A DNA sequence rich in (A+T), located upstream of the -10, -35 region of the Escherichia coli ribosomal RNA promoter rrnB P1 and called the UP element, stimulates transcription by a factor of 30 in vivo, as well as in vitro in the absence of protein factors other than RNA polymerase (RNAP). When fused to other promoters, such as lacUV5, the UP element also stimulates transcription, indicating that it is a separate promoter module. Mutations in the carboxyl-terminal region of the alpha subunit of RNAP prevent stimulation of these promoters by the UP element although the mutant enzymes are effective in transcribing the "core" promoters (those lacking the UP element). Protection of UP element DNA by the mutant RNAPs is severely reduced in footprinting experiments, suggesting that the selective decrease in transcription might result from defective interactions between alpha and the UP element. Purified alpha binds specifically to the UP element, confirming that alpha acts directly in promoter recognition. Transcription of three other promoters was also reduced by the COOH-terminal alpha mutations. These results suggest that UP elements comprise a third promoter recognition region (in addition to the -10, -35 recognition hexamers, which interact with the sigma subunit) and may account for the presence of (A+T)-rich DNA upstream of many prokaryotic promoters. Since the same alpha mutations also block activation by some transcription factors, mechanisms of promoter stimulation by upstream DNA elements and positive control by certain transcription factors may be related.

The α subunit of Escherichia coli RNA polymerase (RNAP) participates in promoter recognition through specific interactions with UP element DNA, a region upstream of the recognition hexamers for the ς subunit (the −10 and −35 hexamers). UP elements have been described in only a small number of promoters, including the rRNA promoter rrnB P1, where the sequence has a very large (30- to 70-fold) effect on promoter activity. Here, we analyzed the effects of upstream sequences from several additional E. coli promoters (rrnD P1, rrnB P2, λp R, lac, merT, and RNA II). The relative effects of different upstream sequences were compared in the context of their own core promoters or as hybrids to thelac core promoter. Different upstream sequences had different effects, increasing transcription from 1.5- to ∼90-fold, and several had the properties of UP elements: they increased transcription in vitro in the absence of accessory protein factors, and transcription stimulation required the C-terminal domain of the RNAP α subunit. The effects of the upstream sequences correlated generally with their degree of similarity to an UP element consensus sequence derived previously. Protection of upstream sequences by RNAP in footprinting experiments occurred in all cases and was thus not a reliable indicator of UP element strength. These data support a modular view of bacterial promoters in which activity reflects the composite effects of RNAP interactions with appropriately spaced recognition elements (−10, −35, and UP elements), each of which contributes to activity depending on its similarity to the consensus.

Five recombinant DNA plasmids have been constructed that contain structural gene sequences for rat tyrosine hydroxylase [TyrOHase; tyrosine 3-monooxygenase; L-tyrosine,tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating), EC 1.14. The peripheral autonomic nervous system provides a useful model of neuronal development because the neurons in the sympathetic ganglia, which arise from the neural crest, become either cholinergic or adrenergic. Both in vivo and in vitro studies (1, 2) have shown that the neurotransmitter choice is labile during a prolonged period of life. Tyrosine hydroxylase [TyrOHase; tyrosine 3-monooxygenase; L-tyrosine,tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2] is a specific marker for adrenergic neurons and has been widely used to study their ontogeny. The regulation of the expression of TyrOHase has been shown to be influenced by the target tissue (1, 2), by neural activity (3, 4) and by chemical or hormonal factors such as glucocorticoids (5-7) and nerve growth factor (7,8). A full understanding of the regulation of this enzyme requires an analysis of the DNA encoding its gene and of its RNA transcripts.As a first step we report here the isolation of recombinant plasmids carrying DNA sequences complementary to a mRNA coding for rat TyrOHase antigen. To carry out this work we have taken advantage of a cloned cell line of rat pheochromocytoma characterized by relatively high levels of catecholamine-synthesizing enzyme and transmitter (9). TyrOHase cDNA clones were selected by the combined use of differential colony hybridization and immunoprecipitation ofthe products ofplasmidselected mRNA translation. The TyrOHase cDNA probes allowed the characterization of TyrOHase mRNA from PC 12 cells and human pheochromocytoma. MATERIALS AND METHODSGrowth of Cells and Tissues and RNA Isolation. Rat pheochromocytoma PC 12 cells, originally obtained through the generosity of L. A. Greene, were grown and harvested after treatment with dexamethasone as described (10). In addition, PC 12 cells (= 107) were used to subcutaneously inoculate nude mice. Tumor nodules could be palpated at the site ofinoculation after 5-7 weeks. Four days before the turnors were collected, the animals were daily subjected to dexamethasone treatment (100 ug/kg). The final dose of dexamethasone (300 ,ug/kg) was given 6 hr before harvesting. Human pheochromocytoma were obtained from J. P. Luton (Hopital Cochin, Paris) 15-30 min after surgery and were stored frozen (-70°C) until RNA extraction. Rat livers were dissected from adult Sprague-Dawley rats killed without anesthesia.RNAs were extracted from tumors as described by Lomedico and Saunders (11) and from PC 12 cells and liver by the method of Auffray and Rougeon (12).Poly(A)-RNA Separation, in Vitro Protein Synthesis, and Immunoprecipitations. Polyadenylylated mRNAs were obtained from total RNA preparations by two cycles of oligo(dT)-cellulose chromatography (13). mRNA coding for TyrOHase antigen was further enriched by centrifugation in a...

rRNA transcription in Escherichia coli is activated by the FIS protein, which binds upstream of rrnp 1promoters and interacts directly with RNA polymerase. Analysis of the contribution of FIS to rrn transcription under changing physiological conditions is complicated by several factors: the wide variation in cellular FIS concentrations with growth conditions, the contributions of several other regulatory systems to rRNA synthesis, and the pleiotropy of fis mutations. In this report, we show by in vivo footprinting and Western blot analysis that occupancy of the rrnBp 1 FIS sites correlates with cellular levels of FIS. We find, using two methods of measurement (pulse induction of a FIS-activated hybrid promoter and primer extension from an unstable transcript made fromrrnBp 1), that the extent of transcription activation by FIS parallels the degree of FIS site occupancy and therefore cellular FIS levels. FIS activates transcription throughout exponential growth at low culture density, butrrnp 1 transcription increases independently of FIS immediately following upshift, before FIS accumulates. These results support the model that FIS is one of a set of overlapping signals that together contribute to transcription fromrrnp 1 promoters during steady-state growth.

The transcription factor FIS has been implicated in the regulation of several stable RNA promoters, including that for the major tRNA Leu species in Escherichia coli, tRNA 1 Leu . However, no evidence for direct involvement of FIS in tRNA 1 Leu expression has been reported. We show here that FIS binds to a site upstream of the leuV promoter (centered at ؊71) and that it directly stimulates leuV transcription in vitro. A mutation in the FIS binding site reduces transcription from a leuV promoter in strains containing FIS but has no effect on transcription in strains lacking FIS, indicating that FIS contributes to leuV expression in vivo. We also find that RNA polymerase forms an unusual heparin-sensitive complex with the leuV promoter, having a downstream protection boundary of ϳ؊7, and that the first two nucleotides of the transcript, GTP and UTP, are required for formation of a heparin-stable complex that extends downstream of the transcription start site. These studies have implications for the regulation of leuV transcription.