Promoter independent abortive transcription assays unravel functional interactions between TFIIB and RNA polymerase

• Published in: Methods in molecular biology (Clifton, N.J.) Vol. 977; p. 217
• Main Authors: Wiesler, Simone C, Werner, Finn, Weinzierl, Robert O J
• Format: Journal Article
• Language: English
• Published: United States 2013
• Subjects: Archaeal Proteins - chemistry; DNA-Directed RNA Polymerases - chemistry; Escherichia coli Proteins - chemistry; Genes, Reporter; Luciferases - biosynthesis; Luciferases - genetics; Methanococcales - enzymology; Methanococcales - genetics; Promoter Regions, Genetic; Protein Binding; Titrimetry; Transcription Factor TFIIB - chemistry; Transcription Initiation, Genetic; Transcription, Genetic; Viral Proteins - chemistry
• ISSN: 1940-6029
• DOI: 10.1007/978-1-62703-284-1_17

TFIIB-like general transcription factors are required for transcription initiation by all eukaryotic and archaeal RNA polymerases (RNAPs). TFIIB facilitates both recruitment and post-recruitment steps of initiation; in particular, TFIIB stimulates abortive initiation. X-ray crystallography of TFIIB-RNAP II complexes shows that the TFIIB linker region penetrates the RNAP active center, yet the impact of this arrangement on RNAP activity and underlying mechanisms remains elusive. Promoter-independent abortive initiation assays exploit the intrinsic ability of RNAP enzymes to initiate transcription from nicked DNA templates and record the formation of the first phosphodiester bonds. These assays can be used to measure the effect of transcription factors such as TFIIB and RNAP mutations on abortive transcription.