Two Ribosomal DNA-Binding Factors Interact with a Cluster of Motifs on the 5' External Transcribed Spacer, Upstream from the Primary Pre-rRNA Processing Site in a Higher Plant

1997 ◽  
Vol 247 (3) ◽  
pp. 981-989 ◽  
Author(s):  
David Caparros-Ruiz ◽  
Sylvie Lahmy ◽  
Sarah Piersanti ◽  
Manuel Echeverria
Keyword(s):  
Dna Binding ◽  
Ribosomal Dna ◽  
Rrna Processing ◽  
Higher Plant ◽  
External Transcribed Spacer ◽  
Dna Binding Factors ◽  
Processing Site

scholarly journals Partially Processed pre-rRNA Is Preserved in Association with Processing Components in Nucleolus-derived Foci during Mitosis

1998 ◽  
Vol 9 (9) ◽  
pp. 2407-2422 ◽  
Author(s):  
Miroslav Dundr ◽  
Mark O.J. Olson
Keyword(s):  
Molecular Weight ◽  
Internal Transcribed Spacer ◽  
Rnase P ◽  
Rrna Processing ◽  
Rnase Mrp ◽  
External Transcribed Spacer ◽  
Early Anaphase ◽  
Late Telophase ◽  
Processing Site

Previous studies showed that components implicated in pre-rRNA processing, including U3 small nucleolar (sno)RNA, fibrillarin, nucleolin, and proteins B23 and p52, accumulate in perichromosomal regions and in numerous mitotic cytoplasmic particles, termed nucleolus-derived foci (NDF) between early anaphase and late telophase. The latter structures were analyzed for the presence of pre-rRNA by fluorescence in situ hybridization using probes for segments of pre-rRNA with known half-lives. The NDF did not contain the short-lived 5′-external transcribed spacer (ETS) leader segment upstream from the primary processing site in 47S pre-rRNA. However, the NDF contained sequences from the 5′-ETS core, 18S, internal transcribed spacer 1 (ITS1), and 28S segments and also had detectable, but significantly reduced, levels of the 3′-ETS sequence. Northern analyses showed that in mitotic cells, the latter sequences were present predominantly in 45S-46S pre-rRNAs, indicating that high-molecular weight processing intermediates are preserved during mitosis. Two additional essential processing components were also found in the NDF: U8 snoRNA and hPop1 (a protein component of RNase MRP and RNase P). Thus, the NDF appear to be large complexes containing partially processed pre-rRNA associated with processing components in which processing has been significantly suppressed. The NDF may facilitate coordinated assembly of postmitotic nucleoli.

scholarly journals Interactions with Single-stranded and Double-stranded DNA-binding Factors and Alternative Promoter Conformation upon Transcriptional Activation of theHtf9-a/RanBP1andHtf9-cGenes

1998 ◽  
Vol 273 (1) ◽  
pp. 495-505 ◽  
Author(s):  
Gigliola Di Matteo ◽  
Massimiliano Salerno ◽  
Giulia Guarguaglini ◽  
Barbara Di Fiore ◽  
Franco Palitti ◽  
...  
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
Alternative Promoter ◽  
Double Stranded Dna ◽  
Dna Binding Factors

scholarly journals Recruitment of TATA-Binding Protein–TAFI Complex SL1 to the Human Ribosomal DNA Promoter Is Mediated by the Carboxy-Terminal Activation Domain of Upstream Binding Factor (UBF) and Is Regulated by UBF Phosphorylation

1999 ◽  
Vol 19 (4) ◽  
pp. 2872-2879 ◽  
Author(s):  
JoAnn C. Tuan ◽  
Weiguo Zhai ◽  
Lucio Comai
Keyword(s):  
Dna Binding ◽  
Ribosomal Dna ◽  
Transcriptional Activation ◽  
Direct Contact ◽  
Binding Protein ◽  
Tata Binding Protein ◽  
Rrna Synthesis ◽  
Binding Factor ◽  
Upstream Binding Factor ◽  
Carboxy Terminal

ABSTRACT Human rRNA synthesis by RNA polymerase I requires at least two auxiliary factors, upstream binding factor (UBF) and SL1. UBF is a DNA binding protein with multiple HMG domains that binds directly to the CORE and UCE elements of the ribosomal DNA promoter. The carboxy-terminal region of UBF is necessary for transcription activation and has been shown to be extensively phosphorylated. SL1, which consists of TATA-binding protein (TBP) and three associated factors (TAFIs), does not have any sequence-specific DNA binding activity, and its recruitment to the promoter is mediated by specific protein interactions with UBF. Once on the promoter, the SL1 complex makes direct contact with the DNA promoter and directs promoter-specific initiation of transcription. To investigate the mechanism of UBF-dependent transcriptional activation, we first performed protein-protein interaction assays between SL1 and a series of UBF deletion mutants. This analysis indicated that the carboxy-terminal domain of UBF, which is necessary for transcriptional activation, makes direct contact with the TBP-TAFI complex SL1. Since this region of UBF can be phosphorylated, we then tested whether this modification plays a functional role in the interaction with SL1. Alkaline phosphatase treatment of UBF completely abolished the ability of UBF to interact with SL1; moreover, incubation of the dephosphorylated UBF with nuclear extracts from exponentially growing cells was able to restore the UBF-SL1 interaction. In addition, DNase I footprinting analysis and in vitro-reconstituted transcription assays with phosphatase-treated UBF provided further evidence that UBF phosphorylation plays a critical role in the regulation of the recruitment of SL1 to the ribosomal DNA promoter and stimulation of UBF-dependent transcription.

scholarly journals Single-strand-DNA-binding factors specifically recognize the pyrimidine element in the chick α2(I) collagen gene promoter

1996 ◽  
Vol 314 (1) ◽  
pp. 293-296 ◽  
Author(s):  
Dashzeveg BAYARSAIHAN ◽  
Lewis N. LUKENS
Keyword(s):  
Dna Binding ◽  
Chick Embryo ◽  
Gene Promoter ◽  
Binding Activity ◽  
Dna Conformation ◽  
Collagen Gene ◽  
Nuclear Extracts ◽  
Binding Factor ◽  
Dna Binding Factors ◽  
Collagen Promoter

A pyrimidine element with mirror repeats centred at position -192 bp of the chick α2(I) collagen promoter interacts with sequence-specific DNA-binding factors. These factors bind to only the pyrimidine strand of this region and have no affinity for the complementary purine strand. Binding activity is also seen with the double-stranded form of this element, but with less affinity than to the single-stranded pyrimidine species. Southwestern blot analyses have shown that proteins of 80 and 134 kDa in chick embryo fibroblast nuclear extracts bind to the pyrimidine strand, whereas only a 134 kDa DNA-binding protein was found in chondrocyte nuclear extracts. The binding mechanism of these nuclear proteins with single-stranded DNA might be based on a non-B-DNA conformation of the pyrimidine element. The position of this binding site in the promoter region, its potential for adopting an unusual secondary structure and the presence of the 80 kDa binding factor in chick embryo fibroblasts, but not in chondrocytes, suggest a possible role for this factor in the expression of the α2(I) collagen gene.

DNA-binding factors of B lymphoid cells are susceptible to limited proteolytic cleavage during nuclear extract preparation

1988 ◽  
Vol 8 (4) ◽  
pp. 1812-1815
Author(s):  
E L Mather
Keyword(s):  
Dna Binding ◽  
Nuclear Extract ◽  
Lymphoid Cells ◽  
Chain Gene ◽  
Immunoglobulin Heavy Chain Gene ◽  
Mobility Shift ◽  
Lymphoid Lines ◽  
Dna Binding Factors ◽  
Mobility Shift Assay ◽  
B Lymphoid

DNA-binding proteins that interact with the 3' end of the mouse mu immunoglobulin heavy chain gene were identified by the electrophoretic mobility shift assay. Complexes of distinctly different mobilities were formed by extracts prepared from B lymphoid lines representing different stages of maturation. The apparent stage-specific differences are shown to be due to proteolytic events that occurred during extract preparation.

petD mRNA maturation in Chlamydomonas reinhardtii chloroplasts: role of 5' endonucleolytic processing

1994 ◽  
Vol 14 (9) ◽  
pp. 6180-6186
Author(s):  
W Sakamoto ◽  
N R Sturm ◽  
K L Kindle ◽  
D B Stern
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Fusion Gene ◽  
Deletion Mutants ◽  
Chloroplast Genes ◽  
Coding Region ◽  
Higher Plant ◽  
Glucuronidase Activity ◽  
Mrna Maturation ◽  
Processing Site ◽  
And Function

Complex processing of primary transcripts occurs during the expression of higher-plant chloroplast genes. In Chlamydomonas reinhardtii, most chloroplast genes appear to possess their own promoters, rather than being transcribed as part of multicistronic operons. By generating specific deletion mutants, we show that petD, which encodes subunit IV of the cytochrome b6/f complex, has an RNA processing site that is required for accumulation of monocistronic petD mRNA in petD promoter deletion mutants; in such mutants, transcription of petD originates from the upstream petA promoter. The 5' ends of transcripts initiated at the petD promoter are probably also generated by processing, since the 5' end of monocistronic petD mRNA is the same in wild-type strains as it is in the petD promoter mutants. The location and function of the processing site were further examined by inserting petD-uidA fusion genes into the chloroplast genome (uidA is an Escherichia coli gene that encodes beta-glucuronidase). When a promoterless petD-uidA fusion gene was inserted downstream of petA, a monocistronic uidA transcript accumulated, which was apparently initiated at the petA promoter and was processed at a site corresponding precisely to the petD mRNA 5' end. When a construct including only sequences downstream of +25 relative to the mature mRNA 5' end was inserted into the same site, a dicistronic petA-uidA transcript accumulated but no monocistronic uidA transcript could be detected, suggesting that a processing site lies at least partially within the region from -1 to +25. Beta-glucuronidase activity was not detected in transformants that accumulated only the dicistronic petA-uidA transcript, suggesting that the first 25 bp of the 5' untranslated region are required for translation initiation. One explanation for this translational defect is that Chlamydomonas chloroplasts cannot translate the second coding region of some dicistronic messages.

scholarly journals E4F and ATF, two transcription factors that recognize the same site, can be distinguished both physically and functionally: a role for E4F in E1A trans activation.

1990 ◽  
Vol 10 (10) ◽  
pp. 5138-5149 ◽  
Author(s):  
R J Rooney ◽  
P Raychaudhuri ◽  
J R Nevins
Keyword(s):  
Dna Binding ◽  
Binding Activity ◽  
Stable Complex ◽  
Small Subset ◽  
Adenovirus Infection ◽  
Binding Assays ◽  
Dna Binding Activity ◽  
Sequence Recognition ◽  
Nuclear Factors ◽  
Dna Binding Factors

Previous experiments have identified an element in the adenovirus E4 promoter that is critical for E1A-dependent trans activation and that can confer inducibility to a heterologous promoter. This DNA element is a recognition site for multiple nuclear factors, including ATF, which is likely a family of DNA-binding factors with similar DNA recognition properties. However, ATF activity was found not to be altered in any demonstrable way as a result of adenovirus infection. In contrast, another factor that recognizes this element, termed E4F, was found at only very low levels in uninfected cells but was increased markedly upon adenovirus infection, as measured in DNA-binding assays. Although both the ATF activity and the E4F activity recognized and bound to the same two sites in the E4 promoter, they differed in their sequence recognition of these sites. Furthermore, E4F bound only to a small subset of the ATF recognition sites; for instance, E4F did not recognize the ATF sites in the E2 or E3 promoters. Various E4F and ATF binding sites were inserted into an expression vector and tested by cotransfection assays for responsiveness to E1A. We found that a sequence capable of binding E4F could confer E1A inducibility. In contrast, a sequence that could bind ATF but not E4F did not confer E1A inducibility. We also found that E4F formed a stable complex with the E4 promoter, whereas the ATF DNA complex was unstable and rapidly dissociated. We conclude that the DNA-binding specificity of E4F as well as the alterations in DNA-binding activity of E4F closely correlates with E1A stimulation of the E4 promoter.

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