scholarly journals Promoter scanning during transcription initiation in Saccharomyces cerevisiae: Pol II in the “shooting gallery”

2019 ◽  
Author(s):  
Chenxi Qiu ◽  
Huiyan Jin ◽  
Irina Vvedenskaya ◽  
Jordi Abante Llenas ◽  
Tingting Zhao ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Transcription Initiation ◽  
Promoter Sequence ◽  
Preinitiation Complex ◽  
Pol Ii ◽  
Transcription Start Sites ◽  
Promoter Architecture ◽  
General Transcription Factors ◽  
Promoter Class ◽  
Shooting Gallery

ABSTRACTBackgroundThe majority of eukaryotic promoters utilize multiple transcription start sites (TSSs). How multiple TSSs are specified at individual promoters across eukaryotes is not understood for most species. In S. cerevisiae, a preinitiation complex comprised of Pol II and conserved general transcription factors (GTFs) assembles and opens DNA upstream of TSSs. Evidence from model promoters indicates that the preinitiation complex (PIC) scans from upstream to downstream to identify TSSs. Prior results suggest that TSS distributions at promoters where scanning occurs shift in a polar fashion upon alteration in Pol II catalytic activity or GTF function.ResultsTo determine extent of promoter scanning across promoter classes in S. cerevisiae, we perturbed Pol II catalytic activity and GTF function and analyzed their effects on TSS usage genome-wide. We find that alterations to Pol II, TFIIB, or TFIIF function widely alter the initiation landscape consistent with promoter scanning operating at all yeast promoters, regardless of promoter class. Promoter architecture, however, can determine extent of promoter sensitivity to altered Pol II activity in ways that are predicted by a scanning model.ConclusionsOur observations coupled with previous data validate key predictions of the scanning model for Pol II initiation in yeast – which we term the “shooting gallery”. In this model, Pol II catalytic activity, and the rate and processivity of Pol II scanning together with promoter sequence determine the distribution of TSSs and their usage.

scholarly journals Structure of the human Mediator-bound transcription preinitiation complex

Science ◽  
2021 ◽  
pp. eabg3074
Author(s):  
R. Abdella ◽  
A. Talyzina ◽  
S. Chen ◽  
C. J. Inouye ◽  
R. Tjian ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Rna Polymerase Ii ◽  
Active Site ◽  
Binding Sites ◽  
Preinitiation Complex ◽  
Eukaryotic Transcription ◽  
Pol Ii ◽  
General Transcription Factors ◽  
Cryo Electron Microscopy ◽  
Multiple Contacts

Eukaryotic transcription requires the assembly of a multi-subunit preinitiation complex (PIC) comprised of RNA polymerase II (Pol II) and the general transcription factors. The co-activator Mediator is recruited by transcription factors, facilitates the assembly of the PIC, and stimulates phosphorylation of the Pol II C-terminal domain (CTD) by the TFIIH subunit CDK7. Here, we present the cryo-electron microscopy structure of the human Mediator-bound PIC at sub-4 Å. Transcription factor binding sites within Mediator are primarily flexibly tethered to the tail module. CDK7 is stabilized by multiple contacts with Mediator. Two binding sites exist for the Pol II CTD, one between the head and middle modules of Mediator and the other in the active site of CDK7, providing structural evidence for Pol II CTD phosphorylation within the Mediator-bound PIC.

scholarly journals Requirements for RNA polymerase II preinitiation complex formation in vivo

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Natalia Petrenko ◽  
Yi Jin ◽  
Liguo Dong ◽  
Koon Ho Wong ◽  
Kevin Struhl
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Associated Factors ◽  
Yeast Cells ◽  
Preinitiation Complex ◽  
Pol Ii ◽  
General Transcription Factors ◽  
Nucleotide Triphosphates

Transcription by RNA polymerase II requires assembly of a preinitiation complex (PIC) composed of general transcription factors (GTFs) bound at the promoter. In vitro, some GTFs are essential for transcription, whereas others are not required under certain conditions. PICs are stable in the absence of nucleotide triphosphates, and subsets of GTFs can form partial PICs. By depleting individual GTFs in yeast cells, we show that all GTFs are essential for TBP binding and transcription, suggesting that partial PICs do not exist at appreciable levels in vivo. Depletion of FACT, a histone chaperone that travels with elongating Pol II, strongly reduces PIC formation and transcription. In contrast, TBP-associated factors (TAFs) contribute to transcription of most genes, but TAF-independent transcription occurs at substantial levels, preferentially at promoters containing TATA elements. PICs are absent in cells deprived of uracil, and presumably UTP, suggesting that transcriptionally inactive PICs are removed from promoters in vivo.

scholarly journals High-Resolution Mapping of Transcription Initiation in the Asexual Stages of Toxoplasma gondii

2021 ◽  
Vol 10 ◽  
Author(s):  
Benedikt M. Markus ◽  
Benjamin S. Waldman ◽  
Hernan A. Lorenzi ◽  
Sebastian Lourido
Keyword(s):  
Toxoplasma Gondii ◽  
Transcription Initiation ◽  
Nucleosome Occupancy ◽  
Mrna Isoforms ◽  
Protein Coding ◽  
Transcription Start Sites ◽  
Promoter Architecture ◽  
Depth Analysis ◽  
Life Threatening ◽  
Specific Alternative

Toxoplasma gondii is a common parasite of humans and animals, causing life-threatening disease in the immunocompromized, fetal abnormalities when contracted during gestation, and recurrent ocular lesions in some patients. Central to the prevalence and pathogenicity of this protozoan is its ability to adapt to a broad range of environments, and to differentiate between acute and chronic stages. These processes are underpinned by a major rewiring of gene expression, yet the mechanisms that regulate transcription in this parasite are only partially characterized. Deciphering these mechanisms requires a precise and comprehensive map of transcription start sites (TSSs); however, Toxoplasma TSSs have remained incompletely defined. To address this challenge, we used 5′-end RNA sequencing to genomically assess transcription initiation in both acute and chronic stages of Toxoplasma. Here, we report an in-depth analysis of transcription initiation at promoters, and provide empirically-defined TSSs for 7603 (91%) protein-coding genes, of which only 1840 concur with existing gene models. Comparing data from acute and chronic stages, we identified instances of stage-specific alternative TSSs that putatively generate mRNA isoforms with distinct 5′ termini. Analysis of the nucleotide content and nucleosome occupancy around TSSs allowed us to examine the determinants of TSS choice, and outline features of Toxoplasma promoter architecture. We also found pervasive divergent transcription at Toxoplasma promoters, clustered within the nucleosomes of highly-symmetrical phased arrays, underscoring chromatin contributions to transcription initiation. Corroborating previous observations, we asserted that Toxoplasma 5′ leaders are among the longest of any eukaryote studied thus far, displaying a median length of approximately 800 nucleotides. Further highlighting the utility of a precise TSS map, we pinpointed motifs associated with transcription initiation, including the binding sites of the master regulator of chronic-stage differentiation, BFD1, and a novel motif with a similar positional arrangement present at 44% of Toxoplasma promoters. This work provides a critical resource for functional genomics in Toxoplasma, and lays down a foundation to study the interactions between genomic sequences and the regulatory factors that control transcription in this parasite.

scholarly journals Kin28 depletion increases association of TFIID subunits Taf1 and Taf4 with promoters in Saccharomyces cerevisiae

2020 ◽  
Vol 48 (8) ◽  
pp. 4244-4255 ◽  
Author(s):  
Elisabeth R Knoll ◽  
Z Iris Zhu ◽  
Debasish Sarkar ◽  
David Landsman ◽  
Randall H Morse
Keyword(s):  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Mediator Complex ◽  
Gene Promoters ◽  
Structural Studies ◽  
Initiation Complex ◽  
Pol Ii ◽  
Promoter Escape ◽  
General Transcription Factors ◽  
Encoding Genes

Abstract Transcription of eukaryotic mRNA-encoding genes by RNA polymerase II (Pol II) begins with assembly of the pre-initiation complex (PIC), comprising Pol II and the general transcription factors. Although the pathway of PIC assembly is well established, the mechanism of assembly and the dynamics of PIC components are not fully understood. For example, only recently has it been shown that in yeast, the Mediator complex normally occupies promoters only transiently, but shows increased association when Pol II promoter escape is inhibited. Here we show that two subunits of TFIID, Taf1 and Taf4, similarly show increased occupancy as measured by ChIP upon depletion or inactivation of Kin28. In contrast, TBP occupancy is unaffected by depletion of Kin28, thus revealing an uncoupling of Taf and TBP occupancy during the transcription cycle. Increased Taf1 occupancy upon Kin28 depletion is suppressed by depletion of TBP, while depletion of TBP in the presence of Kin28 has little effect on Taf1 occupancy. The increase in Taf occupancy upon depletion of Kin28 is more pronounced at TFIID-dominated promoters compared to SAGA-dominated promoters. Our results support the suggestion, based on recent structural studies, that TFIID may not remain bound to gene promoters through the transcription initiation cycle.

scholarly journals Structures of the human Mediator and Mediator-bound preinitiation complex

Science ◽  
2021 ◽  
pp. eabg0635
Author(s):  
Xizi Chen ◽  
Xiaotong Yin ◽  
Jiabei Li ◽  
Zihan Wu ◽  
Yilun Qi ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Atomic Resolution ◽  
Cyclin Dependent Kinase ◽  
Preinitiation Complex ◽  
Pol Ii ◽  
Gating Mechanism ◽  
Almost All

The 1.3-MDa transcription factor IID (TFIID) is required for preinitiation complex (PIC) assembly and RNA polymerase II (Pol II)-mediated transcription initiation on almost all genes. The 26-subunit Mediator stimulates transcription and cyclin-dependent kinase 7 (CDK7)-mediated phosphorylation of Pol II C-terminal domain (CTD). We determined the structures of human Mediator in the Tail module-extended (at near-atomic resolution) and Tail-bent conformations and structures of TFIID-based PIC-Mediator (76 polypeptides, ~4.1 MDa) in four distinct conformations. PIC-Mediator assembly induces concerted reorganization (Head-tilting and Middle-down) of Mediator and creates a Head-Middle sandwich, which stabilizes two CTD segments and brings CTD to CDK7 for phosphorylation, suggesting a CTD-gating mechanism favorable for phosphorylation. The TFIID-based PIC architecture modulates Mediator organization and TFIIH stabilization, underscoring the significance of TFIID in orchestrating PIC-Mediator assembly.

scholarly journals Structure of human Mediator-RNA polymerase II transcription pre-initiation complex

2021 ◽  
Author(s):  
Srinivasan Rengachari ◽  
Sandra Schilbach ◽  
Shintaro Aibara ◽  
Christian Dienemann ◽  
Patrick Cramer
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Complex Structure ◽  
Proximal Part ◽  
Initiation Complex ◽  
Protein Coding ◽  
Pol Ii ◽  
Promoter Escape ◽  
General Transcription Factors

Mediator is a conserved coactivator that enables regulated transcription initiation from eukaryotic protein-coding genes1-3. Mediator is recruited by transcriptional activators and binds the pre-initiation complex (PIC) to stimulate RNA polymerase II (Pol II) phosphorylation and promoter escape1-6. Here we prepare a 20-subunit recombinant human Mediator, reconstitute a 50-subunit Mediator-PIC complex, and resolve the complex structure by cryo-EM at an overall resolution of 4.5 Å. Mediator binds with its head module to the Pol II stalk and the general transcription factors TFIIB and TFIIE, resembling the Mediator-PIC interactions in the corresponding yeast complex7-9. One end of Mediator contains the metazoan-specific subunits MED27-MED30, which associate with exposed regions in MED14 and MED17 to form the proximal part of the tail module that binds activators. The opposite end of Mediator positions the flexibly linked CDK-activating kinase (CAK) of the general transcription factor TFIIH near the C-terminal repeat domain (CTD) of Pol II. The Mediator shoulder domain holds the CAK subunit CDK7, whereas the hook domain contacts a CDK7 element that flanks the kinase active site. The shoulder and hook reside in the Mediator head and middle modules, respectively, which can move relative to each other and may induce an active conformation of CDK7 to allosterically stimulate CTD phosphorylation and Pol II escape from the promoter.

pGR71 plasmid promotor sequence temporally regulated in Bacillus subtilis

1998 ◽  
Vol 44 (12) ◽  
pp. 1186-1192
Author(s):  
Guy Daxhelet ◽  
Philippe Gilot ◽  
Etienne Nyssen ◽  
Philippe Hoet
Keyword(s):  
Bacillus Subtilis ◽  
Binding Site ◽  
Transcription Initiation ◽  
Promoter Sequence ◽  
Initiation Site ◽  
Chloramphenicol Acetyltransferase ◽  
Ribosome Binding Site ◽  
Chloramphenicol Resistance ◽  
E Coli ◽  
Ribosome Binding

pGR71, a composite of plasmids pUB110 and pBR322, replicates in Escherichia coli and in Bacillus subtilis. It carries the chloramphenicol resistance gene (cat) from Tn9, which is not transcribed in either host by lack of a promoter. The cat gene is preceded by a Shine-Dalgarno sequence functional in E. coli but not in B. subtilis. Deleted pGR71 plasmids were obtained in B. subtilis when cloning foreign viral DNA upstream of this cat sequence, as well as by BAL31 exonuclease deletions extending upstream from the cat into the pUB110 moiety. These mutant plasmids expressed chloramphenicol acetyltransferase (CAT), conferring on B. subtilis resistance to high chloramphenicol concentrations. CAT expression peaked at the early postexponential phase of B. subtilis growth. The transcription initiation site of cat, determined by primer extension, was located downstream of a putative promoter sequence within the pUB110 moiety. N-terminal amino acid sequencing showed that native CAT was produced by these mutant plasmids. The cat ribosome-binding site, functional in E. coli, was repositioned within the pUB110 moiety and had consequently an extended homology with B. subtilis 16S rRNA, explaining the production of native enzyme.Key words: chloramphenicol acetyltransferase, Bacillus subtilis, postexponential gene expression, plasmid pUB110, ribosome-binding site, transcriptional promoter.

scholarly journals Discovery of widespread transcription initiation at microsatellites predictable by sequence-based deep neural network

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Mathys Grapotte ◽  
Manu Saraswat ◽  
Chloé Bessière ◽  
Christophe Menichelli ◽  
Jordan A. Ramilowski ◽  
...  
Keyword(s):  
Transcription Initiation ◽  
Tandem Repeats ◽  
Specific Gene ◽  
Rna Seq ◽  
Transcription Start Sites ◽  
Long Read ◽  
Cap Analysis ◽  
Dna Tandem Repeats ◽  
Short Tandem ◽  
Str Polymorphism

AbstractUsing the Cap Analysis of Gene Expression (CAGE) technology, the FANTOM5 consortium provided one of the most comprehensive maps of transcription start sites (TSSs) in several species. Strikingly, ~72% of them could not be assigned to a specific gene and initiate at unconventional regions, outside promoters or enhancers. Here, we probe these unassigned TSSs and show that, in all species studied, a significant fraction of CAGE peaks initiate at microsatellites, also called short tandem repeats (STRs). To confirm this transcription, we develop Cap Trap RNA-seq, a technology which combines cap trapping and long read MinION sequencing. We train sequence-based deep learning models able to predict CAGE signal at STRs with high accuracy. These models unveil the importance of STR surrounding sequences not only to distinguish STR classes, but also to predict the level of transcription initiation. Importantly, genetic variants linked to human diseases are preferentially found at STRs with high transcription initiation level, supporting the biological and clinical relevance of transcription initiation at STRs. Together, our results extend the repertoire of non-coding transcription associated with DNA tandem repeats and complexify STR polymorphism.

The human transforming growth factor alpha promoter directs transcription initiation from a single site in the absence of a TATA sequence

1988 ◽  
Vol 8 (12) ◽  
pp. 5549-5554
Author(s):  
E B Jakobovits ◽  
U Schlokat ◽  
J L Vannice ◽  
R Derynck ◽  
A D Levinson
Keyword(s):  
Growth Factor ◽  
Transcription Initiation ◽  
Transforming Growth Factor ◽  
Promoter Sequence ◽  
Transforming Growth Factor Alpha ◽  
Sequence Motif ◽  
Factor Alpha ◽  
Mammalian Genes ◽  
Tata Sequence ◽  
The Brain

Transforming growth factor alpha (TGF-alpha) is a transformation-responsive mitogenic polypeptide that is expressed in the brain, epithelial cells, and activated macrophages. We isolated and characterized the TGF-alpha promoter and localized the 5' end of the TGF-alpha transcript to a unique position. Surprisingly, no apparent TATA box was present in the promoter sequence, suggesting that transcription from mammalian genes can initiate at unique and specific positions from promoters lacking this sequence motif.

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