Search results for "RNA polymerase"

Biotin-Genomic Run-On (Bio-GRO): A High-Resolution Method for the Analysis of Nascent Transcription in Yeast

2016

Transcription is a highly complex biological process, with extensive layers of regulation, some of which remain to be fully unveiled and understood. To be able to discern the particular contributions of the several transcription steps it is crucial to understand RNA polymerase dynamics and regulation throughout the transcription cycle. Here we describe a new nonradioactive run-on based method that maps elongating RNA polymerases along the genome. In contrast with alternative methodologies for the measurement of nascent transcription, the BioGRO method is designed to minimize technical noise that arises from two of the most common sources that affect this type of strategies: contamination wi…

De novo transcriptome assembly of a facultative parasitic nematode Pelodera (syn. Rhabditis) strongyloides

2018

Pelodera strongyloides is a generally free-living gonochoristic facultative nematode. The whole genomic sequence of P. strongyloides remains unknown but 4 small subunit ribosomal RNA (ssrRNA) gene sequences are available. This project launched a de novo transcriptome assembly with 100 bp paired-end RNA-seq reads from normal, starved and wet-plate cultured animals. Trinity assembly tool generated 104,634 transcript contigs with N50 contig being 2195 bp and average contig length at 1103 bp. Transcriptome BLASTX matching results of five nematodes (C. elegans, Strongyloides stercoralis, Necator americanus, Trichuris trichiura, and Pristionchus pacificus) were consistent with their evolutionary …

Prefoldin-like Bud27 influences the transcription of ribosomal components and ribosome biogenesis in Saccharomyces cerevisiae

2020

Understanding the functional connection that occurs for the three nuclear RNA polymerases to synthesize ribosome components during the ribosome biogenesis process has been the focal point of extensive research. To preserve correct homeostasis on the production of ribosomal components, cells might require the existence of proteins that target a common subunit of these RNA polymerases to impact their respective activities. This work describes how the yeast prefoldin-like Bud27 protein, which physically interacts with the Rpb5 common subunit of the three RNA polymerases, is able to modulate the transcription mediated by the RNA polymerase I, likely by influencing transcription elongation, the …

Zinc(II)—The Overlooked Éminence Grise of Chloroquine’s Fight against COVID-19?

2020

The authors would like to thank Agnieszka Michalczuk for providing us with her artistic vision of SARS-CoV-2.

Different pathways for the nuclear import of yeast RNA polymerase II

2015

Recent studies suggest that RNA polymerase II (Pol II) has to be fully assembled before being imported into the nucleus, while other reports indicate a distinct mechanism to import large and small subunits. In yeast, Iwr1 binds to the holoenzyme assembled in the cytoplasm and directs its nuclear entry. However, as IWR1 is not an essential gene, Iwr1-independent pathway(s) for the nuclear import of Pol II must exist. In this paper, we investigate the transport into the nucleus of several large and small Pol II subunits in the mutants of genes involved in Pol II biogenesis. We also analyse subcellular localization in the presence of drugs that can potentially affect Pol II nuclear import. Our…

2015

The combination of Reverse Transcription (RT) and high-throughput sequencing has emerged as a powerful combination to detect modified nucleotides in RNA via analysis of either abortive RT-products or of the incorporation of mismatched dNTPs into cDNA. Here we simultaneously analyze both parameters in detail with respect to the occurrence of N-1-methyladenosine (m(1)A) in the template RNA. This naturally occurring modification is associated with structural effects, but it is also known as a mediator of antibiotic resistance in ribosomal RNA. In structural probing experiments with dimethylsulfate, m(1)A is routinely detected by RT-arrest. A specifically developed RNA-Seq protocol was tailored…

Corrigendum to “External conditions inversely change the RNA polymerase II elongation rate and density in yeast” [Biochim. Biophys. Acta 1829/11 (201…

2017

Electrochemical probe for the monitoring of DNA-protein interactions.

2010

Self-assembly of thiol-terminated oligonucleotides on gold substrates provides a convenient way for DNA-functionalized surfaces. Here we describe the development of an electrochemical assay for the detection of DNA-protein interactions based on the modification of the electrochemical response of methylene blue (MB) intercalated in the DNA strands. Using a functionalized electrode with double stranded DNA carrying T3 RNA polymerase binding sequence, we show a substantial attenuation of the current upon the DNA-protein interaction. Moreover, a Langmuir binding isotherm for T3 RNA polymerase (T3 Pol) gives a dissociation constant K(D) equal to 0.46+/-0.23 microM. Such value is 100 times lower …

Regulon-Specific Control of Transcription Elongation across the Yeast Genome

2009

Transcription elongation by RNA polymerase II was often considered an invariant non-regulated process. However, genome-wide studies have shown that transcriptional pausing during elongation is a frequent phenomenon in tightly-regulated metazoan genes. Using a combination of ChIP-on-chip and genomic run-on approaches, we found that the proportion of transcriptionally active RNA polymerase II (active versus total) present throughout the yeast genome is characteristic of some functional gene classes, like those related to ribosomes and mitochondria. This proportion also responds to regulatory stimuli mediated by protein kinase A and, in relation to cytosolic ribosomal-protein genes, it is medi…

Cell volume homeostatically controls the rDNA repeat copy number and rRNA synthesis rate in yeast

2021

[Abstract] The adjustment of transcription and translation rates to the changing needs of cells is of utmost importance for their fitness and survival. We have previously shown that the global transcription rate for RNA polymerase II in budding yeast Saccharomyces cerevisiae is regulated in relation to cell volume. Total mRNA concentration is constant with cell volume since global RNApol II-dependent nascent transcription rate (nTR) also keeps constant but mRNA stability increases with cell size. In this paper, we focus on the case of rRNA and RNA polymerase I. Contrarily to that found for RNA pol II, we detected that RNA polymerase I nTR increases proportionally to genome copies and cell s…