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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We examined the ability of purified RNA polymerase (RNAP) II lacking the carboxy-terminal heptapeptide repeat domain (CTD), called RNAP IIB, to transcribe a variety of promoters in HeLa extracts in which endogenous RNAP II activity was inhibited with anti-CTD monoclonal antibodies. Not all promoters were efficiently transcribed by RNAP IIB, and transcription did not correlate with the in vitro strength of the promoter or with the presence of a consensus TATA box. This was best illustrated by the GC-rich, non-TATA box promoters of the bidirectional dihydrofolate reductase (DHFR)-REP-encoding locus. Whereas the REP promoter was transcribed by RNAP IIB, the DHFR promoter remained inactive after addition of RNAP IIB to the antibody-inhibited reactions. However, both promoters were efficiently transcribed when purified RNAP with an intact CTD was added. We analyzed a series of promoter deletions to identify which cis elements determine the requirement for the CTD of RNAP II. All of the promoter deletions of both DHFR and REP retained the characteristics of their respective full-length promoters, suggesting that the information necessary to specify the requirement for the CTD is contained within approximately 65 bp near the initiation site. Furthermore, a synthetic minimal promoter of DHFR, consisting of a single binding site for Sp1 and a binding site for the HIP1 initiator cloned into a bacterial vector sequence, required RNAP II with an intact CTD for activity in vitro. Since the synthetic minimal promoter of DHFR and the smallest REP promoter deletion are both activated by Sp1, the differential response in this assay does not result from upstream activators. However, the sequences around the start sites of DHFR and REP are not similar and our data suggest that they bind different proteins. Therefore, we propose that specific initiator elements are important for determination of the requirement of some promoters for the CTD.
Mol Cell Biol 1992 May
PMID:The HIP1 initiator element plays a role in determining the in vitro requirement of the dihydrofolate reductase gene promoter for the C-terminal domain of RNA polymerase II. 156 52

We have previously confirmed the estrogen-induced protein of rat uterus to be creatine kinase B (CKB), and demonstrated a 1.7-kilobase pair fragment encompassing the promoter and adjoining 5'-flank to be capable of conferring estrogen responsiveness in HeLa cells. In this study we find an element at -550, aGGTCAgaaCACCCt, with limited similarity to the estrogen response element consensus, to be involved in conferring estrogen responsiveness on the CKB promoter. This element can bind estrogen receptor (ER) and is flanked by two GC boxes, which we find capable of binding bacterially expressed Sp1. Additional responsiveness is found closely associated with the CKB promoter at high levels of cotransfected ER construct. No potential response element was identified in this region, but we find the ER DNA-binding domain to be required.
Mol Endocrinol 1992 Feb
PMID:Delineation of sites mediating estrogen regulation of the rat creatine kinase B gene. 156 66

We have demonstrated that the retinoblastoma gene product (Rb) can positively regulate transcription from the fourth promoter of the insulinlike growth factor II gene. Two copies of a motif (the retinoblastoma control element) similar to that found in the human c-fos, transforming growth factor beta 1, and c-myc promoters are responsible for conferring Rb regulation to the fourth promoter of the insulinlike growth factor II gene. We have shown that the transcription factor Sp1 can bind to and stimulate transcription from the retinoblastoma control element motif. Moreover, by using a GAL4-Sp1 fusion protein, we have directly demonstrated that Rb positively regulates Sp1 transcriptional activity in vivo. These results indicate that Rb can function as a positive regulator of transcription and that Sp1 is one potential target, either directly or indirectly, for transcriptional regulation by Rb.
Mol Cell Biol 1992 Jun
PMID:The retinoblastoma gene product regulates Sp1-mediated transcription. 158 49

MCM1 performs several functions necessary for its role in regulating cell type-specific gene expression in the yeast Saccharomyces cerevisiae: DNA binding, transcription activation, and interaction with coregulatory proteins such as alpha 1. We analyzed a set of MCM1 deletion derivatives using in vivo reporter gene assays and in vitro DNA-binding studies to determine which regions of MCM1 are important for its various activities. We also analyzed a set of LexA-MCM1 hybrids to examine the ability of different segments of MCM1 to activate transcription independent of MCM1's DNA-binding function. The first third of MCM1 [MCM1(1-96)], which includes an 80-residue segment homologous to the mammalian serum response factor, was sufficient for high-affinity DNA binding, for activation of reporter gene expression, and for interaction with alpha 1 in vitro and in vivo. However, the ability of MCM1(1-96) to activate transcription and to interact with alpha 1 was somewhat reduced compared with wild-type MCM1 [MCM1(1-286)]. Optimal interaction with alpha 1 required residues 99 to 117, in which 18 of 19 amino acids are acidic in character. Optimal transcription activation required a segment from residues 188 to 286, in which 50% of the amino acids are glutamine. Deletion of this segment from MCM1 reduced expression of reporter genes by about twofold. Moreover, LexA-MCM1 hybrids containing this segment were able to activate expression of reporter genes that rely on LexA binding sites as potential upstream activation sequences. Thus, glutamine-rich regions may contribute to the activation function of yeast transcription activators, as has been suggested for glutamine-rich mammalian proteins such as Sp1.
Mol Cell Biol 1992 Aug
PMID:The N-terminal 96 residues of MCM1, a regulator of cell type-specific genes in Saccharomyces cerevisiae, are sufficient for DNA binding, transcription activation, and interaction with alpha 1. 163 Apr 61

The Thy-1 gene promoter resembles a "housekeeping" promoter in that it is located within a methylation-free island, lacks a canonical TATA box, and displays heterogeneity in the 5'-end termini of the mRNA. Using transgenic mice, we show that this promoter does not confer any tissue specificity and is active only in a position-dependent manner. It can only be activated in a tissue-specific manner by elements that lie downstream of the initiation site. We have analyzed the functional domains of the minimal Thy-1 promoter and show that the dominant promoter elements consist of multiple binding sites for the transcription factor Sp1, an inverted CCAAT box, and sequences proximal to the transcription start site. DNase I and gel mobility shift assays show the binding of a number of nuclear factors to these elements, including Sp1 and CP1. Our results show that the structure of this promoter only permits productive interactions of the two transcription factors Sp1 and CP1 with the basal transcription machinery in the presence of enhancer sequences.
Mol Cell Biol 1991 Apr
PMID:The functional domains of the murine Thy-1 gene promoter. 167 42

Transcription of the human fetal globin genes in erythroid cells is tightly regulated during different stages of development and differentiation. Two naturally occurring mutations 202 base pairs upstream of the duplicated gamma globin genes are associated with incorrectly regulated gamma globin gene gene expression; elevated levels of fetal globin are synthesized during adult life. A C-to-G base substitution upstream of the G gamma-globin gene is highly correlated with a dramatic increase in gene expression. It increases the similarity of the region to the consensus Sp1 recognition site. We determined that the mutated DNA had a 5- to 10-fold-higher affinity for Sp1 than did normal gamma globin gene sequence. We also observed a reduction in normal factor-binding activity. A different substitution at -202, C to T, upstream of the A gamma-globin gene was associated with a more moderate increase in fetal globin expression. This mutation decreased the similarity of the sequence to an Sp1 recognition site. We determined that it did not result in enhanced Sp1 binding but did alter normal factor binding. We suggest that these changes in nuclear protein-binding properties detected in vitro are responsible for the enhanced gamma globin gene expression found in -202 G gamma beta + patients with hereditary persistence of fetal hemoglobin.
Mol Cell Biol 1990 Jan
PMID:A naturally occurring gamma globin gene mutation enhances SP1 binding activity. 168 66

Through analysis of rat genomic cosmid clones, the 5'-most exon of the rat neural cell adhesion molecule (NCAM) gene was identified. This exon, here named exon 0, contained the entire 5' untranslated region and the N-terminal signal sequence of the polypeptide. Exon 0 was isolated from a 1.6-kilobase (kb) EcoRI-HindIII fragment of rat genomic cosmid clone 9 which was 35 kb in length. This fragment was sequenced and found to contain approximately 940 base pairs (bp) of 5'-flanking sequence, exon 0, which was approximately 245 bp in length, and approximately 400 bp of the following intron 0. By using information derived from this fragment and the pR18 rat NCAM cDNA, the transcription initiation sites were determined with two assays. Both primer extensions and nuclease S1 protection assays of postnatal day 7 rat brain RNA identified seven initiation sites within a single 10-bp region at positions -195 to -186 relative to the translation start site. An additional minor site was found at position -329. In the immediate 5' region, no consensus TATA or CCAAT sequences were found. Potential regulatory elements within this region include Sp1 consensus binding sites and also a 178-bp homopurine-homopyrimidine sequence containing several mirror repeats. NCAM has multiple transcripts which are regulated in a developmental and tissue-specific fashion. To determine whether these transcripts are initiated at the same sites, transcription initiation sites were analyzed in postnatal day 7 and adult rat brain and also in cultured cell lines of neuronal, glial, and muscle phenotypes. These tissues and cells exhibited distinct NCAM transcript populations in Northern (RNA) dot blot analysis. In all cases similar transcription start sites were found, suggesting that all major NCAM transcripts have similar or identical initiation sites. These results provide essential information to begin analysis of NCAM regulation in different tissues and during development.
Mol Cell Biol 1990 Jul
PMID:Transcription initiation sites and structural organization of the extreme 5' region of the rat neural cell adhesion molecule gene. 169 9

A genomic DNA fragment containing the Raf-1 promoter region was isolated by using a cDNA extension clone. Nucleotide sequencing of genomic DNA clones, primer extension, and S1 nuclease assays have been used to identify the 5' ends of Raf-1 RNAs. Consistent with its ubiquitous expression, the Raf-1 promoter region had features of a housekeeping gene in that it was GC-rich (HTF-like), lacked TATA and CAAT boxes, and contained heterogeneous RNA start sites and four potential binding sites for the transcription factor SP1. In addition, an octamer motif (ATTTCAT), a potential binding site for the octamer family of transcription factors, was located at -734 base pairs. The Raf-1 promoter region drove reporter gene expression 30-fold over the promoterless reporter in Cos 7 cells.
Mol Cell Biol 1990 Jul
PMID:Molecular organization of the human Raf-1 promoter region. 169 10

We have isolated clones encoding the rat insulin-like growth factor-binding protein-2 (IGFBP-2) gene and determined its organization and nucleotide sequence. The rat IGFBP-2 gene spans at least 8 kilobases and consists of four exons, each of which contains protein-coding sequences. The amino acid sequences of exons 1, 3, and 4 are 32-50% identical to the corresponding exons of human IGFBP-1 and IGFBP-3, and 87-91% identical to those of human IGFBP-2. The 18 cysteines in the mature binding proteins are conserved. Exon 2 shows negligible homology. Primer-extended reverse transcription indicated that the 5' end of IGFBP-2 mRNA is 151 nucleotides up-stream from the translation start site [designated nucleotide (nt) -151]. Consistent with this result, IGFBP-2 mRNA protected a genomic fragment terminating at approximately nt -148, as well as smaller fragments. A 1260 nt fragment containing 1144 nt of 5' flanking region had promoter activity when inserted in the correct orientation into a plasmid containing a promoterless luciferase reporter gene and transiently transfected into BRL-3A rat liver cells, which express IGFBP-2, but not when transfected into H4-II-E cells, which do not express IGFBP-2. The IGFBP-2 gene lacks a TATA box immediately up-stream from the transcription initiation site. It is GC rich (66% between nt -270 and +385) and contains GC boxes that might be recognized by transcription factors Sp1 or ETF. The promoter region contains multiple direct and indirect repeats. One direct repeat contains a variant Sp1 site (-158 to -150) near the consensus Sp1 site at nt -138 to -130. The 5' flanking region also contains motifs that might be recognized by transcription factors AP-1 (Jun/Fos), AP-2, and liver factor B1. The role of these sites in basal and regulated expression of the IGFBP-2 gene remains to be determined.
Mol Endocrinol 1990 Dec
PMID:Cloning of the rat insulin-like growth factor-binding protein-2 gene and identification of a functional promoter lacking a TATA box. 170 31

Tissue plasminogen activator (t-PA) mRNA levels are high in murine brain, lower in kidney, and undetectable in liver. Differences in t-PA mRNA levels are regulated in part at the transcriptional level. Brain, kidney, and liver nuclear extracts direct regulated transcription from the murine t-PA promoter in a manner that reflects the relative levels of t-PA gene expression in these tissues in vivo. Analysis of mutants has defined two GC box motifs as important elements for regulated transcription in vitro. Upon investigation of protein-DNA binding, we detected an activity in brain extracts which was not detected in kidney or liver extracts. An Sp1-like factor also binds to this region in all three tissue types. DNA interference experiments show that the brain-enriched binding activity and the Sp1-like factor contact the same GC-rich sequences. These studies provide additional evidence that brain-enriched DNA-binding activities can interact with sequences also recognized by ubiquitous transcription factors.
Mol Cell Biol 1991 Jun
PMID:In vitro analysis of the tissue plasminogen activator promoter reveals a GC box-binding activity present in murine brain but undetectable in kidney and liver. 171 25


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