scholarly journals Use of a novel S1 nuclease RNA-mapping technique to measure efficiency of transcription termination on polyomavirus DNA.

1986 ◽  
Vol 6 (5) ◽  
pp. 1624-1632 ◽  
Author(s):  
R W Tseng ◽  
N H Acheson
Keyword(s):  
Transcription Initiation ◽  
Transcription Termination ◽  
Late Phase ◽  
Full Length ◽  
Mapping Technique ◽  
S1 Nuclease ◽  
Dna Fragment ◽  
Rna Mapping ◽  
Dna Strand

We devised a strategy to measure the efficiency of transcription termination in vivo by RNA polymerase on polyomavirus DNA. Pulse-labeled nuclear RNA was hybridized with a single-stranded polyomavirus DNA fragment which spans the transcription initiation region. Hybrids were treated with RNase, bound to nitrocellulose filters, eluted with S1 nuclease, and analyzed by gel electrophoresis. The ratio of full-length to less-than-full-length DNA-RNA hybrids was used to calculate transcription termination frequency. We found that 50% of the polymerases terminated per traverse of the L DNA strand during the late phase of infection. The method for mapping in vivo pulse-labeled RNAs which we developed is potentially useful for studying unstable cellular or viral RNAs.

Use of a novel S1 nuclease RNA-mapping technique to measure efficiency of transcription termination on polyomavirus DNA

1986 ◽  
Vol 6 (5) ◽  
pp. 1624-1632
Author(s):  
R W Tseng ◽  
N H Acheson
Keyword(s):  
Transcription Initiation ◽  
Transcription Termination ◽  
Late Phase ◽  
Full Length ◽  
Mapping Technique ◽  
S1 Nuclease ◽  
Dna Fragment ◽  
Rna Mapping ◽  
Dna Strand

We devised a strategy to measure the efficiency of transcription termination in vivo by RNA polymerase on polyomavirus DNA. Pulse-labeled nuclear RNA was hybridized with a single-stranded polyomavirus DNA fragment which spans the transcription initiation region. Hybrids were treated with RNase, bound to nitrocellulose filters, eluted with S1 nuclease, and analyzed by gel electrophoresis. The ratio of full-length to less-than-full-length DNA-RNA hybrids was used to calculate transcription termination frequency. We found that 50% of the polymerases terminated per traverse of the L DNA strand during the late phase of infection. The method for mapping in vivo pulse-labeled RNAs which we developed is potentially useful for studying unstable cellular or viral RNAs.

scholarly journals Termination-altering mutations in the second-largest subunit of yeast RNA polymerase III.

1995 ◽  
Vol 15 (3) ◽  
pp. 1467-1478 ◽  
Author(s):  
S A Shaaban ◽  
B M Krupp ◽  
B D Hall
Keyword(s):  
Amino Acid ◽  
Rna Polymerase ◽  
Transcription Initiation ◽  
Transcription Termination ◽  
Rna Polymerase Iii ◽  
In Vitro Transcription ◽  
The Other ◽  
Polymerase Iii

In order to identify catalytically important amino acid changes within the second-largest subunit of yeast RNA polymerase III, we mutagenized selected regions of its gene (RET1) and devised in vivo assays for both increased and decreased transcription termination by this enzyme. Using as the reporter gene a mutant SUP4-o tRNA gene that in one case terminates prematurely and in the other case fails to terminate, we screened mutagenized RET1 libraries for reduced and increased transcription termination, respectively. The gain in suppression phenotype was in both cases scored as a reduction in the accumulation of red pigment in yeast strains harboring the ade2-1 ochre mutation. Termination-altering mutations were obtained in regions of the RET1 gene encoding amino acids 300 to 325, 455 to 486, 487 to 521, and 1061 to 1082 of the protein. In degree of amino acid sequence conservation, these range from highly variable in the first to highly conserved in the last two regions. Residues 300 to 325 yielded mainly reduced-termination mutants, while in region 1061 to 1082, increased-termination mutants were obtained exclusively. All mutants recovered, while causing gain of suppression with one SUP4 allele, brought about a reduction in suppression with the other allele, thus confirming that the phenotype is due to altered termination rather than an elevated level of transcription initiation. In vitro transcription reactions performed with extracts from several strong mutants demonstrated that the mutant polymerases respond to RNA terminator sequences in a manner that matches their in vivo termination phenotypes.

scholarly journals H3K36 methylation and DNA-binding are critical for Ioc4 recruitment and Isw1b remodeller function

2021 ◽  
Author(s):  
Jian Li ◽  
Lena Bergmann ◽  
Kimberly M Webb ◽  
Madelaine M Gogol ◽  
Philipp Voigt ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Functional Characterization ◽  
Full Length ◽  
Pwwp Domain ◽  
H3k36 Methylation ◽  
Chromatin Remodelling Complex

The Isw1b chromatin-remodelling complex is specifically recruited to gene bodies to help retain pre-existing histones during transcription by RNA polymerase II. Recruitment is dependent on H3K36 methylation and the Isw1b subunit Ioc4, which contains an N-terminal PWWP domain. Here, we present the crystal structure of the Ioc4-PWWP domain including a detailed functional characterization of the domain on its own as well as in the context of full-length Ioc4 and the Isw1b remodeller. Ioc4-PWWP preferentially binds H3K36me3-containing nucleosomes. The ability of the PWWP domain to bind DNA is required for this interaction. It is also promoted by the unique insertion motif present in Ioc4-PWWP. The ability to bind H3K36me3 as well as DNA are also critical for full-length Ioc4 binding to nucleosomes in vitro as well as its recruitment to gene bodies in vivo. Furthermore, a fully functional Ioc4-PWWP domain is necessary for efficient remodelling by Isw1b and the maintenance of ordered chromatin in vivo, thereby preventing intragenic transcription initiation and the production of non-coding RNAs.

scholarly journals The N-terminal Domain of the Yeast Mitochondrial RNA Polymerase Regulates Multiple Steps of Transcription

2011 ◽  
Vol 286 (18) ◽  
pp. 16109-16120 ◽  
Author(s):  
Swaroopa Paratkar ◽  
Aishwarya P. Deshpande ◽  
Guo-Qing Tang ◽  
Smita S. Patel
Keyword(s):  
Amino Acids ◽  
Rna Polymerase ◽  
Transcription Initiation ◽  
Rna Synthesis ◽  
Full Length ◽  
Mitochondrial Rna ◽  
Terminal Domain ◽  
Mitochondrial Rna Polymerase

Transcription of the yeast (Saccharomyces cerevisiae) mitochondrial (mt) genome is catalyzed by nuclear-encoded proteins that include the core RNA polymerase (RNAP) subunit Rpo41 and the transcription factor Mtf1. Rpo41 is homologous to the single-subunit bacteriophage T7/T3 RNAP. Its ∼80-kDa C-terminal domain is highly conserved among mt RNAPs, but its ∼50-kDa N-terminal domain (NTD) is less conserved and not present in T7/T3 RNAP. To understand the role of the NTD, we have biochemically characterized a series of NTD deletion mutants of Rpo41. Our studies show that NTD regulates multiple steps of transcription initiation. Interestingly, NTD functions in an autoinhibitory manner during initiation, and its partial deletion increases the efficiency of RNA synthesis. Deletion of 1–270 amino acids (DN270) reduces abortive synthesis and increases full-length to abortive RNA ratio relative to full-length (FL) Rpo41. A larger deletion of 1–380 amino acids (DN380), decreases RNA synthesis on duplex but not on premelted promoter. We show that DN380 is defective in promoter opening near the transcription start site. Most strikingly, both DN270 and DN380 catalyze highly processive RNA synthesis on the premelted promoter, and unlike the FL Rpo41, the mutants are not inhibited by Mtf1. Both mutants show weaker interactions with Mtf1, which explains many of our results, and particularly the ability of the mutants to efficiently transition from initiation to elongation. We propose that in vivo the accessory proteins that bind NTD may modulate interactions of Rpo41 with the promoter/Mtf1 to activate and allow timely release from Mtf1 for transition into elongation.

scholarly journals Clusters of S1 nuclease-hypersensitive sites induced in vivo by DNA damage.

1997 ◽  
Vol 17 (9) ◽  
pp. 5437-5452 ◽  
Author(s):  
J Legault ◽  
A Tremblay ◽  
D Ramotar ◽  
M E Mirault
Keyword(s):  
Dna Damage ◽  
Single Strand ◽  
S1 Nuclease ◽  
Strand Breaks ◽  
Abasic Sites ◽  
Mouse Tissues ◽  
Hypersensitive Sites ◽  
Dna Strand ◽  
Gamma Irradiated

DNA end-labeling procedures were used to analyze both the frequency and distribution of DNA strand breaks in mammalian cells exposed or not to different types of DNA-damaging agents. The 3' ends were labeled by T4 DNA polymerase-catalyzed nucleotide exchange carried out in the absence or presence of Escherichia coli endonuclease IV to cleave abasic sites and remove 3' blocking groups. Using this sensitive assay, we show that DNA isolated from human cells or mouse tissues contains variable basal levels of DNA strand interruptions which are associated with normal bioprocesses, including DNA replication and repair. On the other hand, distinct dose-dependent patterns of DNA damage were assessed quantitatively in cultured human cells exposed briefly to menadione, methylmethane sulfonate, topoisomerase II inhibitors, or gamma rays. In vivo induction of single-strand breaks and abasic sites by methylmethane sulfonate was also measured in several mouse tissues. The genomic distribution of these lesions was investigated by DNA cleavage with the single-strand-specific S1 nuclease. Strikingly similar cleavage patterns were obtained with all DNA-damaging agents tested, indicating that the majority of S1-hypersensitive sites detected were not randomly distributed over the genome but apparently were clustered in damage-sensitive regions. The parallel disappearance of 3' ends and loss of S1-hypersensitive sites during post-gamma-irradiation repair periods indicates that these sites were rapidly repaired single-strand breaks or gaps (2- to 3-min half-life). Comparison of S1 cleavage patterns obtained with gamma-irradiated DNA and gamma-irradiated cells shows that chromatin structure was the primary determinant of the distribution of the DNA damage detected.

scholarly journals Functional characterization of a full length pregnane X receptor, expression in vivo, and identification of PXR alleles, in Zebrafish (Danio rerio)

2013 ◽  
Vol 142-143 ◽  
pp. 447-457 ◽  
Author(s):  
Afonso C.D. Bainy ◽  
Akira Kubota ◽  
Jared V. Goldstone ◽  
Roger Lille-Langøy ◽  
Sibel I. Karchner ◽  
...  
Keyword(s):  
Danio Rerio ◽  
Functional Characterization ◽  
Pregnane X Receptor ◽  
Full Length ◽  
Receptor Expression

scholarly journals Engineering nucleosomes for generating diverse chromatin assemblies

2021 ◽  
Author(s):  
Zenita Adhireksan ◽  
Deepti Sharma ◽  
Phoi Leng Lee ◽  
Qiuye Bao ◽  
Sivaraman Padavattan ◽  
...  
Keyword(s):  
Dynamic Properties ◽  
Dna Nanostructures ◽  
Macromolecular Assemblies ◽  
Maximum Resolution ◽  
Different Types ◽  
Chromatin Structural ◽  
Dna Fragment

Abstract Structural characterization of chromatin is challenging due to conformational and compositional heterogeneity in vivo and dynamic properties that limit achievable resolution in vitro. Although the maximum resolution for solving structures of large macromolecular assemblies by electron microscopy has recently undergone profound increases, X-ray crystallographic approaches may still offer advantages for certain systems. One such system is compact chromatin, wherein the crystalline state recapitulates the crowded molecular environment within the nucleus. Here we show that nucleosomal constructs with cohesive-ended DNA can be designed that assemble into different types of circular configurations or continuous fibers extending throughout crystals. We demonstrate the utility of the method for characterizing nucleosome compaction and linker histone binding at near-atomic resolution but also advance its application for tackling further problems in chromatin structural biology and for generating novel types of DNA nanostructures. We provide a library of cohesive-ended DNA fragment expression constructs and a strategy for engineering DNA-based nanomaterials with a seemingly vast potential variety of architectures and histone chemistries.

scholarly journals Decoding the temporal nature of brain GR activity in the NFκB signal transition leading to depressive-like behavior

2021 ◽  
Author(s):  
Young-Min Han ◽  
Min Sun Kim ◽  
Juyeong Jo ◽  
Daiha Shin ◽  
Seung-Hae Kwon ◽  
...  
Keyword(s):  
Inhibitory Action ◽  
Time Course ◽  
Molecular Mechanisms ◽  
Late Phase ◽  
Fine Tuning ◽  
Dependent Manner ◽  
Middle Phase ◽  
Temporal Shift

AbstractThe fine-tuning of neuroinflammation is crucial for brain homeostasis as well as its immune response. The transcription factor, nuclear factor-κ-B (NFκB) is a key inflammatory player that is antagonized via anti-inflammatory actions exerted by the glucocorticoid receptor (GR). However, technical limitations have restricted our understanding of how GR is involved in the dynamics of NFκB in vivo. In this study, we used an improved lentiviral-based reporter to elucidate the time course of NFκB and GR activities during behavioral changes from sickness to depression induced by a systemic lipopolysaccharide challenge. The trajectory of NFκB activity established a behavioral basis for the NFκB signal transition involved in three phases, sickness-early-phase, normal-middle-phase, and depressive-like-late-phase. The temporal shift in brain GR activity was differentially involved in the transition of NFκB signals during the normal and depressive-like phases. The middle-phase GR effectively inhibited NFκB in a glucocorticoid-dependent manner, but the late-phase GR had no inhibitory action. Furthermore, we revealed the cryptic role of basal GR activity in the early NFκB signal transition, as evidenced by the fact that blocking GR activity with RU486 led to early depressive-like episodes through the emergence of the brain NFκB activity. These results highlight the inhibitory action of GR on NFκB by the basal and activated hypothalamic-pituitary-adrenal (HPA)-axis during body-to-brain inflammatory spread, providing clues about molecular mechanisms underlying systemic inflammation caused by such as COVID-19 infection, leading to depression.

scholarly journals High Natural Permissivity of Primary Rabbit Cells for HIV-1, with a Virion Infectivity Defect in Macrophages as the Final Replication Barrier

2010 ◽  
Vol 84 (23) ◽  
pp. 12300-12314 ◽  
Author(s):  
Hanna-Mari Tervo ◽  
Oliver T. Keppler
Keyword(s):  
T Cells ◽  
Late Phase ◽  
Small Animal ◽  
Receptor Complex ◽  
Restriction Factors ◽  
Cyclin T1 ◽  
Primary Rabbit ◽  
Species Specific ◽  
Hiv 1

ABSTRACT An immunocompetent, permissive, small-animal model would be valuable for the study of human immunodeficiency virus type 1 (HIV-1) pathogenesis and for the testing of drug and vaccine candidates. However, the development of such a model has been hampered by the inability of primary rodent cells to efficiently support several steps of the HIV-1 replication cycle. Although transgenesis of the HIV receptor complex and human cyclin T1 have been beneficial, additional late-phase blocks prevent robust replication of HIV-1 in rodents and limit the range of in vivo applications. In this study, we explored the HIV-1 susceptibility of rabbit primary T cells and macrophages. Envelope-specific and coreceptor-dependent entry of HIV-1 was achieved by expressing human CD4 and CCR5. A block of HIV-1 DNA synthesis, likely mediated by TRIM5, was overcome by limited changes to the HIV-1 gag gene. Unlike with mice and rats, primary cells from rabbits supported the functions of the regulatory viral proteins Tat and Rev, Gag processing, and the release of HIV-1 particles at levels comparable to those in human cells. While HIV-1 produced by rabbit T cells was highly infectious, a macrophage-specific infectivity defect became manifest by a complex pattern of mutations in the viral genome, only part of which were deamination dependent. These results demonstrate a considerable natural HIV-1 permissivity of the rabbit species and suggest that receptor complex transgenesis combined with modifications in gag and possibly vif of HIV-1 to evade species-specific restriction factors might render lagomorphs fully permissive to infection by this pathogenic human lentivirus.

scholarly journals A 5′ Splice Site Enhances the Recruitment of Basal Transcription Initiation Factors In Vivo

2008 ◽  
Vol 29 (2) ◽  
pp. 271-278 ◽  
Author(s):  
Christian Kroun Damgaard ◽  
Søren Kahns ◽  
Søren Lykke-Andersen ◽  
Anders Lade Nielsen ◽  
Torben Heick Jensen ◽  
...  
Keyword(s):  
Splice Site ◽  
Transcription Initiation ◽  
Basal Transcription ◽  
Initiation Factors
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