Acanthamoeba castellanii RNA polymerase II transcription in vitro: accurate initiation at the adenovirus major late promoter

Kinetic analyses of runoff transcription in a cell-free eucaryotic transcription system revealed that the bacteriophage lambda 4S RNA terminator caused human RNA polymerase II to pause on the template and partially terminate transcription of transcripts initiated by the adenovirus 2 major late promoter. Analogous to the procaryotic RNA polymerase, the eucaryotic enzyme terminated just beyond the guanine-plus-cytosine-rich region of dyad symmetry in the terminator sequence. These results suggest that the eucaryotic RNA polymerase II may respond to transcription termination sequences similar to those used by the procaryotic enzyme. However, similar templates containing lambda tint or lambda tR1 terminators did not elicit pausing or termination, suggesting that other features, such as sequence specificity, may also be involved.

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Transcription initiation complexes and upstream activation with RNA polymerase II lacking the C-terminal domain of the largest subunit

Molecular and Cellular Biology ◽

10.1128/mcb.10.10.5562-5564.1990 ◽

1990 ◽

Vol 10 (10) ◽

pp. 5562-5564

Author(s):

S Buratowski ◽

P A Sharp

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Transcription Initiation ◽

Adenovirus Type ◽

Late Promoter ◽

Terminal Domain ◽

Late Transcription ◽

Major Late Promoter

RNA polymerase II assembles with other factors on the adenovirus type 2 major late promoter to generate pairs of transcription initiation complexes resolvable by nondenaturing gel electrophoresis. The pairing of the complexes is caused by the presence or absence of the C-terminal domain of the largest subunit. This domain is not required for transcription stimulation by the major late transcription factor in vitro.

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[38] RNA polymerase II transcription in vitro

Guide to Yeast Genetics and Molecular Biology - Methods in Enzymology ◽

10.1016/0076-6879(91)94041-a ◽

1991 ◽

pp. 545-550 ◽

Cited By ~ 15

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

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Pausing and termination of human RNA polymerase II transcription at a procaryotic terminator.

Molecular and Cellular Biology ◽

10.1128/mcb.3.10.1687 ◽

1983 ◽

Vol 3 (10) ◽

pp. 1687-1693 ◽

Cited By ~ 6

Author(s):

G W Hatfield ◽

J A Sharp ◽

M Rosenberg

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Sequence Specificity ◽

Late Promoter ◽

Transcription System ◽

A Cell ◽

Major Late Promoter ◽

Dyad Symmetry ◽

Rna Polymerase Ii Transcription ◽

Terminator Sequence

Kinetic analyses of runoff transcription in a cell-free eucaryotic transcription system revealed that the bacteriophage lambda 4S RNA terminator caused human RNA polymerase II to pause on the template and partially terminate transcription of transcripts initiated by the adenovirus 2 major late promoter. Analogous to the procaryotic RNA polymerase, the eucaryotic enzyme terminated just beyond the guanine-plus-cytosine-rich region of dyad symmetry in the terminator sequence. These results suggest that the eucaryotic RNA polymerase II may respond to transcription termination sequences similar to those used by the procaryotic enzyme. However, similar templates containing lambda tint or lambda tR1 terminators did not elicit pausing or termination, suggesting that other features, such as sequence specificity, may also be involved.

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Role of the mammalian transcription factors IIF, IIS, and IIX during elongation by RNA polymerase II.

Molecular and Cellular Biology ◽

10.1128/mcb.11.3.1195 ◽

1991 ◽

Vol 11 (3) ◽

pp. 1195-1206 ◽

Cited By ~ 94

Author(s):

E Bengal ◽

O Flores ◽

A Krauskopf ◽

D Reinberg ◽

Y Aloni

Keyword(s):

Transcription Factors ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Gel Filtration ◽

Late Promoter ◽

Elongation Complex ◽

Cell Extracts ◽

Adenovirus Major Late Promoter ◽

Major Late Promoter ◽

Transcription Complexes

We have used a recently developed system that allows the isolation of complexes competent for RNA polymerase II elongation (E. Bengal, A. Goldring, and Y. Aloni, J. Biol. Chem. 264:18926-18932, 1989). Pulse-labeled transcription complexes were formed at the adenovirus major late promoter with use of HeLa cell extracts. Elongation-competent complexes were purified from most of the proteins present in the extract, as well as from loosely bound elongation factors, by high-salt gel filtration chromatography. We found that under these conditions the nascent RNA was displaced from the DNA during elongation. These column-purified complexes were used to analyze the activities of different transcription factors during elongation by RNA polymerase II. We found that transcription factor IIS (TFIIS), TFIIF, and TFIIX affected the efficiency of elongation through the adenovirus major late promoter attenuation site and a synthetic attenuation site composed of eight T residues. These factors have distinct activities that depend on whether they are added before RNA polymerase has reached the attenuation site or at the time when the polymerase is pausing at the attenuation site. TFIIS was found to have antiattenuation activity, while TFIIF and TFIIX stimulated the rate of elongation. In comparison with TFIIF, TFIIS is loosely bound to the elongation complex. We also found that the activities of the factors are dependent on the nature of the attenuator. These results indicate that at least three factors play a major role during elongation by RNA polymerase II.

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Heterogeneous nuclear ribonucleoprotein K is a transcription factor.

Molecular and Cellular Biology ◽

10.1128/mcb.16.5.2350 ◽

1996 ◽

Vol 16 (5) ◽

pp. 2350-2360 ◽

Cited By ~ 243

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.

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