PRE Promoter Research Articles

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The transcription activator of the λ phage, CII, determines whether the phage will undergo the lytic or the lysogenic pathway. In a report by Zhao etal. in this issue of Structure, the cryo-EM structure of the λCII-dependent transcription activation complex reveals how λCII activates the PRE promoter to turn on the lysogenic pathway.

Abstract P3-04-10: Progesterone receptor (PR) blockade by antiprogestin, CDB4124 in hormone receptor positive breast cancer cells leads to significant inhibition of G2/M cell cycle genes

Abstract Background: Several lines of evidence suggest that progesterone signaling is important in the breast cancer development, particularly in young women. Therefore, we sought to establish the effects of the antiprogestin CDB4124 (telapristone), and to identify PR related signature genes in hormone receptor positive (ER+ PR+) breast cancer cells. Methods: The PR expressing breast cancer cell line T47D was used to evaluate responses to PR ligands (P4, MPA and R5020, 10nM) alone or in combination with estradiol (E2, 1nM). The effects of the antiprogestin CDB4124 (0.1 or 1μM) were tested using varying hormonal conditions. The effect on (a) PRE promoter activity by dual luciferase assay; (b) cell proliferation using the MTT assay; (c) cell cycle by flow cytometry; (d) determination of gene expression signatures related to active PR responses. For the gene array experiment, cells were treated with either vehicle or R5020 (10nM) or R5020 (10nM) plus CDB4124 (1μM) in triplicate for 24hr. Total RNA was isolated and converted to cDNA and human Illumina chip microarray was performed. Data obtained from the microarray was further analyzed by Metacore Gene GO and Ingenuity Pathway Analysis. Real time PCR was performed in triplicate to confirm the expression of those genes related to the cell cycle and proliferation. ANOVA analysis and post-hoc Sidak test were used to determined the statistical significance of the data. Results: The PRE reporter activity resulting from P4, MPA and R5020 stimulation was inhibited by 80-90% in the presence of CDB4124 at 10 to 1000nM (p< 0.001). Cell proliferation was increased by PR ligands (P4, MPA and R5020) in the presence of E2; the addition of CDB4124 caused 50% inhibition of proliferation (p< 0.01) at 72 hours. Cell cycle analysis of T47D cells treated with P4, MPA and R5020 alone or in combination with E2 showed significant increases in S and G2/M phase and decreases in G0/G1. These were blocked by CDB4124 at 0.1 or 1.0μM (p<0.05 for all). Gene GO metacore analysis of genes identified in the microarray revealed significant enrichment of cell cycle pathways (FDR, p< 1.0X10-11 ) upon treatment with R5020. The addition of CDB4124 to R5020 treated samples showed inhibition of the same cell cycle pathways (FDR,p<1.0X10-14). A 16-gene panel related to G2/M phase of cell cycle was selected based on >1.5 fold upregulation (p<0.001) during treatment with R5020,10nM and blockade by CD4124. Real time PCR confirmed upregulation of this 16 gene panel ≥2.0 (p<0.05) in the presence of PR ligands alone or in combination with E2 which were significantly blocked by the addition of CDB4124. Conclusion: These data demonstrate that PR mediated cell proliferation occurs upon treatment with three different ligands of PR (P4, MPA and R5020); that PR actively engages key genes involved in the G2/M phase of the cell cycle to drive proliferation of ER+ and PR+ cells; and that the antiprogestin, CBD4124 is a potent transcriptional inhibitor for blockade of PR mediated cell proliferation in hormone receptor positive breast cancer cells. Citation Format: Akash Gupa, Mi Ran Choi, Susan E Clare, Jun Wang, Oukseub Lee, David Z Ivancic, J Julie Kim, Seema A Khan. Progesterone receptor (PR) blockade by antiprogestin, CDB4124 in hormone receptor positive breast cancer cells leads to significant inhibition of G2/M cell cycle genes [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P3-04-10.

Interactions among CII protein, RNA polymerase and the lambda PRE promoter: contacts between RNA polymerase and the -35 region of PRE are identical in the presence and absence of CII protein.

The DNA recognition sequence for the transcriptional activator, CII protein, which is critical for lysogenization by bacteriophage lambda, overlaps the -35 region of the P(RE) promoter. Data presented here show that activation by CII does not change the pattern of cleavage of the -35 region of P(RE) by iron (S)-1-(p-bromoacetamidobenzyl)-EDTA (Fe-BABE) conjugated to the sigma subunit of RNA polymerase (RNAP). Thus, the overall interaction between sigma and the -35 region of P(RE) is not significantly altered by CII. Therefore, the effects of the activator on RNAP binding to the promoter and formation of open complexes do not reflect a large-scale qualitative change in the nature of the interaction between RNAP and promoter DNA. The ability of CII to stimulate lysogenization is reduced in the presence of plasmid-borne rpoA variants encoding alanine substitutions at several positions in the C-terminal domain of the alpha subunit. However, it has not been possible to identify residues that directly affect the interaction between the activator and RNA polymerase.

Stability of CII is a key element in the cold stress response of bacteriophage lambda infection.

Bacteria are known to adapt to environmental changes such as temperature fluctuations. It was found that temperature affects the lysis-lysogeny decision of lambda such that at body temperature (37 degrees C) the phage can select between the lytic and lysogenic pathways, while at ambient temperature (20 degrees C) the lytic pathway is blocked. This temperature-dependent discriminatory developmental pathway is governed mainly by the phage CII activity as a transcriptional activator. Mutations in cII or point mutations at the pRE promoter lead to an over-1,000-fold increase in mature-phage production at low temperature while mutations in cI cause a smaller increase in phage production. Interference with CII activity can restore lytic growth at low temperature. We found that at low temperature the stability of CII in vivo is greatly increased. It was also found that phage DNA replication is blocked at 20 degrees C but can be restored by supplying O and P in trans. It is proposed that CII hampers transcription of the rightward pR promoter, thus reducing the levels of the lambda O and P proteins, which are necessary for phage DNA replication. Our results implicate CII itself or host proteins affecting CII stability as a "molecular thermometer".

A single-base-pair mutation changes the specificities of both a transcription activation protein and its binding site.

The C1 protein of bacteriophage P22 binds to a unique site in the -35 region of the PRE promoter and activates transcription of the phage c2 repressor gene. This -35 target has an approximate direct repeat that overlaps the 5' end of the c1 coding region. We have isolated a single-base-pair mutation in this region that changes the PRE -35 target as well as the amino-terminal region of the C1 protein. Although the mutant C1 protein activates the mutant PRE promoter, it fails to activate the wild-type PRE promoter. This suggests that a single-base-pair mutation changes the specificities of both a protein and its target site. These studies also indicate that C1 binding to DNA is influenced by contacts made through residues near the amino terminus.

Activation defects caused by mutations in Escherichia coli rpoA are promoter specific.

Escherichia coli RNA polymerases containing mutated alpha subunits were tested for their ability to respond to three different positive regulators (activators) in vitro. The two alpha (rpoA) mutants, alpha-256 and alpha-235, have deletions of the C-terminal 73 and 94 amino acids, respectively. In runoff transcription assays catalyzed by reconstituted holoenzyme, the effects of the mutations on each of three promoters tested were different: activation of the lambda pRM promoter by cI protein (repressor) was nearly normal, activation of the lambda pRE promoter by cII protein was reduced approximately fivefold, and direct activation of the trpPB promoter of Pseudomonas aeruginosa was completely inhibited. We also found that the reconstituted mutant enzyme was defective in recognition of trpPI in the absence of activator. The differential responses of the three promoters to their activators in the presence of the mutant enzymes indicate that the location of an activator-binding site does not by itself determine the region of RNA polymerase with which the activator interacts.

Constitutive function of a positively regulated promoter reveals new sequences essential for activity.

A consensus "-10" recognition sequence for RNA polymerase was created at the positively regulated lambda Pre promoter by introducing three single base pair mutations. This altered promoter, Pre*, functions constitutively in vivo and in vitro at high efficiency despite very poor consensus "-35" region sequence homology. We examined the influence of the -35 region sequence information on promoter function by shifting the wild type -35 region +/- 2 base pairs relative to the -10 region consensus sequence and by completely replacing it with alternative DNA sequences. In every case, the altered Pre* promoters retained transcriptional activity although differences in their transcriptional efficiencies were observed. Apparently the Pre* promoter does not require specific -35 region sequences for constitutive promoter activity, although the -35 region sequences can modulate overall promoter strength. In addition, by point mutation analysis we have identified bases immediately upstream of the -10 hexamer which are essential for constitutive function of the Pre* promoter. We propose that these mutants define an extended -10 region at Pre* that compensates for its poor -35 region sequence information by providing critical contacts that stabilize productive RNA polymerase binding.

Mutations that improve the pRE promoter of coliphage lambda.

The dya5 mutation, a C----T change at position -43 of the lambda pRE promoter, results in a twofold increase in pRE activity in vivo. Smaller increases in pRE activity are found for the dya2 mutation, a T----C change at position -1 of pRE, and the dya3 mutation, an A----G change at +5 of pRE. The mutant pRE promoters retain complete dependence on cII protein for activity. These observations argue, at least for pRE-like promoters, that promoter activities are influenced by nucleotide sequences at least eight nucleotides to the 5'-side of the conventional -35 region consensus sequence, and by nucleotide sequences near the start-site of transcription. Although Hawley and McClure (1983) found A-T pairs more frequently than G-TC pairs in the region of -40 to -45 of prokaryotic promoters, other mutations that change a G-TC pair to an A-T pair at positions -41, -44 and -45 of pRE do not result in increased promoter activity. We also found that a T----C change at positions -42 results in a mild decrease in promoter activity. These observations argue that Ts at positions -42 and -43 of pRE are required for maximum promoter activity, but do not support the hypothesis that As and Ts in the -40 and -45 region generally lead to higher promoter activities.

Mutations that affect the efficiency of translation of mRNA for the cII gene of coliphage lambda.

Starting with the lambda pRE-strain lambda ctr1 cy3008, which forms clear plaques, we have isolated two mutant strains, lambda dya2 ctr1 cy3008 and lambda dya3 ctr1 cy3008, that form plaques with very slightly turbid centers. The dya2 and dya3 mutations lie in the region of overlap between the PRE promoter and the ribosome recognition region of the cII gene, and have nucleotide alterations at positions -1 and +5 of pRE, and alterations in cII mRNA at -16 and -21 nucleotides before the initial AUG codon of the gene. Both mutations destabilize a stem structure that may be formed by cII mRNA, and dya2 also changes the sequence on cII mRNA that is complementary to the 3'-end of 16 S rRNA from 5'-UAAGGA-3' to 5'-UGAGGA-3'. --The dya2 and dya3 mutations, along with the ctr1 mutation, which destabilizes either of two alternate stem structures which may be formed by cII mRNA (these being more stable stem structures than the one affected by dya2 and dya3), were tested for their ability to reverse two cII-mutations that are characterized by inefficient translation of cII mRNA. These are cII3088, an A----G mutation four bases before the initial AUG codon, and cII3059, a GUU----GAU (Val2----Asp) second codon mutation. It was found that ctr1 completely reverses the translation defects of these two mutations, while dya2 partially reverses these translation defects. The dya3 mutation has no effect on translation efficiency under any condition tested.(ABSTRACT TRUNCATED AT 250 WORDS)

Repression of a mutant derivative of the pRE promoter of bacteriophage lambda by its activator, CII

A 2-bp insertion between the −10 and −35 regions of the p RE promoter of bacteriophage λ reverses the effect of the activator protein, CII, on transcription from p RE in vitro. The mutant promoter is weakly constitutive in the absence of cII protein and repressed in its presence. This is in sharp contrast to wild-type p RE which is inactive in the absence of cII protein and stimulated at least 1000-fold in its presence (Shih and Gussin, 1984a; McClure and Hoopes, 1985). These effects are explained by the creation of a new −35 region with weak homology to the −35 consensus sequence for Escherichia coli promoters, and by the altered spatial relationship between the −35 region and the CII-binding site. This interpretation was confirmed by analysis of double mutants containing known cy ( p RE) mutations together with the 2-bp insertion. Insertion of 4 bp or deletion of 2 bp completely inactivates p RE in the presence or absence of cII protein, again indicating that activation is dependent upon proper spacing between the −35 region and the transcription start point.

CII-dependent activation of the pRE promoter of coliphage lambda fused to the Escherichia coli galK gene

Using a cloning vector designed for the study of prokaryotic promoters by fusion to the Escherichia coli galactokinase gene ( galK), we have constructed a plasmid in which the λp re promoter controls galactokinase expression. A galK − host containing this plasmid has a Gal − phenotype since transcription from P RE requires activation by the A CII protein. When CII protein is provided by a prophage, galactokinase is synthesized at a rate dependent on the concentration of CII protein. A second plasmid was constructed in which the P RE promoter from phage21 controls galactokinase expression. Transcription of the galK gene in this plasmid requires the phage 21 CII protein. Using this system, we demonstrate that the λ and 21 P RE promoters are highly selective for their corresponding CII proteins. However, a cross-reaction between 21 P RE and the λ CII protein was observed. In addition, we transferred the P RE - galK fusion unit from the plasmid to a phage, and then to the host chromosome in single copy. Galactokinase expression in this single copy P RE - galK system is also dependent on CII protein, which may be provided from a multicopy plasmid. The high concentration of CII protein provided by the plasmid results in maximal expression of the P RE - galK transcription unit. In this second system low levels of CII activity from CII − mutants are amplified and can be readily detected.

Kinetic analysis of mutations affecting the cII activation site at the PRE promoter of bacteriophage lambda.

Abortive initiation and run-off transcription assays were used to study the effects of cy mutations on activation of the phage lambda PRE promoter by cII gene product. Six point mutations in the repeated T-T-G-C sequences that flank the -35 consensus region of PRE decreased the apparent affinity of the promoter for cII protein by factors of 4-16 relative to the wild-type affinity. Kinetic analyses of transcription initiation in the presence and absence of cII protein demonstrated that five of the six mutations did not significantly affect the intrinsic interaction of RNA polymerase with PRE. Thus, these mutations differ from other cy mutations, including those in the -35 consensus region, which affect the formation of polymerase-PRE closed complexes or the isomerization of closed complexes to open complexes but do not affect the binding of cII protein. A sixth T-T-G-C mutation, cy3001, may affect intrinsic initiation by RNA polymerase as well as cII binding.

Role of cII protein in stimulating transcription initiation at the λ PRE promoter : Enhanced formation and stabilization of open complexes

Abortive and productive initiation assays were used to study transcription initiation at the PRE promoter of phage lambda in vitro. Two parameters were measured: k2, the rate constant for the transition between closed and open complexes; and KB, the equilibrium constant for the initial binding of RNA polymerase to promoter DNA. In the absence of cII protein (which activates PRE) the PRE promoter was extremely weak as expected, with k2 = 4.0 X 10(-4) S-1 and KB = 1.0 X 10(7) M-1. The addition of cII protein resulted in about a 15-fold increase in KB and a 40-fold increase in k2. Thus, cII activation of PRE results both in enhanced binding of RNA polymerase to DNA to form closed complexes and in an enchanced rate of isomerization of closed to open complexes. In addition, we found that open complexes formed in the presence of cII protein were at least four times as stable as those formed in its absence. This suggests that RNA polymerase and cII protein may remain in close contact even after complexes are formed.

Mutational analysis of a regulatory region in bacteriophage lambda that has overlapping signals for the initiation of transcription and translation.

The positively regulated PRE promoter of phage lambda structurally overlaps with the ribosome-binding and NH2-terminal coding region of the regulatory protein (cII) that activates PRE transcription. We have isolated and characterized 27 different point mutations that occur within the 36-base-pair overlapping region. A comparison of genetic crossover data with nucleotide separations as determined by DNA sequence analysis reveals that recombination frequencies are greatly depressed at very short distances. Moreover, recombination frequency is critically dependent upon the precise nucleotide sequence of the crossover region for distances of five nucleotides or less. The mutations define precise positions and sequences that are important to (i) PRE promoter function, (ii) translation of the cII gene, and (iii) cII gene function. Mutational changes that affect the function of one element in this region concomitantly define phenotypically silent alterations in the other two elements. Mutations deficient in promoter function (P-RE or cy) are clustered in two regions that lie approximately equal to 10 and approximately equal to 35 nucleotides before the initial base of PRE mRNA, analogous to mutations in other promoters. P-RE mutations in the -10 region alter bases that are conserved in prokaryotic promoters, but P-RE mutations in the -35 region do not affect bases that are normally conserved in other promoters. Several mutations deficient in cII gene activity affect the initiation of cII protein synthesis, including an A leads to G change four bases outside the cII coding region, and AUG leads to GUG, AUG leads to ACG, and AUG leads to AUA mutations in the initiation codon. In the region of overlap between the PRE promoter and the NH2-terminal region of the cII gene, most amino acid substitutions in the cII protein do not result in a loss of cII function, indicating that this region of the gene does not contain essential information for cII function. We suggest that the overlap itself is an evolutionarily conserved structure and that it somehow coordinates the bidirectional transcriptional and translational events that occur in this region.

Control of lambda repressor prophage and establishment transcription by the product of gene tof.

Control of expression of the bacteriophage lambda (lambda) repressor was studied by measuring repressor transcription in noninduced and derepressed lambda lysogens. Three distinct modes of leftward transcription were observed from cI and the adjacent genes associated with the control of repressor synthesis: The prophage or maintenance mode Prm-cI-rex-ti repressor transcript occurs from repressed lysogens; the oop (Po-oop-to) transcript, and the lit (lit-ti) RNA, from the distal half of gene rex, both occur from induced tof+ prophage; the repressor establishment mode of transcription is observed throughout the rex-cI-tof-y-cII-oop interval between Po and ti from induced tof- prophage. The overall level of establishment mRNA synthesis is partially template dependent. However, the actual initiation step for repressor establishment transcription requires the participation of the lambda cIII, cII products, and also either requires the activity of Escherichia coli replication proteins, or is triggered by a replication initiation event. The cII cIII products do not positively stimulate de novo initiation of establishment transcription, but rather act after an initial replication-dependent step. Initiation of the establishment mode of repressor transcription is totally inhibited by more than 125-fold, in an all or none fashion, by the lambda antirepressor (Tof), the product of gene tof (cro). Since Tof only reduces the in vivo rightward transcription of cII from Pr by about 2-fold, we suggest that Tof inhibits repressor establishment transcription by either uncoupling the replication and cII-cIII dependent events, or by inhibiting the activity rather than the expression of the cIII, cII products. Our results do not fully support either of the present hypotheses that establishment transcription is initiated from the hypothetical Pre promoter in the y-interval, or arises through antitermination of the oop RNA. Since the initiation and control of the establishment mode of repressor transcription parallels the control of lit RNA synthesis, we propose a common mechanism underlies the initiation of these transcripts.

Repressor synthesis in vivo after infection of E. coli by a prm− mutant of bacteriophage λ

Genetic-complementation experiments have shown previously that the mutation prm116, which maps near the right operator in phage lambda, inactivates the prm promoter but not the pre promoter for cI (repressor) gene expression. Direct assays for repressor synthesis in vivo confirm these conclusions: first, λprm116 does not synthesize repressor after infection of an immune lysogen, when prm should normally be active; second, activation of pre by cII and cIII proteins after infection of nonlysogenic cells is not affected by the prm116 mutation. Unexpectedly, however, λprm116 does not shut off repressor synthesis at the normal time after infection of a nonlysogen, so that, in fact, repressor levels late in infection with λprm116 are actually greater than those observed after wild-type infection. Since cro gene product is required to shut off repressor synthesis under these conditions, we speculate that the prm116 mutation may also alter a cro target site near the right operator.