Biochemical methods to characterize RNA polymerase II elongation complexes

J. Brooks Crickard, Joseph C. Reese

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

Transcription of DNA into RNA is critical for all life, and RNA polymerases are enzymes tasked with this activity. In eukaryotes, RNA Polymerase II (RNAPII) is responsible for transcription of all protein coding genes and many non-coding RNAs. RNAPII carries out the remarkable feat of unwinding the stable double-stranded DNA template, synthesizing the transcript and re-forming the double helix behind it with great precision and speed. In vitro, RNAPII is capable of carrying out templated RNA chain elongation in the absence of any accessory proteins. However, in cells, the transcription of genes is influenced by several factors, including DNA structure, chromatin, co-transcriptional processes, and DNA binding proteins, which impede the smooth progression of RNAPII down the template. Many transcription elongation proteins have evolved to mitigate the complications and barriers encountered by polymerase during transcription. Many of these elongation factors physically interact with components of the RNAPII elongation complex, including the growing RNA transcript and the DNA template entering and exiting RNAPII. To better understand how transcription elongation factors (EFs) regulate RNAPII, elegant methods are required to probe the structure of the elongation complex. Here, we describe a collection of biochemical assays to interrogate the structure of the RNAPII elongation complex of Saccharomyces cerevisiae that are capable of providing insights into the function of EFs and the elongation process.

Original languageEnglish (US)
Pages (from-to)70-81
Number of pages12
JournalMethods
Volume159-160
DOIs
StatePublished - Apr 15 2019

Fingerprint

RNA Polymerase II
Elongation
Transcription
Peptide Elongation Factors
DNA
RNA
Genes
Untranslated RNA
Proteins
DNA-Binding Proteins
Accessories
DNA-Directed RNA Polymerases
Eukaryota
Yeast
Chromatin
Saccharomyces cerevisiae
Assays
Transcription Factors
Enzymes

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Biochemistry, Genetics and Molecular Biology(all)

Cite this

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abstract = "Transcription of DNA into RNA is critical for all life, and RNA polymerases are enzymes tasked with this activity. In eukaryotes, RNA Polymerase II (RNAPII) is responsible for transcription of all protein coding genes and many non-coding RNAs. RNAPII carries out the remarkable feat of unwinding the stable double-stranded DNA template, synthesizing the transcript and re-forming the double helix behind it with great precision and speed. In vitro, RNAPII is capable of carrying out templated RNA chain elongation in the absence of any accessory proteins. However, in cells, the transcription of genes is influenced by several factors, including DNA structure, chromatin, co-transcriptional processes, and DNA binding proteins, which impede the smooth progression of RNAPII down the template. Many transcription elongation proteins have evolved to mitigate the complications and barriers encountered by polymerase during transcription. Many of these elongation factors physically interact with components of the RNAPII elongation complex, including the growing RNA transcript and the DNA template entering and exiting RNAPII. To better understand how transcription elongation factors (EFs) regulate RNAPII, elegant methods are required to probe the structure of the elongation complex. Here, we describe a collection of biochemical assays to interrogate the structure of the RNAPII elongation complex of Saccharomyces cerevisiae that are capable of providing insights into the function of EFs and the elongation process.",
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Biochemical methods to characterize RNA polymerase II elongation complexes. / Brooks Crickard, J.; Reese, Joseph C.

In: Methods, Vol. 159-160, 15.04.2019, p. 70-81.

Research output: Contribution to journalArticle

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