Biotin-Genomic Run-On (Bio-GRO): A High-Resolution Method for the Analysis of Nascent Transcription in Yeast.
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 with mature RNA and amplification-based technical biasing. The method is strand-specific, compatible with commercial microarrays, and has been successfully applied to both yeasts Saccharomyces cerevisiae and Candida albicans. BioGRO profiling provides powerful insights not only into the biogenesis and regulation of canonical gene transcription but also into the noncoding and antisense transcriptomes.
Departamento de Bioquímica y Biología Molecular and ERI Biotecmed, Facultad de Biológicas, Universitat de València, C/Dr. Moliner 50. E46100 Burjassot, València, Spain.|Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, Stockholm, Sweden.
Antonio
A
Jordán-Pla
Departamento de Bioquímica y Biología Molecular and ERI Biotecmed, Facultad de Biológicas, Universitat de València, C/Dr. Moliner 50. E46100 Burjassot, València, Spain.
Ana
A
Miguel
Servicio de Análisis Multigénico, INCLIVA, Universitat de València, València, Spain.
Eva
E
Serna
European Molecular Biology Laboratory (EMBL), Genome Biology Unit, Heidelberg, Germany.
Vicent
V
Pelechano
Departamento de Bioquímica y Biología Molecular and ERI Biotecmed, Facultad de Biológicas, Universitat de València, C/Dr. Moliner 50. E46100 Burjassot, València, Spain. jose.e.perez@uv.es.
José E
JE
Pérez-Ortín
D001710:Biotin / Q000235:genetics; D020869:Gene Expression Profiling / Q000379:methods*; D016681:Genome, Fungal; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D012319:RNA Polymerase II / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000235:genetics; D014158:Transcription, Genetic; D059467:Transcriptome / Q000235:genetics
D001710:Biotin; D012319:RNA Polymerase II
Candida albicans; Nascent RNA; Nascent transcription; RNA polymerase II; RNA polymerase II I; Saccharomyces cerevisiae; Yeast