Transcription of the sea urchin U6 gene in vitro requires a TATA-like box, a proximal sequence element, and sea urchin USF, which binds an essential E box.

Abstract

The tandemly repeated gene set encoding the sea urchin U6 gene has been cloned from the sea urchin Strongylocentrotus purpuratus. The U6 gene is transcribed by RNA polymerase III in a sea urchin nuclear extract. Like that of the vertebrate U6 genes, transcription of the sea urchin U6 gene does not require any internal sequences or 3' sequences but requires onl 5' flanking sequences. Only 88 nucleotides of 5' flanking sequence are required for maximal expression in vitro. Mutagenesis experiments demonstrated the requirement for three elements, a CACGTG element at -80, a proximal sequence element at about -55, and the TATA-like box at -25. The major protein in sea urchin extracts that interacts with the CACGTG element is sea urchin USF, and immunodepletion of sea urchin USF greatly reduces transcription. The USF binding site in the U6 gene is highly homologous (11 of 13 nucleotides) with the USF binding sites found in the promoter of the S. purpuratus spec genes.The genes encoding the spliceosomal small nuclear RNAs (snRNAs) and some other snRNAs in metazoans have unusual promoter structures (27) Here we report the sequence requirements for expression of the sea urchin U6 snRNA gene. As with the vertebrate U6 snRNA genes, there is no internal promoter element, and there is a required TATA-like box. There are also two other elements absolutely required for transcription in vitro, a sequence at the expected position for a PSE between -45 and -65 and an E-box sequence at -80. The E-box sequence binds to the sea urchin homolog of USF (suUSF), and immunodepletion experiments demonstrate that suUSF is required for U6 transcription by RNA polymerase III. MATERUILS AND METHODSPreparation of sea urchin nuclear extract. Sea urchins (Strongylocentrotus purpuratus or Lytechinus variegatus) were grown to the hatching blastula stage, and nuclei were prepared from embryos 1 to 2 h after hatching. Nuclei were prepared exactly as previously described (23). The nuclei were stored in liquid N2. Nuclear extract was prepared as previously described (23) except for the final dialysis step, which was done for 4 h against 80 mM KCl-25 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) (pH 7.5)-0.1 mM EDTA-20% glycerol-1 mM dithiothreitol (DTT)-0.1 mM phenylmethylsulfonyl fluoride. For some of the mobility shift experiments, nuclei were prepared from frozen embryos processed by the method of Calzone et al. (4). These nuclei were also purified by centrifugation through 2 M sucrose as described previously (23)

The dual role of helix – loop – helix-zipper protein USF in ribosomal RNA gene transcription in vivo

“…The pol I promoter, therefore, belongs to a family of other E-box containing USF-activated cellular promoters that include those of alcohol dehydrogenase (Potter et al, 1991), mouse metallothionein-I (Carthew et al, 1987;Mueller et al, 1988), human heme oxygenase (Sato et al, 1990), human insulin (Read et al, 1993) and human CD2 (Outram and Owen, 1994). In addition to activation of alcohol dehydrogenase, human insulin and CD2 genes, USF is also known to stimulate pol III transcription of U6 small nuclear RNA gene (Li et al, 1994). USF thus appears to be a common transcription factor for all three RNA polymerases.…”

Identification and Topological Arrangement of Drosophila Proximal Sequence Element (PSE)-Binding Protein Subunits That Contact the PSEs of U1 and U6 Small Nuclear RNA Genes

“…The U6 gene promoter is representative of an unusual class of RNAP III promoters that contain a TATA box but lack internal promoter elements (5,15,19,30). Promoters of snRNA genes transcribed by either RNAP II or RNAP III contain an essential proximal sequence element (PSE) at a conserved location approximately 40 to 65 bp upstream of the transcription start site that is required for the initiation of snRNA transcription (4,9,18,26,29,30,32,36).…”

Common Factors Direct Transcription through the Proximal Sequence Elements (PSEs) of the Embryonic Sea Urchin U1, U2, and U6 Genes despite Minimal Sequence Similarity among the PSEs

“…The construction of the U6 maxigene has been previously described (10). The starting gene for all the constructs contained 856 nucleotides (nt) of 5Ј flanking sequence and was cloned in pGEM-5zf(ϩ) (Promega, Madison, Wis.).…”

“…There are three important elements: an E box which binds sea urchin USF (10) located at position Ϫ80, the suPSE located at about position Ϫ55, and a TATA-like element located at position Ϫ25 from the start of transcription. All three elements are necessary for efficient expression in vitro (10). We report here additional properties of the U6 PSE and show that the U1 and U2 PSEs can substitute for the U6 PSE despite the fact that there is little sequence similarity between the U6 PSE and the U1 and U2…”

“…There is little sequence similarity among the PSEs of the U1, U2, and U6 genes. There are also other elements required for efficient expression of the sea urchin snRNA genes, but these elements are not shared among the U1, U2, and U6 genes (10,26,30). In addition, both the embryonic and constitutive sea urchin U2 snRNA genes are unique among metazoan snRNA genes transcribed by Pol II, since they contain a required TATA box (26).…”