scholarly journals Engineering Transcriptional Interference through RNA Polymerase Processivity Control

2020 ◽  
Author(s):  
Nolan J. O’Connor ◽  
Antoni E. Bordoy ◽  
Anushree Chatterjee
Keyword(s):  
Rna Polymerase ◽  
Logic Gates ◽  
Antisense Transcription ◽  
Gene Repression ◽  
Transcriptional Interference ◽  
Bacterial Genomes ◽  
Phage Protein ◽  
In Cis ◽  
Transcriptional Process ◽  
Influence Gene Expression

ABSTRACTAntisense transcription is widespread in all kingdoms of life and has been shown to influence gene expression through transcriptional interference (TI), a phenomenon in which one transcriptional process negatively influences another in cis. The processivity, or uninterrupted transcription, of an RNA Polymerase (RNAP) is closely tied to levels of antisense transcription in bacterial genomes, but its influence on TI, while likely important, is not well-characterized. Here we show that TI can be tuned through processivity control via three distinct antitermination strategies: the antibiotic bicyclomycin, phage protein Psu, and ribosome-RNAP coupling. We apply these methods toward TI and tune ribosome-RNAP coupling to produce 38-fold gene repression due to RNAP collisions. We then couple protein roadblock and RNAP collisions to design minimal genetic NAND and NOR logic gates. Together these results show the importance of processivity control for strong TI and demonstrate the potential for TI to create sophisticated switching responses.

scholarly journals iRAPs curb antisense transcription in E. coli

2019 ◽  
Vol 47 (20) ◽  
pp. 10894-10905 ◽  
Author(s):  
Andrés Magán ◽  
Fabian Amman ◽  
Fatinah El-Isa ◽  
Natascha Hartl ◽  
Ilya Shamovsky ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Antisense Transcription ◽  
Rna Aptamers ◽  
Antisense Strand ◽  
Genomic Context ◽  
Reporter System ◽  
E Coli ◽  
Polymerase Binding ◽  
In Cis

Abstract RNA polymerase-binding RNA aptamers (RAPs) are natural RNA elements that control transcription in cis by directly contacting RNA polymerase. Many RAPs inhibit transcription by inducing Rho-dependent termination in Escherichia coli. Here, we studied the role of inhibitory RAPs (iRAPs) in modulation of antisense transcription (AT) using in silico and in vivo approaches. We revisited the antisense transcriptome in cells with impaired AT regulators (Rho, H-NS and RNaseIII) and searched for the presence of RAPs within antisense RNAs. Many of these RAPs were found at key genomic positions where they terminate AT. By exploring the activity of several RAPs both in a reporter system and in their natural genomic context, we confirmed their significant role in AT regulation. RAPs coordinate Rho activity at the antisense strand and terminate antisense transcripts. In some cases, they stimulated sense expression by alleviating ongoing transcriptional interference. Essentially, our data postulate RAPs as key determinants of Rho-mediated AT regulation in E. coli.

scholarly journals Construction of two-input logic gates using Transcriptional Interference

2019 ◽  
Author(s):  
Antoni E. Bordoy ◽  
Nolan J. O’Connor ◽  
Anushree Chatterjee
Keyword(s):  
Gene Expression ◽  
Rna Polymerase ◽  
Molecular Mechanisms ◽  
Logic Gates ◽  
Transcriptional Interference ◽  
Input Devices ◽  
New Strategy ◽  
Multiple Characteristics ◽  
Single Input ◽  
Genetic Responses

ABSTRACTTranscriptional Interference (TI) has been shown to regulate gene expression at the DNA level via different molecular mechanisms. The obstacles present on the DNA that a transcribing RNA Polymerase might encounter, e.g. a DNA-bound protein or another RNA Polymerase, can result in TI causing termination of transcription, thus reducing gene expression. However, the potential of TI as a new strategy to engineer complex gene expression modules has not been fully explored yet. Here we created a series of two-input devices using the presence of a roadblocking protein using both experimental and mathematical modeling approaches. We explore how multiple characteristics affect the response of genetic devices engineered to act like either AND, OR, or Single Input logic gates. We show that the dissociation constant of the roadblocking protein, inducer activation of promoter and operator sites, and distance between tandem promoters tune gate behavior. This work highlights the potential of rationally creating different types of genetic responses using the same transcription factors in subtly different genetic architectures.

scholarly journals Initiation and elongation of polyribonucleotide chains on rat ventral-prostate chromatin transcribed by homologous ribonucleic acid polymerase B.

1977 ◽  
Vol 166 (2) ◽  
pp. 189-198 ◽  
Author(s):  
P Thomas ◽  
P Davies ◽  
K Griffiths
Keyword(s):  
Rna Polymerase ◽  
Rna Synthesis ◽  
Prostate Gland ◽  
Ventral Prostate ◽  
Control Of Gene Expression ◽  
Rat Ventral Prostate ◽  
Associated Proteins ◽  
Ribonucleoside Triphosphate ◽  
Androgenic Steroids ◽  
Transcriptional Process

The characteristics of initiation of RNA synthesis and the elongation of RNA chains on rat ventral-prostate chromatin by RNA polymerase B were investigated by two methods. 1. Initiation was carried out under low-salt conditions with three ribonucleoside triphosphates, and elongation was begun in the absence of reinitiation by the addition of the fourth ribonucleoside triphosphate and increasing the salt concentration. 2. Stable initiation complexes were formed by preincubation of enzyme with template at 37 degrees C, elongation was started by the addition of all four ribonucleoside triphosphates and reinitiation or spurious RNA synthesis was prevented by rifamycin AF/013. The latter method gave more reliable results. The dependence of those parameters on the androgenic status of the animal was studied. During the first 24h after castration, elongation was mainly affected, whereas after 72h a smaller number of initiation sites for RNA polymerase B on chromatin was evident. Considerable diurnal variations in the various parameters were observed. Changes in the relative concentrations of the chromatin-associated proteins were also observed after castration. In the rat ventral-prostate gland androgenic steroids may not only influence one stage of the transcriptional process, but may affect many factors involved in the control of gene expression.

Microinjection of anti-topoisomerase I immunoglobulin G into nuclei of Chironomus tentans salivary gland cells leads to blockage of transcription elongation

1987 ◽  
Vol 7 (12) ◽  
pp. 4308-4316
Author(s):  
E Egyházi ◽  
E Durban
Keyword(s):  
Salivary Gland ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Immunoglobulin G ◽  
Topoisomerase I ◽  
Chironomus Tentans ◽  
Balbiani Ring ◽  
Gland Cells ◽  
Salivary Gland Cells ◽  
Transcriptional Process

Purified anti-topoisomerase I immunoglobulin G (IgG) was microinjected into nuclei of Chironomus tentans salivary gland cells, and the effect on DNA transcription was investigated. Synthesis of nucleolar preribosomal 38S RNA by RNA polymerase I and of chromosomal Balbiani ring RNA by RNA polymerase II was inhibited by about 80%. The inhibitory action of anti-topoisomerase I IgG could be reversed by the addition of exogenous topoisomerase I. Anti-topoisomerase I IgG had less effect on RNA polymerase II-promoted activity of other less efficiently transcribing heterogeneous nuclear RNA genes. The pattern of inhibition of growing nascent Balbiani ring chains indicated that the transcriptional process was interrupted at the level of chain elongation. The highly decondensed state of active Balbiani ring chromatin, however, remained unaffected after injection of topoisomerase I antibodies. These data are consistent with the interpretation that topoisomerase I is an essential component in the transcriptional process but not in the maintenance of the decondensed state of active chromatin.

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 Diverse digital and fuzzy composite transcriptional elements are prevalent features of mammalian cis-regulomes

2021 ◽  
Author(s):  
Virendra K Chaudhri ◽  
Harinder Singh
Keyword(s):  
Logic Gates ◽  
Bimodal Distribution ◽  
Open Chromatin ◽  
Regulatory Sequences ◽  
Systematic Analysis ◽  
Dna Motif ◽  
Transcriptional Regulatory ◽  
Regulatory Dna ◽  
In Cis ◽  
Transcriptional Elements

Mammalian transcriptional regulatory sequences are comprised of complex combinations of simple transcription factor (TF) motifs. Stereospecific juxta-positioning of simple TF motifs generates composite elements (CEs), that increase combinatorial and regulatory specificity of TF-DNA interactions. Although a small number of CEs and their cooperative or anti-cooperative modes of TF binding have been thoroughly characterized, a systematic analysis of CE diversity, prevalence and properties in cis-regulomes has not been undertaken. We developed a computational pipeline termed CEseek to discover >20,000 CEs in open chromatin regions of diverse immune cells and validated many using CAP-SELEX, ChIP-Seq and STARR-seq datasets. Strikingly, the CEs manifested a bimodal distribution of configurations, termed digital and fuzzy, based on their stringent or relaxed stereospecific constraints, respectively. Digital CEs mediate cooperative as well as anti-cooperative binding of structurally diverse TFs that likely reflect AND/OR genomic logic gates. In contrast, fuzzy CEs encompass a less diverse set of TF motif pairs that are selectively enriched in p300 associated, multi-genic enhancers. The annotated CEs greatly expand the regulatory DNA motif lexicon and the universe of TF-TF interactions that underlie combinatorial logic of gene regulation.

Sign in / Sign up

Export Citation Format

Share Document