Phosphorylation of Human General Transcription Factors TATA-Binding Protein and Transcription Factor IIB by DNA-Dependent Protein Kinase. Synergistic Stimulation of RNA Polymerase II Basal Transcription In Vitro

1997 ◽  
Vol 247 (3) ◽  
pp. 1166-1173 ◽  
Author(s):  
Taku Chibazakura ◽  
Fumiaki Watanabe ◽  
Shigetaka Kitajima ◽  
Kinji Tsukada ◽  
Yukio Yasukochi ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Rna Polymerase Ii ◽  
Tata Binding Protein ◽  
Dependent Protein Kinase ◽  
General Transcription Factors ◽  
Transcription Factor Iib ◽  
Dependent Protein ◽  
Transcription In Vitro ◽  
Stimulation Of

scholarly journals Mutational analysis of the D1/E1 core helices and the conserved N-terminal region of yeast transcription factor IIB (TFIIB): identification of an N-terminal mutant that stabilizes TATA-binding protein-TFIIB-DNA complexes.

1997 ◽  
Vol 17 (12) ◽  
pp. 6784-6793 ◽  
Author(s):  
C S Bangur ◽  
T S Pardee ◽  
A S Ponticelli
Keyword(s):  
Transcription Factor ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Binding Protein ◽  
Tata Binding Protein ◽  
Terminal Region ◽  
Amino Terminal ◽  
Transcription Factor Iib

The general transcription factor IIB (TFIIB) plays an essential role in transcription of protein-coding genes by RNA polymerase II. We have used site-directed mutagenesis to assess the role of conserved amino acids in several important regions of yeast TFIIB. These include residues in the highly conserved amino-terminal region and basic residues in the D1 and E1 core domain alpha-helices. Acidic substitutions of residues K190 (D1) and K201 (E1) resulted in growth impairments in vivo, reduced basal transcriptional activity in vitro, and an inability to form stable TFIIB-TATA-binding protein-DNA (DB) complexes. Significantly, these mutants retained the ability to respond to acidic activators in vivo and to the Gal4-VP16 activator in vitro, supporting the view that these basic residues play a role in basal transcription. In addition, 14 single-amino-acid substitutions were introduced in the conserved amino-terminal region. Three of these mutants, the L50D, R64E, and R78L mutants, displayed altered growth properties in vivo and were compromised for supporting transcription in vitro. The L50D mutant was impaired for RNA polymerase II interaction, while the R64E mutant exhibited altered transcription start site selection both in vitro and in vivo and, surprisingly, was more active than the wild type in the formation of stable DB complexes. These results support the view that the amino-terminal domain is involved in the direct interaction between yeast TFIIB and RNA polymerase II and suggest that this domain may interact with DNA and/or modulate the formation of a DB complex.

The general transcription factor RAP30 binds to RNA polymerase II and prevents it from binding nonspecifically to DNA

1992 ◽  
Vol 12 (1) ◽  
pp. 30-37
Author(s):  
M T Killeen ◽  
J F Greenblatt
Keyword(s):  
Transcription Factor ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Small Subunit ◽  
Glutathione S Transferase ◽  
General Transcription Factor ◽  
Indirect Consequence ◽  
Transcription In Vitro ◽  
Sigma 70

RAP30/74 is a human general transcription factor that binds to RNA polymerase II and is required for initiation of transcription in vitro regardless of whether the promoter has a recognizable TATA box (Z. F. Burton, M. Killeen, M. Sopta, L. G. Ortolan, and J. F. Greenblatt, Mol. Cell. Biol. 8:1602-1613, 1988). Part of the amino acid sequence of RAP30, the small subunit of RAP30/74, has limited homology with part of Escherichia coli sigma 70 (M. Sopta, Z. F. Burton, and J. Greenblatt, Nature (London) 341:410-414, 1989). To determine which sigmalike activities of RAP30/74 could be attributed to RAP30, we purified human RAP30 and a RAP30-glutathione-S-transferase fusion protein that had been produced in E. coli. Bacterially produced RAP30 bound to RNA polymerase II in the absence of RAP74. Both partially purified natural RAP30/74 and recombinant RAP30 prevented RNA polymerase II from binding nonspecifically to DNA. In addition, nonspecific transcription by RNA polymerase II was greatly inhibited by RAP30-glutathione-S-transferase. DNA-bound RNA polymerase II could be removed from DNA by partially purified RAP30/74 but not by bacterially expressed RAP30. Thus, the ability of RAP30/74 to recruit RNA polymerase II to a promoter-bound preinitiation complex may be an indirect consequence of its ability to suppress nonspecific binding of RNA polymerase II to DNA.

Interaction of the TFIIB zinc ribbon with RNA polymerase II

2008 ◽  
Vol 36 (4) ◽  
pp. 595-598 ◽  
Author(s):  
Laura M. Elsby ◽  
Stefan G.E. Roberts
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Structural Models ◽  
Initiation Complex ◽  
General Transcription Factors ◽  
Transcription Factor Iib ◽  
General Transcription Factor ◽  
Zinc Ribbon

Transcription by RNA polymerase II requires the assembly of the general transcription factors at the promoter to form a pre-initiation complex. The general transcription factor TF (transcription factor) IIB plays a central role in the assembly of the pre-initiation complex, providing a bridge between promoter-bound TFIID and RNA polymerase II/TFIIF. We have characterized a series of TFIIB mutants in their ability to support transcription and recruit RNA polymerase II to the promoter. Our analyses identify several residues within the TFIIB zinc ribbon that are required for RNA polymerase II assembly. Using the structural models of TFIIB, we describe the interface between the TFIIB zinc ribbon region and RNA polymerase II.

scholarly journals Heterogeneous nuclear ribonucleoprotein K is a transcription factor.

1996 ◽  
Vol 16 (5) ◽  
pp. 2350-2360 ◽  
Author(s):  
E F Michelotti ◽  
G A Michelotti ◽  
A I Aronsohn ◽  
D Levens
Keyword(s):  
Transcription Factor ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Hnrnp K ◽  
Heterogeneous Nuclear Ribonucleoprotein ◽  
Transcription In Vitro ◽  
Nuclear Ribonucleoprotein ◽  
Rna Polymerase Ii Transcription

The CT element is a positively acting homopyrimidine tract upstream of the c-myc gene to which the well-characterized transcription factor Spl and heterogeneous nuclear ribonucleoprotein (hnRNP) K, a less well-characterized protein associated with hnRNP complexes, have previously been shown to bind. The present work demonstrates that both of these molecules contribute to CT element-activated transcription in vitro. The pyrimidine-rich strand of the CT element both bound to hnRNP K and competitively inhibited transcription in vitro, suggesting a role for hnRNP K in activating transcription through this single-stranded sequence. Direct addition of recombinant hnRNP K to reaction mixtures programmed with templates bearing single-stranded CT elements increased specific RNA synthesis. If hnRNP K is a transcription factor, then interactions with the RNA polymerase II transcription apparatus are predicted. Affinity columns charged with recombinant hnRNP K specifically bind a component(s) necessary for transcription activation. The depleted factors were biochemically complemented by a crude TFIID phosphocellulose fraction, indicating that hnRNP K might interact with the TATA-binding protein (TBP)-TBP-associated factor complex. Coimmunoprecipitation of a complex formed in vivo between hnRNP K and epitope-tagged TBP as well as binding in vitro between recombinant proteins demonstrated a protein-protein interaction between TBP and hnRNP K. Furthermore, when the two proteins were overexpressed in vivo, transcription from a CT element-dependent reporter was synergistically activated. These data indicate that hnRNP K binds to a specific cis element, interacts with the RNA polymerase II transcription machinery, and stimulates transcription and thus has all of the properties of a transcription factor.

scholarly journals Downstream promoter interactions of TFIID TAFs facilitate transcription reinitiation

2017 ◽  
Author(s):  
Yoo Jin Joo ◽  
Scott B. Ficarro ◽  
Luis M. Soares ◽  
Yujin Chun ◽  
Jarrod A. Marto ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Rna Polymerase Ii ◽  
In Vitro Transcription ◽  
Tata Binding Protein ◽  
Transcription Reinitiation ◽  
Promoter Dna ◽  
Basal Transcription Factors ◽  
Necessary And Sufficient

AbstractTFIID binds promoter DNA to recruit RNA polymerase II and other basal factors for transcription. Although the TATA-Binding Protein (TBP) subunit of TFIID is necessary and sufficient for in vitro transcription, the TBP-Associated Factor (TAF) subunits recognize downstream promoter elements, act as co-activators, and interact with nucleosomes. Here we show that transcription induces stable TAF binding to downstream promoter DNA, independent of upstream contacts, TBP, or other basal transcription factors. This transcription-dependent TAF complex promotes subsequent activator-independent transcription, and promoter response to TAF mutations in vivo correlates with the level of downstream, rather than overall, Taf1 crosslinking. We propose a new model in which TAFs function as reinitiation factors, accounting for the differential responses of promoters to various transcription factor mutations.

scholarly journals Influence of the phosphorylation state of neurofilament proteins on the interactions between purified filaments in vitro

1988 ◽  
Vol 252 (3) ◽  
pp. 655-660 ◽  
Author(s):  
J Eyer ◽  
J F Leterrier
Keyword(s):  
Direct Relationship ◽  
Functional Heterogeneity ◽  
Neurofilament Proteins ◽  
Dependent Protein Kinase ◽  
Phosphorylation Level ◽  
Progressive Loss ◽  
Dependent Protein ◽  
Phosphate Groups ◽  
Stimulation Of

The extensive enzymic dephosphorylation of neurofilaments determined the progressive loss of their capacity to interconnect in vitro into a reticulated network, measured by the formation of highly viscous gels in purified preparations of neurofilaments [Leterrier & Eyer (1987) Biochem. J. 245, 93-101]. Conversely, a cyclic AMP-dependent activation of the gelation process was obtained by phosphorylation of the neurofilament proteins by the cyclic-nucleotide-dependent protein kinase added to the preparation. These findings argue for a direct relationship between the high phosphorylation level of the neurofilament subunits and the cross-bridging of the polymers in vitro. However, a transient stimulation of the neurofilament viscosity kinetics was also observed during the early steps of dephosphorylation with acid phosphatase, which, moreover, disappeared with longer incubation times before the net inhibition was obtained. In the same way, the calmodulin-dependent brain phosphatase, calcineurin, induced a permanent activation of the phenomenon, correlated with a low dephosphorylation capacity of the neurofilament molecules. Taken together, these results suggest a functional heterogeneity of the numerous phosphate groups of the neurofilament subunits and raise the hypothesis of a highly controlled regulation of the neurofilament cross-bridging by selective phosphorylation-dephosphorylation mechanisms.

scholarly journals Activation of protein kinase B β and γ isoforms by insulin in vivo and by 3-phosphoinositide-dependent protein kinase-1 in vitro: comparison with protein kinase B α

1998 ◽  
Vol 331 (1) ◽  
pp. 299-308 ◽  
Author(s):  
Kay S. WALKER ◽  
Maria DEAK ◽  
Andrew PATERSON ◽  
Kevin HUDSON ◽  
Philip COHEN ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Protein Kinase ◽  
Protein Kinase B ◽  
Rat Hepatocytes ◽  
Dependent Protein Kinase ◽  
L6 Myotubes ◽  
Dependent Protein ◽  
Stimulation Of

The regulatory and catalytic properties of the three mammalian isoforms of protein kinase B (PKB) have been compared. All three isoforms (PKBα, PKBβ and PKBγ) were phosphorylated at similar rates and activated to similar extents by 3-phosphoinositide-dependent protein kinase-1 (PDK1). Phosphorylation and activation of each enzyme required the presence of PtdIns(3,4,5)P3 or PtdIns(3,4)P2, as well as PDK1. The activation of PKBβ and PKBγ by PDK1 was accompanied by the phosphorylation of the residues equivalent to Thr308 in PKBα, namely Thr309 (PKBβ) and Thr305 (PKBγ). PKBγ which had been activated by PDK1 possessed a substrate specificity identical with that of PKBα and PKBβ towards a range of peptides. The activation of PKBγ and its phosphorylation at Thr305 was triggered by insulin-like growth factor-1 in 293 cells. Stimulation of rat adipocytes or rat hepatocytes with insulin induced the activation of PKBα and PKBβ with similar kinetics. After stimulation of adipocytes, the activity of PKBβ was twice that of PKBα, but in hepatocytes PKBα activity was four-fold higher than PKBβ. Insulin induced the activation of PKBα in rat skeletal muscle in vivo, with little activation of PKBβ. Insulin did not induce PKBγ activity in adipocytes, hepatocytes or skeletal muscle, but PKBγ was the major isoform activated by insulin in rat L6 myotubes (a skeletal-muscle cell line).

Direct cleavage of human TATA-binding protein by poliovirus protease 3C in vivo and in vitro

1993 ◽  
Vol 13 (2) ◽  
pp. 1232-1237
Author(s):  
M E Clark ◽  
P M Lieberman ◽  
A J Berk ◽  
A Dasgupta
Keyword(s):  
Transcription Factor ◽  
Host Cell ◽  
Rna Polymerase Ii ◽  
Binding Protein ◽  
Tata Binding Protein ◽  
Significant Inhibition ◽  
Pol Ii ◽  
Poliovirus Infection

Host cell RNA polymerase II (Pol II)-mediated transcription is inhibited by poliovirus infection. This inhibition is correlated to a specific decrease in the activity of a chromatographic fraction which contains the transcription factor TFIID. To investigate the mechanism by which poliovirus infection results in a decrease of TFIID activity, we have analyzed a component of TFIID, the TATA-binding protein (TBP). Using Western immunoblot analysis, we show that TBP is cleaved in poliovirus-infected cells at the same time postinfection as when Pol II transcription is inhibited. Further, we show that one of the cleaved forms of TBP can be reproduced in vitro by incubating TBP with cloned, purified poliovirus encoded protease 3C. Protease 3C is a poliovirus-encoded protease that specifically cleaves glutamine-glycine bonds in the viral polyprotein. The cleavage of TBP by protease 3C occurs directly. Finally, incubation of an uninfected cell-derived TBP-containing fraction (TFIID) with protease 3C results in significant inhibition of Pol II-mediated transcription in vitro. These results demonstrate that a cellular transcription factor can be directly cleaved both in vitro and in vivo by a viral protease and suggest a role of the poliovirus proteinase 3C in host cell Pol II-mediated transcription shutoff.

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