ArticleProtein expression and purification2010
Site-directed mutagenesis, purification and assay of Saccharomyces cerevisiae RNA polymerase II.
Article in Protein expression and purification, 2010. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 13 papers.
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Who cites it
13 citing papers in PubMed, 20 citations in OpenAlex.
- Structural basis of transcription-coupled RNA damage by incorporation of oxidized ribonucleotides.Proceedings of the National Academy of Sciences of the United States of America · 2026Article
- RNA Polymerase II Activity Control of Gene Expression and Involvement in Disease.Journal of molecular biology · 2025Review
- Structural basis of transcription: RNA polymerase II substrate binding and metal coordination using a free-electron laser.Proceedings of the National Academy of Sciences of the United States of America · 2024Article
- Nucleotide Loading Modes of Human RNA Polymerase II as Deciphered by Molecular Simulations.Biomolecules · 2020Article
- Kinetics of nucleotide entry into RNA polymerase active site provides mechanism for efficiency and fidelity.Biochimica et biophysica acta. Gene regulatory mechanisms · 2017Article
- Computational simulation strategies for analysis of multisubunit RNA polymerases.Chemical reviews · 2013Review
- Dismantling promoter-driven RNA polymerase II transcription complexes in vitro by the termination factor Rat1.The Journal of biological chemistry · 2013Article
- Basic mechanisms of RNA polymerase II activity and alteration of gene expression in Saccharomyces cerevisiae.Biochimica et biophysica acta · 2013Review
- Molecular dynamics and mutational analysis of the catalytic and translocation cycle of RNA polymerase.BMC biophysics · 2012Article
- Interaction of RNA polymerase II fork loop 2 with downstream non-template DNA regulates transcription elongation.The Journal of biological chemistry · 2011Article
- Initiation complex structure and promoter proofreading.Science (New York, N.Y.) · 2011Article
- RNA polymerase II with open and closed trigger loops: active site dynamics and nucleic acid translocation.Biophysical journal · 2010Article
- Conformational coupling, bridge helix dynamics and active site dehydration in catalysis by RNA polymerase.Biochimica et biophysica acta · 2010Article
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Authors and funding
6 authors at 3 institutions in 2 countries.
Funding
Abstract
In order to analyze the structure-function of multi-subunit RNA polymerases (RNAPs), it is necessary to make site-directed mutations in key residues. Because Saccharomyces cerevisiae RNAP II is isolated as a 12 subunit enzyme that has not been amenable to in vitro reconstitution, making site-directed mutations in a particular subunit presents technical issues. In this work, we demonstrate a method to generate and purify site-directed mutants in the second largest (Rpb2) RNAP II subunit from yeast, using a tandem affinity purification tag. Mutants are analyzed for growth defects in vivo and for defects in transcriptional elongation in vitro. We show that Rpb2 R512A/C located just C-terminal to fork loop 2 (Rpb2 500-511) has transcriptional defects that are distinct from surrounding fork loop 2 region mutants. Rpb2 E529A/D replacements are faster and E529Q is slower than wild type RNAP II in elongation. E529 appears to form an ion pair with K987, an essential active site residue. Mutations are also analyzed within the active site region indicating key residues for catalysis and the importance of a Rpb2 R983-E1028 ion pair. Rpb2 R983Q and E1028Q are defective in escape from a transcriptional stall.
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