Synthetic octapeptide pyroGLU-ASP-ASP-SER-ASP-GLU-GLU-ASN controls DNA transcription in vitro by RNA polymerase II

1993 ◽  
Vol 49 (10) ◽  
pp. 902-905 ◽  
Author(s):  
A. Angiolillo ◽  
A. Desgro ◽  
V. Marsili ◽  
F. Panara ◽  
G. L. Gianfranceschi
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Dna Transcription ◽  
Transcription In Vitro

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.

[38] RNA polymerase II transcription in vitro

1991 ◽  
pp. 545-550 ◽  
Author(s):  
Neal F. Lue ◽  
Peter M. Flanagan ◽  
Raymond J. Kelleher ◽  
Aled M. Edwards ◽  
Roger D. Kornberg
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription In Vitro ◽  
Rna Polymerase Ii Transcription

Mechanism of RNA polymerase II-specific initiation of transcription in vitro: ATP requirement and uncapped runoff transcripts

Cell ◽  
1982 ◽  
Vol 29 (3) ◽  
pp. 877-886 ◽  
Author(s):  
David Bunick ◽  
Ruben Zandomeni ◽  
Steven Ackerman ◽  
Roberto Weinmann
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Initiation Of Transcription ◽  
Transcription In Vitro

scholarly journals In vitro initiation of transcription by RNA polymerase II on in vivo-assembled chromatin templates.

1992 ◽  
Vol 12 (4) ◽  
pp. 1639-1651 ◽  
Author(s):  
S C Batson ◽  
R Sundseth ◽  
C V Heath ◽  
M Samuels ◽  
U Hansen
Keyword(s):  
Transcription Factors ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Viral Dna ◽  
Dna Templates ◽  
Initiation Of Transcription ◽  
Transcription In Vitro ◽  
Alpha Amanitin

We have studied the initiation of transcription in vitro by RNA polymerase II on simian virus 40 (SV40) minichromosomal templates isolated from infected cells. The efficiency and pattern of transcription from the chromatin templates were compared with those from viral DNA templates by using two in vitro transcription systems, either HeLa whole-cell extract or basal transcription factors, RNA polymerase II, and one of two SV40 promoter-binding transcription factors, LSF and Sp1. Dramatic increases in numbers of transcripts upon addition of transcription extract and different patterns of usage of the multiple SV40 initiation sites upon addition of Sp1 versus LSF strongly suggested that transcripts were being initiated from the minichromosomal templates in vitro. That the majority of transcripts from the minichromosomes were due to initiation de novo was demonstrated by the efficient transcription observed in the presence of alpha-amanitin, which inhibited minichromosome-associated RNA polymerase II, and an alpha-amanitin-resistant RNA polymerase II, which initiated transcription in vitro. The pattern of transcription from the SV40 late and early promoters on the minichromosomal templates was similar to the in vivo pattern of transcription during the late stages of viral infection and was distinct from the pattern of transcription generated from viral DNA in vitro. In particular, the late promoter of the minichromosomal templates was transcribed with high efficiency, similar to viral DNA templates, while the early-early promoter of the minichromosomal templates was inhibited 10- to 15-fold. Finally, the number of minichromosomes competent to initiate transcription in vitro exceeded the amount actively being transcribed in vivo.

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 Uncoupling of initiation and reinitiation rates during HeLa RNA polymerase II transcription in vitro.

1993 ◽  
Vol 13 (8) ◽  
pp. 4572-4577 ◽  
Author(s):  
Y Jiang ◽  
J D Gralla
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Promoter Activity ◽  
Atp Hydrolysis ◽  
Preinitiation Complex ◽  
Transcription In Vitro ◽  
Rna Polymerase Ii Transcription

RNA polymerase II transcription is influenced both by how rapidly a gene is induced and by the rate at which continuous reinitiation occurs after induction. We show here that in vitro the rates of these two critical steps need not be the same. For activator GAL-AH-dependent HeLa transcription, the rate of assembling a preinitiation complex is significantly slower than the rate of reinitiation. Although reinitiation is rapid, it still requires ATP hydrolysis. This unexpected uncoupling of the rates of initiation and reinitiation implies that in regulating mammalian promoter activity, one must consider separately the controls on initiation during induction and the controls on the subsequent reinitiation events.

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