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. 2010 Jan;69(1):83-90.
doi: 10.1016/j.pep.2009.06.016. Epub 2009 Jun 28.

Site-directed mutagenesis, purification and assay of Saccharomyces cerevisiae RNA polymerase II

Céline Domecq  1 , Maria Kireeva, Jacques Archambault, Mikhail Kashlev, Benoit Coulombe, Zachary F Burton
Affiliations

Site-directed mutagenesis, purification and assay of Saccharomyces cerevisiae RNA polymerase II

Céline Domecq et al. Protein Expr Purif. 2010 Jan.

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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Figures

Figure 1
Figure 1
In vivo tests of RNAP II strains. Growth of strains on galactose, glucose, 5-fluoro-orotic acid (5FOA), 6-azauracil (6AU) and growth of strains in a Δdst1 (TFIIS knockout; KO DST1_glucose) strain.
Figure 2
Figure 2
TAP-purification of RNAP II proteins. Proteins are stained with silver.
Figure 3
Figure 3
RNAP II proteins were assembled into RNA9 (G9) TECs. ATP, GTP and CTP were added for 1–3 min to stall the TEC at A20, and then all 4 NTPs were supplied at 10 or 200 μM for the times shown. Persistence of the A20 TEC during the chase for Rpb2 R983Q, indicates long-term stalling or arrest at A20.
Figure 4
Figure 4
Structural summary of mutational analysis. The bridge helix (Rpb1 810 to 845) is yellow; Rpb2 segments are orange; RNA is purple; the DNA template strand is tan; the i+1 active site GMPCPP is mauve; Mg2+ is green; apparent ionic interactions are indicated by solid lines; apparent hydrogen bonds are indicated by dashed lines. The image is from the Sc RNAP II TEC structure PDB 2NVT, which has an open trigger loop conformation, but contacts are very similar in 2E2H, which has a closed trigger helix (catalytic) conformation (not shown). Rpb2 R504 and D505 are not visible because they are located within a disordered segment of fork loop 2. The image was generated using Visual Molecular Dynamics (27) software.
See this image and copyright information in PMC

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