Transcription Factor Analysis in Trypanosomatids.
Extract preparation
In vitro transcription
Primer extension
Promoter pull-down assay
Radiolabeling of DNA
Journal
Methods in molecular biology (Clifton, N.J.)
ISSN: 1940-6029
Titre abrégé: Methods Mol Biol
Pays: United States
ID NLM: 9214969
Informations de publication
Date de publication:
2020
2020
Historique:
entrez:
30
3
2020
pubmed:
30
3
2020
medline:
2
2
2021
Statut:
ppublish
Résumé
Known transcription factors of trypanosomatid organisms are extremely divergent in amino acid sequence to their counterparts in other eukaryotes. Sequence similarity is so limited that factors have been primarily identified by functional and structural studies. In addition, trypanosomatids may have evolved factors that are specific to this group of organisms. Under these circumstances, an in vitro transcription system is invaluable as it allows for unambiguous determination of a factor's transcriptional role. Here we describe procedures for the preparation of transcriptionally active extracts, detail in vitro transcription reactions, and specify the particular strategy necessary to detect template-derived RNA in this system. As examples of how to use this system, we describe factor depletion from extract and antibody-mediated interference with a factor's transcriptional function. Furthermore, we detail a promoter pull-down assay that makes use of the extracts and facilitates analysis of a factor's interaction with promoter DNA.
Identifiants
pubmed: 32221925
doi: 10.1007/978-1-0716-0294-2_16
pmc: PMC7337167
mid: NIHMS1600339
doi:
Substances chimiques
Protozoan Proteins
0
RNA, Protozoan
0
Transcription Factors
0
Types de publication
Journal Article
Research Support, N.I.H., Extramural
Langues
eng
Sous-ensembles de citation
IM
Pagination
265-284Subventions
Organisme : NIAID NIH HHS
ID : R01 AI028798
Pays : United States
Organisme : NIAID NIH HHS
ID : R21 AI142149
Pays : United States
Organisme : NIAID NIH HHS
ID : R56 AI130747
Pays : United States
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