstream of a promoter and RNAP will facilitate transcription, arguing that (I) is correct -eg. if the C-terminal dimerization domain of lcI is fused to the NTD of α in place of the native CTD, lcI can function as an activator of transcription if a binding site is placed upstream of a promoter COMPLEX ACTIVATION -transcription activation involving more than one activator protein -often, one will be a GLOBAL REGULATOR that controls expression of large numbers of genes in response to a global metabolic signal, while the other will be a SPECIFIC REGULATOR, triggering expression of a small number of genes in response to a specific inducer molecule -several mechanisms of complex regulation: I. nucleoprotein structures -binding of multiple activators forms a nucleoprotein complex that triggers transcription activation II. repositioning -binding of a second activator repositions an activator that is already bound to DNA, such that it is now in position to activate transcription III. simultaneous touching -multiple activators interact with distinct regions of RNAP to facilitate transcription activation -common with regulators that interact with σ70 IV. recruitment of bystanders/DNA looping -IHF or other DNA bending protein facilitates contact of RNAP with transcription factors bound far upstream of the promoter -eg. σ54 promoters