Specific small nuclear RNAs from SV40-transformed cells stimulate transcription initiation in nontransformed isolated nuclei

1982 ◽  
Vol 60 (3) ◽  
pp. 252-262 ◽  
Author(s):  
Maurice J. Ringuette ◽  
Karen Gordon ◽  
Jolanta Szyszko ◽  
Margarida O. Krause
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Transformed Cells ◽  
Regulation Of Transcription ◽  
Cell Nuclei ◽  
Small Nuclear Rnas ◽  
Mouse Cells ◽  
3T3 Cell Lines

Previous studies in our laboratory have implicated small nuclear RNAs (SnRNA) in the regulation of transcription in isolated mammalian cell nuclei. The present investigation was designed to develop a transcription assay system using isolated intact nuclei with optimized RNA polymerase II activity which would be capable of reinitiation in vitro to study the mode of action of the "active" RNA.We used nuclei isolated from either human WI38 or Balb 3T3 mouse cells to test the activity of SnRNA purified from SV40-transformed WI38 or 3T3 cell lines. These systems were found to support transcription up to 60 min, 40–60% of which was polymerase II dependent. In vitro initiations were detected by [γ-32P]ATP incorporation as well as by Hg-Sepharose chromatography using (γ-S)ATP as substrate. Results supported the following conclusions: (a) SnRNA from transformed cells stimulates the transcriptional activity of nontransformed nuclei while homologous SnRNA has little or no activity; (b) the stimulation is NaOH-sensitive and is dependent on RNA polymerase II since it is eliminated by 1 μg/mL α-amanitin; (c) the active subfraction of SnRNA from mouse cells was found to be of identical size (320–350 nucleotides) to that previously identified in human and monkey cells; and (d) analysis of the transcripts obtained from control and stimulated cell nuclei revealed that SnRNA activity is due primarily to an increase in the number of initiated chains.

Non-coding RNA in transcription initiation

2006 ◽  
Vol 73 ◽  
pp. 131-140 ◽  
Author(s):  
William O'Gorman ◽  
Kon Yew Kwek ◽  
Benjamin Thomas ◽  
Alexandre Akoulitchev
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
New Technologies ◽  
Initiation Factor ◽  
Regulation Of Transcription ◽  
Transcriptional Elongation ◽  
Ribonucleoprotein Complexes ◽  
U1 Snrna

Diverse classes of non-coding RNAs, including snRNAs (small nuclear RNAs), play fundamental regulatory roles in gene expression. For example, 7SK RNA and the components of the splicing apparatus U1–U6 snRNAs are implicated in the regulation of transcriptional elongation. The first evidence for the involvement of RNA in the regulation of transcriptional initiation is now emerging. TFIIH (transcription factor IIH), a general transcription initiation factor, appears to associate specifically with U1 snRNA, a core splicing component. Reconstituted transcription in vitro demonstrates an increase in the rate of formation of the first phosphodiester bond by RNA polymerase II in presence of U1 snRNA. Reconstituted re-initiation is also stimulated by U1 snRNA. These results suggest that U1 snRNA functions in the regulation of transcription by RNA polymerase II in addition to its role in RNA processing. The implications of these data extend to the development of new technologies that will allow the identification and analysis of diverse RNA species present as regulatory components in transcription-related ribonucleoprotein complexes.

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

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.

scholarly journals A Nonconserved Surface of the TFIIB Zinc Ribbon Domain Plays a Direct Role in RNA Polymerase II Recruitment

2004 ◽  
Vol 24 (7) ◽  
pp. 2863-2874 ◽  
Author(s):  
Thomas C. Tubon ◽  
William P. Tansey ◽  
Winship Herr
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Terminal Region ◽  
General Role ◽  
Pol Ii ◽  
Amino Terminal ◽  
Zinc Ribbon

ABSTRACT The general transcription factor TFIIB is a highly conserved and essential component of the eukaryotic RNA polymerase II (pol II) transcription initiation machinery. It consists of a single polypeptide with two conserved structural domains: an amino-terminal zinc ribbon structure (TFIIBZR) and a carboxy-terminal core (TFIIBCORE). We have analyzed the role of the amino-terminal region of human TFIIB in transcription in vivo and in vitro. We identified a small nonconserved surface of the TFIIBZR that is required for pol II transcription in vivo and for different types of basal pol II transcription in vitro. Consistent with a general role in transcription, this TFIIBZR surface is directly involved in the recruitment of pol II to a TATA box-containing promoter. Curiously, although the amino-terminal human TFIIBZR domain can recruit both human pol II and yeast (Saccharomyces cerevisiae) pol II, the yeast TFIIB amino-terminal region recruits yeast pol II but not human pol II. Thus, a critical process in transcription from many different promoters—pol II recruitment—has changed in sequence specificity during eukaryotic evolution.

scholarly journals RNA polymerase II elongation complexes paused after the synthesis of 15- or 35-base transcripts have different structures

1991 ◽  
Vol 11 (3) ◽  
pp. 1508-1522
Author(s):  
S C Linn ◽  
D S Luse
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Adenovirus Type ◽  
Dnase I ◽  
Transcription Complexes ◽  
Adenovirus Type 2

We have purified specific RNA polymerase II elongation intermediates initiated at the adenovirus type 2 major late promoter and paused either 15 or 35 to 36 bases downstream of the transcription initiation site. Transcription was arrested at these two sites by combining modification of the promoter sequence with limitation of appropriate nucleotide concentrations in the in vitro reaction. The resultant complexes were remarkably stable and could be purified away from free DNA and contaminating protein-DNA complexes, without loss of activity, by the use of sucrose gradient sedimentation and low-ionic-strength polyacrylamide gel electrophoresis. The complexes were characterized by both DNase I and o-phenanthroline-copper ion nuclease protection assays. The DNase I footprints revealed that the structures of the 15- and 35- to 36-nucleotide transcription complexes differed from those previously reported for an adenovirus type 2 major late preinitiation complex and a subsequent intermediate formed upon addition of ATP. Furthermore, the 35- to 36-nucleotide complex protected a significantly smaller portion of the template than the 15-nucleotide species and migrated at a slightly higher rate in polyacrylamide gels. These observations suggest that changes in structural organization may continue to occur in transcription complexes which are already committed to elongation.

scholarly journals ScRpb4, Encoding an RNA Polymerase Subunit from Sugarcane, Is Ubiquitously Expressed and Resilient to Changes in Response to Stress Conditions

Agriculture ◽  
2022 ◽  
Vol 12 (1) ◽  
pp. 81
Author(s):  
Taehoon Kim ◽  
Fábio Ometto Dias ◽  
Agustina Gentile ◽  
Marcelo Menossi ◽  
Kevin Begcy
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Transcriptional Response ◽  
Stress Conditions ◽  
Initiation Complex ◽  
Initial Characterization ◽  
Transcription Initiation Complex

RNA polymerase II is an essential multiprotein complex that transcribes thousands of genes, being a fundamental component of the transcription initiation complex. In eukaryotes, RNA polymerase II is formed by a 10-multisubunit conserved core complex, and two additional peripheral subunits, Rpb4 and Rpb7, form the Rpb4/7 subcomplex. Although transcription is vital for cell and organismal viability, little is known about the transcription initiation complex in sugarcane. An initial characterization of the sugarcane RNA polymerase subunit IV (ScRpb4) was performed. Our results demonstrate that ScRpb4 is evolutionarily conserved across kingdoms. At the molecular level, ScRpb4 expression was found in vegetative and reproductive tissues. Furthermore, the expression of ScRpb4 remained stable under various stress conditions, most likely to ensure a proper transcriptional response. Optimal conditions to express ScRpb4 in vitro for further studies were also identified. In this study, an initial characterization of the sugarcane polymerase II subunit IV is presented. Our results open the window to more specific experiments to study ScRpb4 function, for instance, crystal structure determination and pull-down assays as well as their function under biotic and abiotic stresses.

The C-terminal domain of the largest subunit of RNA polymerase II and transcription initiation

1989 ◽  
Vol 9 (12) ◽  
pp. 5750-5753
Author(s):  
M Moyle ◽  
J S Lee ◽  
W F Anderson ◽  
C J Ingles
Keyword(s):  
Monoclonal Antibodies ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Initiation Factor ◽  
Transcription Initiation Factor ◽  
Evolutionarily Conserved ◽  
Repeat Domain ◽  
Initiation Of Transcription

Monoclonal antibodies specific for the evolutionarily conserved C-terminal heptapeptide repeat domain of the largest subunit of RNA polymerase II inhibited the initiation of transcription from mammalian promoters in vitro. Since these antibodies did not inhibit elongation and randomly initiated transcription, the heptapeptide repeats may function by binding class II transcription initiation factor(s).

Assembly of RNA polymerase II preinitiation complexes before assembly of nucleosomes allows efficient initiation of transcription on nucleosomal templates

1988 ◽  
Vol 8 (8) ◽  
pp. 3114-3121
Author(s):  
J A Knezetic ◽  
G A Jacob ◽  
D S Luse
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Chromatin Assembly ◽  
Nucleosome Assembly ◽  
Preinitiation Complex ◽  
Naked Dna ◽  
Initiation Of Transcription ◽  
Dna Template

We have previously shown that assembly of nucleosomes on the DNA template blocks transcription initiation by RNA polymerase II in vitro. In the studies reported here, we demonstrate that assembly of a complete RNA polymerase II preinitiation complex before nucleosome assembly results in nucleosomal templates which support initiation in vitro as efficiently as naked DNA. Control experiments prove that our observations are not the result of slow displacement of nucleosomes by the transcription machinery during chromatin assembly, nor are they an artifact of inefficient nucleosome deposition on templates already bearing an RNA polymerase. Thus, the RNA polymerase II preinitiation complex appears to be resistant to disruption by subsequent nucleosome assembly.

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