scholarly journals 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

1994 ◽  
Vol 14 (3) ◽  
pp. 2191-2200
Author(s):  
J M Li ◽  
R A Parsons ◽  
W F Marzluff
Keyword(s):  
Sea Urchin ◽  
Rna Polymerase Iii ◽  
Nuclear Extract ◽  
Major Protein ◽  
In Vitro Mutagenesis ◽  
Flanking Sequence ◽  
Sequence Element ◽  
Proximal Sequence Element ◽  
U6 Gene

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 only 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.

scholarly journals 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.

1994 ◽  
Vol 14 (3) ◽  
pp. 2191-2200 ◽  
Author(s):  
J M Li ◽  
R A Parsons ◽  
W F Marzluff
Keyword(s):  
Sea Urchin ◽  
Rna Polymerase Iii ◽  
Nuclear Extract ◽  
Major Protein ◽  
In Vitro Mutagenesis ◽  
Flanking Sequence ◽  
Sequence Element ◽  
Proximal Sequence Element ◽  
U6 Gene

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 only 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.

scholarly journals Common factors direct transcription through the proximal sequence elements (PSEs) of the embryonic sea urchin U1, U2, and U6 genes despite minimal similarity among the PSEs.

1996 ◽  
Vol 16 (3) ◽  
pp. 1275-1281 ◽  
Author(s):  
J M Li ◽  
R P Haberman ◽  
W F Marzluff
Keyword(s):  
Sea Urchin ◽  
Affinity Column ◽  
Rrna Gene ◽  
Start Site ◽  
Small Nuclear Rna ◽  
Mobility Shift ◽  
Sequence Element ◽  
Nuclear Rna ◽  
U6 Gene

The proximal sequence element (PSE) for the sea urchin U6 small nuclear RNA gene has been defined. The most critical nucleotides for expression, located 61 to 64 nucleotides (nt) from the transcription start site, are 4 nt, AACT, at the 5' end of the PSE. Two nucleotide mutations in this region abolish transcription of the sea urchin U6 gene in vitro. The same two nucleotide mutations greatly reduce the binding of specific factors detected by an electrophoretic mobility shift assay. There is also a conserved AC dinucleotide 57 nt from the start site of the sea urchin U1 and U2 PSEs. The sea urchin U1 and U2 PSEs were substituted for the sea urchin U6 PSE, with the conserved AC sequences aligned with those of the U6 PSE. Both of these genes were expressed at levels higher than those observed with the wild-type U6 gene. Similar complexes are formed on the U1 and U2 PSEs, and formation of the complexes is inhibited efficiently by the U6 PSE. In addition, the E-box sequence present upstream of the PSE enhances U6 transcription from both the U1 and U2 PSEs. Finally, depletion of a nuclear extract with a DNA affinity column containing the U6 PSE sequence reduces expression of the U6 genes driven by the U6, U1, or U2 PSE but does not affect expression of the 5S rRNA gene. These data support the possibility that the same factor(s) interacts with the PSE sequences of the U1, U2, and U6 small nuclear RNA genes expressed in early sea urchin embryogenesis.

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

1998 ◽  
Vol 18 (3) ◽  
pp. 1570-1579 ◽  
Author(s):  
Yan Wang ◽  
William E. Stumph
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Sea Urchins ◽  
Binding Protein ◽  
Rna Polymerase Iii ◽  
Specific Protein ◽  
Cross Linking ◽  
Sequence Element ◽  
Proximal Sequence Element ◽  
U6 Gene

ABSTRACT Most small nuclear RNAs (snRNAs) are synthesized by RNA polymerase II, but U6 and a few others are synthesized by RNA polymerase III. Transcription of snRNA genes by either polymerase is dependent on a proximal sequence element (PSE) located upstream of position −40 relative to the transcription start site. In contrast to findings in vertebrates, sea urchins, and plants, the RNA polymerase specificity ofDrosophila snRNA genes is intrinsically encoded in the PSE sequence itself. We have investigated the differential interaction of the Drosophila melanogaster PSE-binding protein (DmPBP) with U1 and U6 gene PSEs. By using a site specific protein-DNA photo-cross-linking assay, we identified three polypeptide subunits of DmPBP with apparent molecular masses of 95, 49, and 45 kDa that are in close proximity to the DNA and two additional putative polypeptides of 230 and 52 kDa that may be integral to the complex. The 95-kDa subunit cross-linked at positions spanning the entire length of the PSE, but the 49- and 45-kDa subunits cross-linked only to the 3′ half of the PSE. The same polypeptides cross-linked to both the U1 and U6 PSE sequences. However, there were significant differences in the cross-linking patterns of these subunits at a subset of the phosphate positions, depending on whether binding was to a U1 or U6 gene PSE. These data suggest that RNA polymerase specificity is associated with distinct modes of interaction of DmPBP with the DNA at U1 and U6 promoters.

UHF-1, a factor required for maximal transcription of early and late sea urchin histone H4 genes: analysis of promoter-binding sites

1991 ◽  
Vol 11 (2) ◽  
pp. 1048-1061
Author(s):  
I J Lee ◽  
L Tung ◽  
D A Bumcrot ◽  
E S Weinberg
Keyword(s):  
Binding Site ◽  
Sea Urchin ◽  
Transcriptional Start Site ◽  
Sodium Dodecyl ◽  
Nuclear Extract ◽  
Dnase I ◽  
Histone H4 ◽  
Band Shift

A protein, denoted UHF-1, was found to bind upstream of the transcriptional start site of both the early and late H4 (EH4 and LH4) histone genes of the sea urchin Strongylocentrotus purpuratus. A nuclear extract from hatching blastulae contained proteins that bind to EH4 and LH4 promoter fragments in a band shift assay and produced sharp DNase I footprints upstream of the EH4 gene (from -133 to -106) and the LH4 gene (from -94 to -66). DNase I footprinting performed in the presence of EH4 and LH4 promoter competitor DNAs indicated that UHF-1 binds more strongly to the EH4 site. A sequence match of 11 of 13 nucleotides was found within the two footprinted regions: [sequence: see text]. Methylation interference and footprinting experiments showed that UHF-1 bound to the two sites somewhat differently. DNA-protein UV cross-linking studies indicated that UHF-1 has an electrophoretic mobility on sodium dodecyl sulfate-acrylamide gels of approximately 85 kDa and suggested that additional proteins, specific to each promoter, bind to each site. In vitro and in vivo assays were used to demonstrate that the UHF-1-binding site is essential for maximal transcription of the H4 genes. Deletion of the EH4 footprinted region resulted in a 3-fold decrease in transcription in a nuclear extract and a 2.6-fold decrease in expression in morulae from templates that had been injected into eggs. In the latter case, deletion of the binding site did not grossly disrupt the temporal program of expression from the injected EH4 genes. LH4 templates containing a 10-bp deletion in the consensus region or base substitutions in the footprinted region were transcribed at 14 to 58% of the level of the wild-type LH4 template. UHF-1 is therefore essential for maximal expression of the early and late H4 genes.

Transcriptional properties of BmX, a moderately repetitive silkworm gene that is an RNA polymerase III template

1988 ◽  
Vol 8 (2) ◽  
pp. 624-631
Author(s):  
E T Wilson ◽  
D P Condliffe ◽  
K U Sprague
Keyword(s):  
Control Region ◽  
Transcription Initiation ◽  
Rna Polymerase Iii ◽  
Sequence Element ◽  
Direct Transcription ◽  
Factor Binding ◽  
Large Gene ◽  
Polymerase Iii ◽  
Downstream Control

We analyzed the transcriptional properties of a repetitive sequence element, BmX, that belongs to a large gene family (approximately 2 x 10(4) copies) in the genome of the Bombyx mori silkworm. We discovered BmX elements because of their ability to direct transcription by polymerase III in vitro and used them to test the generality of the properties of previously identified silkworm polymerase III control elements. We found that the signals that act in cis to control BmX transcription strongly resemble those that direct transcription of other silkworm polymerase III templates. As with silkworm tRNA and 5S RNA genes, transcription of BmX requires sequence signals located both upstream and downstream from the site of transcription initiation. The critical upstream sequences are structurally as well as functionally similar in the three kinds of templates. The downstream control region of BmX resembles the corresponding part of a silkworm alanine tRNA gene in that it provides a large (greater than 100 base pairs) region that influences transcription factor binding. Moreover, the factor-binding regions of both tRNA(Ala) and BmX genes are remarkable in that under certain conditions, key elements within them (the B boxes, for example) appear dispensable. This behavior can be understood if, in both of these templates, the downstream control region acts as a large target for interaction with a multifactor complex.

Multiple hepatic trans-acting factors are required for in vitro transcription of the human alpha-1-antitrypsin gene

1988 ◽  
Vol 8 (10) ◽  
pp. 4362-4369
Author(s):  
Y Li ◽  
R F Shen ◽  
S Y Tsai ◽  
S L Woo
Keyword(s):  
Rat Liver ◽  
Nuclear Extract ◽  
In Vitro Transcription ◽  
Flanking Sequence ◽  
Tissue Specific ◽  
Hepatic Expression ◽  
Nuclear Extracts ◽  
Nuclear Factors ◽  
Alpha 1 Antitrypsin

The human alpha-1-antitrypsin (AAT) gene is expressed in the liver, and its deficiency causes pulmonary emphysema. We have demonstrated that its 5'-flanking region contains cis-acting elements capable of directing proper transcription in the presence of rat liver nuclear extract. The in vitro transcription system is tissue-specific in that the AAT promoter is functional in nuclear extracts prepared from the liver but not from HeLa cells. Experiments in which rat liver and HeLa nuclear extracts were mixed suggested the presence of a specific activator(s) in hepatocytes rather than a repressor(s) in nonproducing cells. Two protected regions were detected in the promoter by DNase I footprinting analysis with rat liver nuclear extracts. Region one spanned -78 to -52 and region two spanned -125 to -100 in the 5'-flanking sequence of the gene. By gel retardation assays with synthetic oligonucleotides, at least two distinct liver nuclear factors were identified, HNF-1 and HNF-2 (hepatocyte nuclear factors), which bound specifically to the first and second region, respectively. We present evidence that HNF-1 and HNF-2 are positively acting, tissue-specific transcription factors that regulate hepatic expression of the human AAT gene.

Mutation of the c-fos gene dyad symmetry element inhibits serum inducibility of transcription in vivo and the nuclear regulatory factor binding in vitro

1987 ◽  
Vol 7 (3) ◽  
pp. 1217-1225
Author(s):  
M E Greenberg ◽  
Z Siegfried ◽  
E B Ziff
Keyword(s):  
Growth Factor ◽  
Nuclear Protein ◽  
Symmetry Element ◽  
Regulatory Sequence ◽  
In Vitro Mutagenesis ◽  
Regulatory Factor ◽  
Mobility Shift ◽  
Sequence Element ◽  
Dyad Symmetry

In vitro mutagenesis of a 61-base-pair DNA sequence element that is necessary for induction of the c-fos proto-oncogene by growth factors revealed that a small region of dyad symmetry within the sequence element is critical for c-fos transcriptional activation. The same c-fos dyad symmetry element was found to bind a nuclear protein in vitro, causing a specific mobility shift of this c-fos regulatory sequence. An analysis of insertion and deletion mutants established a strict correlation between the ability of the dyad symmetry element to promote serum activation of c-fos transcription and in vitro nuclear protein binding. These experiments suggest that the DNA mobility shift assay detects a nuclear protein that mediates growth factor stimulation of c-fos expression. In vitro competition experiments indicate that the c-fos regulatory factor also binds to sequences within another growth factor-inducible gene, the beta-actin gene.

scholarly journals Silk gland-specific tRNA(Ala) genes interact more weakly than constitutive tRNA(Ala) genes with silkworm TFIIIB and polymerase III fractions.

1994 ◽  
Vol 14 (3) ◽  
pp. 1806-1814 ◽  
Author(s):  
H S Sullivan ◽  
L S Young ◽  
C N White ◽  
K U Sprague
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Iii ◽  
Silk Gland ◽  
Flanking Sequence ◽  
Transcription Machinery ◽  
Polymerase Iii ◽  
Active Transcription ◽  
The Difference ◽  
Transcription Complexes

Constitutive and silk gland-specific tRNA(Ala) genes from silkworms have very different transcriptional properties in vitro. Typically, the constitutive type, which encodes tRNA(AlaC), directs transcription much more efficiently than does the silk gland-specific type, which encodes tRNA(AlaSG). We think that the inefficiency of the tRNA(AlaCG) gene underlies its capacity to be turned off in non-silk gland cells. An economical model is that the tRNA(AlaSG) promoter interacts poorly, relative to the tRNA(AlaC) promoter, with one or more components of the basal transcription machinery. As a consequence, the tRNA(AlaSG) gene directs the formation of fewer transcription complexes or of complexes with reduced cycling ability. Here we show that the difference in the number of active transcription complexes accounts for the difference in tRNA(AlaC) and tRNA(AlaSG) transcription rates. To determine whether a particular component of the silkworm transcription machinery is responsible for reduced complex formation on the tRNA(AlaSG) gene, we measured competition by templates for defined fractions of this machinery. We find that the tRNA(AlaSG) gene is greatly impaired, in comparison with the tRNA(AlaC) gene, in competition for either TFIIIB or RNA polymerase III. Competition for each of these fractions is also strongly influenced by the nature of the 5' flanking sequence, the promoter element responsible for the distinctive transcriptional properties of tRNA(AlaSG) and tRNA(AlaC) genes. These results suggest that differential interaction with TFIIIB or RNA polymerase III is a critical functional distinction between these genes.

scholarly journals A nucleosome positioned in the distal promoter region activates transcription of the human U6 gene.

1997 ◽  
Vol 17 (8) ◽  
pp. 4397-4405 ◽  
Author(s):  
W Stünkel ◽  
I Kober ◽  
K H Seifart
Keyword(s):  
Micrococcal Nuclease ◽  
Deletion Mutants ◽  
Dna Fragments ◽  
Wild Type ◽  
Sequence Element ◽  
Proximal Sequence Element ◽  
Experimental Approaches ◽  
Type Gene ◽  
Distal Promoter ◽  
U6 Gene

To investigate the consequences of chromatin reconstitution for transcription of the human U6 gene, we assembled nucleosomes on both plasmids and linear DNA fragments containing the U6 gene. Initial experiments with DNA fragments revealed that U6 sequences located between the distal sequence element (DSE) and the proximal sequence element (PSE) lead to the positioning of a nucleosome partially encompassing these promoter elements. Furthermore, indirect end-labelling analyses of the reconstituted U6 wild-type plasmids showed strong micrococcal nuclease cuts near the DSE and PSE, indicating that a nucleosome is located between these elements. To investigate the influence that nucleosomes exert on U6 transcription, we used two different experimental approaches for chromatin reconstitution, both of which resulted in the observation that transcription of the U6 wild-type gene was enhanced after chromatin assembly. To ensure that the facilitated transcription of the nucleosomal templates is in fact due to a positioned nucleosome, we constructed mutants of the U6 gene in which the sequences between the DSE and PSE were progressively deleted. In contrast to what was observed with the wild-type genes, transcription of these deletion mutants was significantly inhibited when they were packaged into nucleosomes.

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