P1 Start Site Research Articles

Open complex scrunching before nucleotide addition accounts for the unusual transcription start site of E. coli ribosomal RNA promoters

Most Escherichia coli promoters initiate transcription with a purine 7 or 8 nt downstream from the -10 hexamer, but some promoters, including the ribosomal RNA promoter rrnB P1, start 9 nt from the -10 element. We identified promoter and RNA polymerase determinants of this noncanonical rrnB P1 start site using biochemical and genetic approaches including mutational analysis of the promoter, Fe(2+) cleavage assays to monitor template strand positions near the active-site, and Bpa cross-linking to map the path of open complex DNA at amino acid and nucleotide resolution. We find that mutations in several promoter regions affect transcription start site (TSS) selection. In particular, we show that the absence of strong interactions between the discriminator region and σ region 1.2 and between the extended -10 element and σ region 3.0, identified previously as a determinant of proper regulation of rRNA promoters, is also required for the unusual TSS. We find that the DNA in the single-stranded transcription bubble of the rrnB P1 promoter complex expands and is "scrunched" into the active site channel of RNA polymerase, similar to the situation in initial transcribing complexes. However, in the rrnB P1 open complex, scrunching occurs before RNA synthesis begins. We find that the scrunched open complex exhibits reduced abortive product synthesis, suggesting that scrunching and unusual TSS selection contribute to the extraordinary transcriptional activity of rRNA promoters by increasing promoter escape, helping to offset the reduction in promoter activity that would result from the weak interactions with σ.

Transrepression of the estrogen receptor promoter by calcitriol in human breast cancer cells via two negative vitamin D response elements

Calcitriol (1,25-dihydroxyvitamin D3), the hormonally active metabolite of vitamin D, exerts its anti-proliferative activity in breast cancer (BCa) cells by multiple mechanisms including the downregulation of the expression of estrogen receptor α (ER). We analyzed an ∼3.5 kb ER promoter sequence and demonstrated the presence of two potential negative vitamin D response elements (nVDREs), a newly identified putative nVDRE upstream at -2488 to -2473 bp (distal nVDRE) and a previously published sequence (proximal nVDRE) at -94 to -70 bp proximal to the P1 start site. Transactivation analysis using ER promoter deletion constructs and heterologous promoter-reporter constructs revealed that both nVDREs functioned to mediate calcitriol transrepression. In the electrophoretic mobility shift assay, the vitamin D receptor (VDR) showed strong binding to both nVDREs in the presence of calcitriol, and the chromatin immunoprecipitation assay demonstrated the recruitment of the VDR to the distal nVDRE site. Mutations in the 5' hexameric DNA sequence of the distal nVDRE resulted in the loss of calcitriol-mediated transrepression and the inhibition of protein-DNA complex formation, demonstrating the importance of these nucleotides in VDR DNA binding and transrepression. A putative nuclear factor-Y (NFY) binding site, identified within the distal nVDRE, led to the findings that NFY bound to the distal nVDRE site interfered with the binding of the VDR at the site and reduced calcitriol-mediated transrepression. In conclusion, the ER promoter region contains two negative VDREs that act in concert to bind to the VDR and both nVDREs are required for the maximal inhibition of ER expression by calcitriol. The suppression of ER expression and estrogen-mediated signaling by calcitriol in BCa cells suggests that vitamin D may be useful in the treatment of ER+ BCa.

The Proinflammatory Cytokine, Interleukin-6, Up-regulates Calcium-sensing Receptor Gene Transcription via Stat1/3 and Sp1/3

Increased expression of the calcium-sensing receptor (CASR), which controls blood calcium homeostasis, leads to a decrease in the extracellular calcium set-point, thereby reducing parathyroid hormone secretion and renal calcium reabsorption and increasing calcitonin secretion resulting in reduced circulating calcium levels. Critically ill patients with elevated proinflammatory cytokine levels commonly have hypocalcemia, although the mechanism is not known. After intraperitoneal injection of interleukin (IL)-6 in the rat, circulating levels of parathyroid hormone, 1,25-dihydroxyvitamin D, and calcium fell within hours and remained low at 24 h. Expression of CASR (mRNA and protein) increased within hours in parathyroid, thyroid, and kidney and remained elevated at 24 h. The CASR gene has two promoters (P1 and P2) yielding transcripts having alternative 5'-untranslated regions but encoding the same receptor protein. Activities of P1 and P2 promoter/luciferase reporter constructs were stimulated approximately 2-3-fold by IL-6 in proximal tubule HKC cells and TT thyroid C-cells. Studies with P1 deleted and mutated promoter-reporter and Stat1 and/or Stat3 dominant-negative constructs showed that a Stat1/3 element downstream of the P1 start site accounted for the IL-6 induction. There are no Stat elements in the P2 promoter, but Sp1/3 elements are clustered at the transcription start site. A series of transfection P2 promoter-reporter analyses showed that Sp1 together with Stat1/3 was critical for IL-6 responsiveness of P2. By oligonucleotide precipitation assay, IL-6 rapidly promoted a complex containing both Sp1/3 and Stat1/3 on the Sp1/3 elements. In conclusion, Stat1/3 directly controls promoter P1, and the Stats indirectly regulate promoter P2 via Sp1/3 in response to IL-6. By this mechanism, the cytokine likely contributes to altered extracellular calcium homeostasis.

Architecture and transcriptional activity of the initiator element of the TATA-less RPL21 gene.

The nuclear RPL21 gene coding for the plastid ribosomal protein L21 is a TATA-less gene that is overexpressed in a leaf-dependent manner by the specific usage of a strong initiator called P1. We have previously shown that the RPL21 core promoter spanning from -23 to +104 relative to P1 start site activates transcription in the same manner as does the full promoter. Here, we present results of experiments aimed at deciphering the RPL21 core promoter architecture. Results of transient expression using various 5' deletions of the core promoter fused to a chloramphenicol acetyl transferase (CAT) reporter gene show that 34 bp encompassing the P1 initiation site (from -23 to +11) are required for full transcription activation. Gel-shift analysis shows that five DNA/protein complexes (C1-C5) are formed on this 34-bp fragment with protein extracts from green tissues. C1 is the major complex present during seed germination. The other complexes are present in young leaf tissues suggesting a role in transcription activation. Linker scanning mutagenesis experiments show that the five complexes form two independent groups: I (C1-C3) and II (C4 and C5), with a common binding site located on P1. Using transgenic plants, we show that three nucleotides encompassing the P1 start site and three trinucleotides necessary for group I binding are determinant for RPL21 activation. These results identify an unusually compact core structure, which is centred on P1 initiation site and is responsible for transcription activation. A model of the architecture of this region is presented.

Modulation of DNA-protein interactions in the P1 and P2 c-myc promoters by two intercalating drugs.

Regulation of transcription from the oncogene c-myc has an important role in the genesis of various tumors. Therefore, c-myc is a potential target for chemotherapy by drugs which are able to modify its activity directly. In this article, we identify the binding sites in the P1 and P2 promoter regions of c-myc for the intercalating antibiotics actinomycin D and elsamicin A. Gel retardation experiments indicate that actinomycin D or elsamicin A binding can inhibit the formation of several DNA-protein complexes. However, relatively low concentrations of elsamicin A, but not actinomycin D, appear to increase the level of binding to the P1 promoter of a protein factor. Using pure Sp1 transcription factor and an oligonucleotide containing the Sp1 putative binding site, we determined that the binding enhancement induced by small amounts of elsamicin was on the Sp1-DNA complex. Run-off transcription experiments in vitro showed that the effect of elsamicin A on Sp1 binding is followed by the maintenance or a relative rise in transcription levels from the P1 promoter of c-myc, while actinomycin D always inhibited the transcription from the P1 c-myc promoter in a concentration-dependent manner. Higher concentrations of elsamicin acted as an inhibitor of the transcription from the P1 start site but not from the P2.

Dual regulation of the paraquat-inducible gene pqi-5 by SoxS and RpoS in Escherichia coli.

The pqi-5 gene, producing a probable membrane protein of unknown function, has been reported to be a member of the soxRS regulon. The SoxRS-dependent induction of pqi-5 by paraquat occurs only during the exponential phase. The expression of pqi-5 increased in the absence of paraquat during the stationary phase or under conditions of carbon or phosphate starvation. This increase was regulated at the transcriptional level by RpoS (sigma S), which recognized the second promoter (P2) approx. 5 nucleotides upstream from the promoter (P1) used at the exponential phase. Studies with a series of 5' deletions revealed that the paraquat-responsive element resides between -52 and -42 nucleotides upstream from the P1 start site, whose nucleotide sequence matches closely to other SoxS-binding sequences. The stationary-phase induction required sequences up to position -42, which correspond to the 5' border of the putative -35 hexamer for the P2 promoter. The binding of the purified SoxS protein to the pqi-5 promoter upstream sequences was demonstrated by gel mobility-shift and DNase I protection assays. The transcription from P1 promoter by E sigma D was activated by purified SoxS in vitro, as was observed in vivo. The dual regulation of pqi-5 by SoxS at the exponential phase and RpoS at the stationary phase is the first to be reported among the members of the soxRS regulon, suggesting that this gene might indeed play some role under stressful conditions.

Alternative processing of the tryptophanyl-tRNA synthetase mRNA from interferon-treated human cells.

We have analysed the structure of mRNA isoforms of the human gene encoding tryptophanyl-tRNA synthetase (Trp-tRNA synthetase) expressed in the epithelial CaOv cells and MT-4 lymphocytes. The Trp-tRNA synthetase gene is induced by interferon-gamma in both lines and, in MT-4 lymphocytes, also by interferon-alpha. Four Trp-tRNA synthetase mRNA isoforms have different combinations of the first exons IA, IB and II. Two transcription initiation sites (P1 and P2) were detected 90 bp from each other. Processing of the primary transcript initiated from the P1 start site generates the mRNA isoform where exon IA joins to exon II. The other three isoforms are produced by alternative splicing of the primary transcript produced from the P2 start site. Isoform 2 has a 3'-end fragment of exon IA joined to exon II. Isoform 3 contains exons IA and IB. Isoform 4 contains exon IA and exon III and lacks exon II encoding the N-terminus of the Trp-tRNA synthetase. Therefore, the two primary transcripts of the Trp-tRNA synthetase gene differ only in the 5' flank sequence between P1 and P2, and this fragment regulates their processing. Both interferon-alpha and interferon-gamma induce exon IA-containing and exon IB-containing isoforms of the Trp-tRNA synthetase mRNA.

Xenopus laevisc-myc I and II genes: molecular structure and developmental expression

The structure of the two Xenopus laevis c-myc I and c-myc II genes has been investigated by isolating and sequencing genomic and cDNAs clones. In oocytes, c-myc I mRNAs represent 80-90% of the overall amount of c-myc transcripts. The c-myc I expression is controlled primarily by two differentially regulated tandem promoters P1 and P2 which are separated by 50 bases. During oogenesis, maternal c-myc I mRNAs, are transcribed from both promoters whereas zygotic transcripts seem to initiate only from the P2 promoter. Sequence comparison between the promoter regions of c-myc I and II genes reveals the insertion in the c-myc I promoter region, between positions -831 and -389 relative to the P1 start site of a repetitive element. Comparison of X.laevis and mammalian c-myc promoter sequences reveals furthermore the conservation of cis-regulatory elements, including a motif known to be a negative regulator of the human c-myc transcription, a purine rich region, a binding site for the E2-F transcription factor and three SP1 binding sites. Finally, we report characterization of a new c-myc I mRNA which differ at the 5' end. Transcripts are possibly initiated at a putative alternative promoter located further upstream in the genome, and undergoes alternative splicing.

Interaction of an NF-kappa B-like factor with a site upstream of the c-myc promoter.

The c-myc protooncogene has been implicated in control of growth and differentiation of mammalian cells. For instance, growth arrest is often preceded by reduction in c-myc mRNA and gene transcription. To elucidate the mechanisms of control of c-myc gene transcription, we have begun to characterize the interaction of nuclear factors with the 719-base-pair (bp) c-myc regulatory domain, located 1139-421 bp upstream of the P1 start site of the mouse gene. Nuclear extracts from exponentially growing WEHI 231 murine B-lymphoma cells formed multiple complexes in mobility-shift assays. Changes in complex distribution were observed in growth-arrested WEHI 231 cells, and a major site of this interaction mapped to a 21-bp sequence that is similar to the sequences recognized by the NF-kappa B family of proteins. Binding of NF-kappa B-like factors was demonstrated by oligonucleotide competition. Induction of complex formation upon 70Z/3 pre-B- to B-cell differentiation, enhancement of binding by GTP, and detergent-induced release of inhibitor protein suggested that NF-kappa B itself is one member of the family that can bind. Transfection of thymidine kinase-chloramphenicol acetyltransferase constructs containing the 21-bp c-myc sequence into Jurkat cells demonstrated increased chloramphenicol acetyltransferase activity upon phorbol ester and phytohemagglutinin treatment. These results suggest the involvement of NF-kappa B-like factors in the regulation of c-myc transcription.

Transcription of the isoamylase gene (iam) in Pseudomonas amyloderamosa SB-15.

S1 nuclease mapping of RNA prepared from Pseudomonas amyloderamosa SB-15 suggested that the iam gene coding for isoamylase (glycogen 6-glucanohydrolase [EC 3.2.1.68]) is transcribed from two promoters. The transcription start site for the upstream promoter (termed P1) was located -182 base pairs from the first nucleotide of the initiation codon of iam, whereas the start site for the downstream promoter (termed P2) was 99 base pairs downstream of the P1 start site. Transcriptions from these promoters were induced by maltose and were not repressed by glucose. The promoter regions contained sequences homologous to the consensus sequence recognized by sigma 54 RNA polymerase of enteric bacteria and found in promoters of other Pseudomonas species. Northern (RNA) hybridization provided evidence that the iam gene is transcribed as monocistronic mRNAs with an approximate size of 2.6 kilobases.