Remodeling of the σ70 Subunit Non-template DNA Strand Contacts During the Final Step of Transcription Initiation

2005 ◽  
Vol 350 (5) ◽  
pp. 930-937 ◽  
Author(s):  
Konstantin Brodolin ◽  
Nikolay Zenkin ◽  
Konstantin Severinov
Keyword(s):  
Transcription Initiation ◽  
Dna Strand ◽  
Template Dna

scholarly journals Modeling DNA Opening in the Eukaryotic Transcription Initiation Complexes via Coarse-Grained Models

2021 ◽  
Vol 8 ◽  
Author(s):  
Genki Shino ◽  
Shoji Takada
Keyword(s):  
Transcription Initiation ◽  
Molecular Mechanisms ◽  
Md Simulations ◽  
Coarse Grained ◽  
Dynamic Processes ◽  
Eukaryotic Transcription ◽  
Pol Ii ◽  
Promoter Dna ◽  
Dna Strand ◽  
Template Dna

Recently, the molecular mechanisms of transcription initiation have been intensively studied. Especially, the cryo-electron microscopy revealed atomic structure details in key states in the eukaryotic transcription initiation. Yet, the dynamic processes of the promoter DNA opening in the pre-initiation complex remain obscured. In this study, based on the three cryo-electron microscopic yeast structures for the closed, open, and initially transcribing complexes, we performed multiscale molecular dynamics (MD) simulations to model structures and dynamic processes of DNA opening. Combining coarse-grained and all-atom MD simulations, we first obtained the atomic model for the DNA bubble in the open complexes. Then, in the MD simulation from the open to the initially transcribing complexes, we found a previously unidentified intermediate state which is formed by the bottleneck in the fork loop 1 of Pol II: The loop opening triggered the escape from the intermediate, serving as a gatekeeper of the promoter DNA opening. In the initially transcribing complex, the non-template DNA strand passes a groove made of the protrusion, the lobe, and the fork of Rpb2 subunit of Pol II, in which several positively charged and highly conserved residues exhibit key interactions to the non-template DNA strand. The back-mapped all-atom models provided further insights on atomistic interactions such as hydrogen bonding and can be used for future simulations.

scholarly journals modeling DNA opening in the eukaryotic transcription initiation complexes

2021 ◽  
Author(s):  
Genki Shino ◽  
Shoji Takada
Keyword(s):  
Transcription Initiation ◽  
Molecular Mechanisms ◽  
Md Simulations ◽  
Coarse Grained ◽  
Dynamic Processes ◽  
Eukaryotic Transcription ◽  
Pol Ii ◽  
Promoter Dna ◽  
Dna Strand ◽  
Template Dna

Recently, the molecular mechanisms of transcription initiation have been intensively studied. Especially, the cryo-electron microscopy revealed atomic structure details in key states in the eukaryotic transcription initiation. Yet, the dynamic processes of the promoter DNA opening in the pre-initiation complex remain obscured. In this study, based on the three cryo-electron microscopic yeast structures for the closed, open, and initially transcribing complexes, we performed multiscale molecular dynamics (MD) simulations to model structures and dynamic processes of DNA opening. Combining coarse-grained and all-atom MD simulations, we first obtained the atomic model for the DNA bubble in the open complexes. Then, in the MD simulation from the open to the initially transcribing complexes, we found a previously unidentified intermediate state which is formed by the bottleneck in the fork loop 1 of Pol II: The loop opening triggered the escape from the intermediate, serving as a gatekeeper of the promoter DNA opening. In the initially transcribing complex, the non-template DNA strand passes a groove made of the protrusion, the lobe, and the fork of Rpb2 subunit of Pol II, in which several positively charged and highly conserved residues exhibit key interactions to the non-template DNA strand.

scholarly journals The universally-conserved transcription factor RfaH is recruited to a hairpin structure of the non-template DNA strand

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Philipp K Zuber ◽  
Irina Artsimovitch ◽  
Monali NandyMazumdar ◽  
Zhaokun Liu ◽  
Yuri Nedialkov ◽  
...  
Keyword(s):  
Virulence Genes ◽  
Complex Structure ◽  
Transcription Regulator ◽  
Transcription Bubble ◽  
Template Strand ◽  
Domains Of Life ◽  
Dna Strand ◽  
Functional Analyses ◽  
Template Dna

RfaH, a transcription regulator of the universally conserved NusG/Spt5 family, utilizes a unique mode of recruitment to elongating RNA polymerase to activate virulence genes. RfaH function depends critically on an ops sequence, an exemplar of a consensus pause, in the non-template DNA strand of the transcription bubble. We used structural and functional analyses to elucidate the role of ops in RfaH recruitment. Our results demonstrate that ops induces pausing to facilitate RfaH binding and establishes direct contacts with RfaH. Strikingly, the non-template DNA forms a hairpin in the RfaH:ops complex structure, flipping out a conserved T residue that is specifically recognized by RfaH. Molecular modeling and genetic evidence support the notion that ops hairpin is required for RfaH recruitment. We argue that both the sequence and the structure of the non-template strand are read out by transcription factors, expanding the repertoire of transcriptional regulators in all domains of life.

scholarly journals Transcription by an Archaeal RNA Polymerase Is Slowed but Not Blocked by an Archaeal Nucleosome

2004 ◽  
Vol 186 (11) ◽  
pp. 3492-3498 ◽  
Author(s):  
Yunwei Xie ◽  
John N. Reeve
Keyword(s):  
Chromatin Remodeling ◽  
Transcription Initiation ◽  
Regulatory Region ◽  
Nucleosome Assembly ◽  
Eukaryotic Chromatin ◽  
Transcript Elongation ◽  
Chromatin Remodeling Complexes ◽  
Methanothermobacter Thermautotrophicus ◽  
Tata Box Binding Protein ◽  
Template Dna

ABSTRACT Archaeal RNA polymerases (RNAPs) are closely related to eukaryotic RNAPs, and in Euryarchaea, genomic DNA is wrapped and compacted by histones into archaeal nucleosomes. In eukaryotes, transcription of DNA bound into nucleosomes is facilitated by histone tail modifications and chromatin remodeling complexes, but archaeal histones do not have histone tails and archaeal genome sequences provide no evidence for archaeal homologs of eukaryotic chromatin remodeling complexes. We have therefore investigated the ability of an archaeal RNAP, purified from Methanothermobacter thermautotrophicus, to transcribe DNA bound into an archaeal nucleosome by HMtA2, an archaeal histone from M. thermautotrophicus. To do so, we constructed a template that allows transcript elongation to be separated from transcription initiation, on which archaeal nucleosome assembly is positioned downstream from the site of transcription initiation. At 58°C, in the absence of an archaeal nucleosome, M. thermautotrophicus RNAP transcribed this template DNA at a rate of ∼20 nucleotides per second. With an archaeal nucleosome present, transcript elongation was slowed but not blocked, with transcription pausing at sites before and within the archaeal nucleosome. With additional HMtA2 binding, complexes were obtained that also incorporated the upstream regulatory region. This inhibited transcription presumably by preventing archaeal TATA-box binding protein, general transcription factor TFB, and RNAP access and thus inhibiting transcription initiation.

scholarly journals Saccharomyces cerevisiae CYC1 mRNA 5'-end positioning: analysis by in vitro mutagenesis, using synthetic duplexes with random mismatch base pairs.

1985 ◽  
Vol 5 (12) ◽  
pp. 3545-3551 ◽  
Author(s):  
J B McNeil ◽  
M Smith
Keyword(s):  
Saccharomyces Cerevisiae ◽  
In Vitro Mutagenesis ◽  
Base Pairs ◽  
Positioning Analysis ◽  
Dna Strand ◽  
Template Dna ◽  
Double Stranded Oligonucleotide ◽  
Selection Of

Expression of the Saccharomyces cerevisiae CYC1 gene produces mRNA with more than 20 different 5' ends. A derivative of the CYC1 gene (CYC1-157) was constructed with a deletion of a portion of the CYC1 5'-noncoding region, which includes the sites at which many of the CYC1 mRNAs 5' ends map. A 54-mer double-stranded oligonucleotide homologous with the deleted sequence of CYC1-157 and which included a low level of random base pair mismatches (an average of two mismatches per duplex) was used to construct mutants of the CYC1 gene and examine the role of the DNA sequence at and immediately adjacent to the mRNA 5' ends in specifying their locations. The effect of these mutations on the site selection of mRNA 5' ends was examined by primer extension. Results indicate that there is a strong preference for 5' ends which align with an A residue (T in the template DNA strand) preceded by a short tract of pyrimidine residues.

Enhanced basepair dynamics pre-disposes protein-assisted flips of key bases in DNA strand separation during transcription initiation

2018 ◽  
Vol 20 (14) ◽  
pp. 9449-9459 ◽  
Author(s):  
Neeladri Sekhar Roy ◽  
Subrata Debnath ◽  
Abhijit Chakraborty ◽  
Prasenjit Chakraborty ◽  
Indrani Bera ◽  
...  
Keyword(s):  
Transcription Initiation ◽  
Duplex Dna ◽  
Strand Separation ◽  
Dna Strand ◽  
Dependent Processes ◽  
Transcription And Replication

Localized separation of strands of duplex DNA is a necessary step in many DNA-dependent processes, including transcription and replication.

scholarly journals A Thermus phage protein inhibits host RNA polymerase by preventing template DNA strand loading during open promoter complex formation

2017 ◽  
Vol 46 (1) ◽  
pp. 431-441 ◽  
Author(s):  
Wei-Yang Ooi ◽  
Yuko Murayama ◽  
Vladimir Mekler ◽  
Leonid Minakhin ◽  
Konstantin Severinov ◽  
...  
Keyword(s):  
Complex Formation ◽  
Rna Polymerase ◽  
Promoter Complex ◽  
Phage Protein ◽  
Dna Strand ◽  
Template Dna

scholarly journals Proper control of R-loop homeostasis is required for maintenance of gene expression and neuronal function during aging

2021 ◽  
Author(s):  
Juan Jauregui-Lozano ◽  
Alyssa N Easton ◽  
Spencer E Escobedo ◽  
Nadia A Lanman ◽  
Vikki Marie Weake ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Death ◽  
Genome Instability ◽  
Disease Onset ◽  
Visual Response ◽  
Neuronal Function ◽  
Age Related ◽  
Dna Strand ◽  
Photoreceptor Neurons ◽  
Template Dna

Age-related loss of cellular function and increased cell death are characteristic hallmarks of aging. While defects in gene expression and RNA metabolism have been linked with age-associated human neuropathies, it is not clear how the changes that occur during aging contribute to loss of gene expression homeostasis. R-loops are DNA-RNA hybrids that typically form co-transcriptionally via annealing of the nascent RNA to the template DNA strand, displacing the non-template DNA strand. Dysregulation of R-loop homeostasis has been associated with both transcriptional impairment and genome instability. Importantly, a growing body of evidence links R-loop accumulation with cellular dysfunction, increased cell death and chronic disease onset. Here, we characterized the R-loop landscape in aging Drosophila melanogaster photoreceptor neurons. Our data shows that transcribed genes in Drosophila photoreceptor neurons accumulate R-loops during aging. Further, our data reveals an association between age-related R-loop accumulation and decreased expression of long and highly expressed genes. Lastly, we show that photoreceptor-specific depletion of Top3β, a DNA/RNA topoisomerase associated with R-loop resolution, leads to both downregulation of of long genes with neuronal function and decreased visual response in flies. Together, our studies present novel data showing increased levels of R-loop in aging photoreceptor neurons, highlighting the link between dysregulation of R-loop homeostasis, gene expression and visual function.

scholarly journals Asymmetrical template-DNA strand segregation can explain density-associated mutation-rate plasticity

2018 ◽  
Author(s):  
Duur K. Aanen ◽  
Alfons J.M. Debets
Keyword(s):  
Cell Division ◽  
Mutation Rate ◽  
High Density ◽  
Wide Range ◽  
Dna Strands ◽  
Unicellular Organisms ◽  
Dna Strand ◽  
Mother Cells ◽  
Template Dna ◽  
Mutant Cells

SummaryThe mutation rate is a fundamental factor in evolutionary genetics. Recently, mutation rates were found to be strongly reduced at high density in a wide range of unicellular organisms, prokaryotic and eukaryotic. Independently, cell division was found to become more asymmetrical at increasing density in diverse organisms; in yeast, some ‘mother’ cells continue dividing, while their ‘offspring’ cells do not divide further. Here, we investigate how this increased asymmetry in cell division at high density can be reconciled with reduced mutation-rate estimates. We calculated the expected number of mutant cells due to replication errors under various modes of segregation of template-DNA strands and copy-DNA strands, both under exponential and under linear growth. We show that the observed reduction in the mutation rate at high density can be explained if mother cells preferentially retain the template-DNA strands, since new mutations are then confined to non-dividing daughter cells thus reducing the spread of mutant cells. Any other inheritance mode results in anincreasein the number of mutant cells at higher density. The proposed hypothesis that patterns of DNA-strand segregation are density dependent fundamentally challenges our current understanding of mutation-rate estimates and extends the distinction between germline and soma to unicellular organisms.

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