scholarly journals The Human Isoform of RNA Polymerase II Subunit hRPB11bα Specifically Interacts with Transcription Factor ATF4

2019 ◽  
Vol 21 (1) ◽  
pp. 135 ◽  
Author(s):  
Sergey A. Proshkin ◽  
Elena K. Shematorova ◽  
George V. Shpakovski
Keyword(s):  
Transcription Factor ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Fetal Brain ◽  
Gene Promoters ◽  
Α Subunit ◽  
Promoter Recognition ◽  
Rnap Ii ◽  
Human Specific

Rpb11 subunit of RNA polymerase II of Eukaryotes is related to N-terminal domain of eubacterial α subunit and forms a complex with Rpb3 subunit analogous to prokaryotic α2 homodimer, which is involved in RNA polymerase assembly and promoter recognition. In humans, a POLR2J gene family has been identified that potentially encodes several hRPB11 proteins differing mainly in their short C-terminal regions. The functions of the different human specific isoforms are still mainly unknown. To further characterize the minor human specific isoform of RNA polymerase II subunit hRPB11bα, the only one from hRPB11 (POLR2J) homologues that can replace its yeast counterpart in vivo, we used it as bait in a yeast two-hybrid screening of a human fetal brain cDNA library. By this analysis and subsequent co-purification assay in vitro, we identified transcription factor ATF4 as a prominent partner of the minor RNA polymerase II (RNAP II) subunit hRPB11bα. We demonstrated that the hRPB11bα interacts with leucine b-Zip domain located on the C-terminal part of ATF4. Overexpression of ATF4 activated the reporter more than 10-fold whereas co-transfection of hRPB11bα resulted in a 2.5-fold enhancement of ATF4 activation. Our data indicate that the mode of interaction of human RNAP II main (containing major for of hRPB11 subunit) and minor (containing hRPB11bα isoform of POLR2J subunit) transcription enzymes with ATF4 is certainly different in the two complexes involving hRPB3–ATF4 (not hRPB11a–ATF4) and hRpb11bα–ATF4 platforms in the first and the second case, respectively. The interaction of hRPB11bα and ATF4 appears to be necessary for the activation of RNA polymerase II containing the minor isoform of the hRPB11 subunit (POLR2J) on gene promoters regulated by this transcription factor. ATF4 activates transcription by directly contacting RNA polymerase II in the region of the heterodimer of α-like subunits (Rpb3–Rpb11) without involving a Mediator, which provides fast and highly effective activation of transcription of the desired genes. In RNA polymerase II of Homo sapiens that contains plural isoforms of the subunit hRPB11 (POLR2J), the strength of the hRPB11–ATF4 interaction appeared to be isoform-specific, providing the first functional distinction between the previously discovered human forms of the Rpb11 subunit.

scholarly journals Functional dissection of the catalytic mechanism of mammalian RNA polymerase II

2005 ◽  
Vol 83 (4) ◽  
pp. 497-504 ◽  
Author(s):  
Benoit Coulombe ◽  
Marie-France Langelier
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Catalytic Mechanism ◽  
Mutational Analysis ◽  
Structural Elements ◽  
Catalytic Mechanisms ◽  
Rnap Ii ◽  
Affinity Peptide

High resolution X-ray crystal structures of multisubunit RNA polymerases (RNAP) have contributed to our understanding of transcriptional mechanisms. They also provided a powerful guide for the design of experiments aimed at further characterizing the molecular stages of the transcription reaction. Our laboratory used tandem-affinity peptide purification in native conditions to isolate human RNAP II variants that had site-specific mutations in structural elements located strategically within the enzyme's catalytic center. Both in vitro and in vivo analyses of these mutants revealed novel features of the catalytic mechanisms involving this enzyme.Key words: RNA polymerase II, transcriptional mechanisms, mutational analysis, mRNA synthesis.

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.

scholarly journals Human Cyclin K, a Novel RNA Polymerase II-Associated Cyclin Possessing Both Carboxy-Terminal Domain Kinase and Cdk-Activating Kinase Activity

1998 ◽  
Vol 18 (7) ◽  
pp. 4291-4300 ◽  
Author(s):  
Michael C. Edwards ◽  
Calvin Wong ◽  
Stephen J. Elledge
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Cell Cycle Progression ◽  
Large Subunit ◽  
Terminal Domain ◽  
Gene Coding ◽  
Acid Protein ◽  
Rnap Ii ◽  
Carboxy Terminal

ABSTRACT The gene coding for human cyclin K was isolated as aCPR (cell-cycle progression restoration) gene by virtue of its ability to impart a Far− phenotype to the budding yeast Saccharomyces cerevisiae and to rescue the lethality of a deletion of the G1 cyclin genes CLN1,CLN2, and CLN3. The cyclin K gene encodes a 357-amino-acid protein most closely related to human cyclins C and H, which have been proposed to play a role in regulating basal transcription through their association with and activation of cyclin-dependent kinases (Cdks) that phosphorylate the carboxyl-terminal domain (CTD) of the large subunit of RNA polymerase II (RNAP II). Murine and Drosophila melanogaster homologs of cyclin K have also been identified. Cyclin K mRNA is ubiquitously expressed in adult mouse and human tissues, but is most abundant in the developing germ cells of the adult testis and ovaries. Cyclin K is associated with potent CTD kinase and Cdk kinase (CAK) activity in vitro and coimmunoprecipitates with the large subunit of RNAP II. Thus, cyclin K represents a new member of the “transcription” cyclin family which may play a dual role in regulating Cdk and RNAP II activity.

scholarly journals FCP1, a Phosphatase Specific for the Heptapeptide Repeat of the Largest Subunit of RNA Polymerase II, Stimulates Transcription Elongation

2002 ◽  
Vol 22 (21) ◽  
pp. 7543-7552 ◽  
Author(s):  
Subhrangsu S. Mandal ◽  
Helen Cho ◽  
Sungjoon Kim ◽  
Kettly Cabane ◽  
Danny Reinberg
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Elongation ◽  
Carboxy Terminal Domain ◽  
Transcription Factor Iif ◽  
Transcript Elongation ◽  
Rnap Ii ◽  
Carboxy Terminal

ABSTRACT FCP1, a phosphatase specific for the carboxy-terminal domain of RNA polymerase II (RNAP II), was found to stimulate transcript elongation by RNAP II in vitro and in vivo. This activity is independent of and distinct from the elongation-stimulatory activity associated with transcription factor IIF (TFIIF), and the elongation effects of TFIIF and FCP1 were found to be additive. Genetic experiments resulted in the isolation of several distinct fcp1 alleles. One of these alleles was found to suppress the slow-growth phenotype associated with either the reduction of intracellular nucleotide concentrations or the inhibition of other transcription elongation factors. Importantly, this allele of fcp1 was found to be lethal when combined individually with two mutations in the second-largest subunit of RNAP II, which had been shown previously to affect transcription elongation.

scholarly journals Repression of TFIIH Transcriptional Activity and TFIIH-Associated cdk7 Kinase Activity at Mitosis

1998 ◽  
Vol 18 (3) ◽  
pp. 1467-1476 ◽  
Author(s):  
John J. Long ◽  
Anne Leresche ◽  
Richard W. Kriwacki ◽  
Joel M. Gottesfeld
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Kinase Inhibitor ◽  
Large Subunit ◽  
Cyclin B ◽  
Viral Gene ◽  
Gene Promoters ◽  
Nuclear Extracts ◽  
Carboxy Terminal

ABSTRACT Nuclear transcription is repressed when eukaryotic cells enter mitosis. Mitotic repression of transcription of various cellular and viral gene promoters by RNA polymerase II can be reproduced in vitro either with extracts prepared from cells arrested at mitosis with the microtubule polymerization inhibitor nocodazole or with nuclear extracts prepared from asynchronous cells and the mitotic protein kinase cdc2/cyclin B. Purified cdc2/cyclin B kinase is also sufficient to inhibit transcription in reconstituted transcription reactions with biochemically purified and recombinant basal transcription factors and RNA polymerase II. The cyclin-dependent kinase inhibitor p21 Waf1/Cip1/Sdi1 can reverse the effect of cdc2/cyclin B kinase, indicating that repression of transcription is due to protein phosphorylation. Transcription rescue and inhibition experiments with each of the basal factors and the polymerase suggest that multiple components of the transcription machinery are inactivated by cdc2/cyclin B kinase. For an activated promoter, targets of repression are TFIID and TFIIH, while for a basal promoter, TFIIH is the major target for mitotic inactivation of transcription. Protein labeling experiments indicate that the p62 and p36 subunits of TFIIH are in vitro substrates for mitotic phosphorylation. Using the carboxy-terminal domain of the large subunit of RNA polymerase II as a test substrate for phosphorylation, the TFIIH-associated kinase, cdk7/cyclin H, is inhibited concomitant with inhibition of transcription activity. Our results suggest that there exist multiple phosphorylation targets for the global shutdown of transcription at mitosis.

scholarly journals A Fully Recombinant System for Activator-dependent Archaeal Transcription

2004 ◽  
Vol 279 (50) ◽  
pp. 51719-51721 ◽  
Author(s):  
Mohamed Ouhammouch ◽  
Finn Werner ◽  
Robert O. J. Weinzierl ◽  
E. Peter Geiduschek
Keyword(s):  
Transcription Factor ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcriptional Regulators ◽  
The Core ◽  
General Transcription Factor ◽  
Core Components ◽  
Archaeal Transcription ◽  
Bacterial Type

The core components of the archaeal transcription apparatus closely resemble those of eukaryotic RNA polymerase II, while the DNA-binding transcriptional regulators are predominantly of bacterial type. Here we report the construction of an entirely recombinant system for positively regulated archaeal transcription. By omitting individual subunits, or sets of subunits, from thein vitroassembly of the 12-subunit RNA polymerase from the hyperthermophileMethanocaldococcus jannaschii, we describe a functional dissection of this RNA polymerase II-like enzyme, and its interactions with the general transcription factor TFE, as well as with the transcriptional activator Ptr2.

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.

The HIP1 initiator element plays a role in determining the in vitro requirement of the dihydrofolate reductase gene promoter for the C-terminal domain of RNA polymerase II

1992 ◽  
Vol 12 (5) ◽  
pp. 2250-2259
Author(s):  
A B Buermeyer ◽  
N E Thompson ◽  
L A Strasheim ◽  
R R Burgess ◽  
P J Farnham
Keyword(s):  
Rna Polymerase ◽  
Binding Site ◽  
Rna Polymerase Ii ◽  
Dihydrofolate Reductase ◽  
Initiation Site ◽  
Tata Box ◽  
Minimal Promoter ◽  
Rnap Ii ◽  
Promoter Deletions

We examined the ability of purified RNA polymerase (RNAP) II lacking the carboxy-terminal heptapeptide repeat domain (CTD), called RNAP IIB, to transcribe a variety of promoters in HeLa extracts in which endogenous RNAP II activity was inhibited with anti-CTD monoclonal antibodies. Not all promoters were efficiently transcribed by RNAP IIB, and transcription did not correlate with the in vitro strength of the promoter or with the presence of a consensus TATA box. This was best illustrated by the GC-rich, non-TATA box promoters of the bidirectional dihydrofolate reductase (DHFR)-REP-encoding locus. Whereas the REP promoter was transcribed by RNAP IIB, the DHFR promoter remained inactive after addition of RNAP IIB to the antibody-inhibited reactions. However, both promoters were efficiently transcribed when purified RNAP with an intact CTD was added. We analyzed a series of promoter deletions to identify which cis elements determine the requirement for the CTD of RNAP II. All of the promoter deletions of both DHFR and REP retained the characteristics of their respective full-length promoters, suggesting that the information necessary to specify the requirement for the CTD is contained within approximately 65 bp near the initiation site. Furthermore, a synthetic minimal promoter of DHFR, consisting of a single binding site for Sp1 and a binding site for the HIP1 initiator cloned into a bacterial vector sequence, required RNAP II with an intact CTD for activity in vitro. Since the synthetic minimal promoter of DHFR and the smallest REP promoter deletion are both activated by Sp1, the differential response in this assay does not result from upstream activators. However, the sequences around the start sites of DHFR and REP are not similar and our data suggest that they bind different proteins. Therefore, we propose that specific initiator elements are important for determination of the requirement of some promoters for the CTD.

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