Transient reversal of RNA polymerase II active site closing controls fidelity of transcription elongation

Abstract

To study fidelity of RNA polymerase II (Pol II), we analyzed properties of the 6-azauracil-sensitive and TFIIS-dependent E1103G mutant of rbp1 (rpo21), the gene encoding the catalytic subunit of Pol II in Saccharomyces cerevisiae. Using an in vivo retrotransposition-based transcription fidelity assay, we observed that rpb1-E1103G causes a 3-fold increase in transcription errors. This mutant showed a 10-fold decrease in fidelity of transcription elongation in vitro. The mutation does not appear to significantly affect translocation state equilibrium of Pol II in a stalled elongation complex. Primarily, it promotes NTP sequestration in the polymerase active center. Furthermore, pre-steady-state analyses revealed that the E1103G mutation shifted the equilibrium between the closed and the open active center conformations toward the closed form. Thus, open conformation of the active center emerges as an intermediate essential for preincorporation fidelity control. Similar mechanisms may control fidelity of DNA-dependent DNA polymerases and RNA-dependent RNA polymerases.

Figure 1.. The E1103G Substitution in Rpb1 Confers a Transcription Fidelity Defect In Vivo

(A) The experimental setup was designed to determine the frequency of errors in a TRP1 reporter gene during retrotransposition and is shown at the top of the figure. (B) Frequency of TRP1 inactivation in the strain his3-Δ200 trp1-Δ361 ura3–167 leu2Δ::G418 spt3–101 rpb1-Δ::NAT [pJS366] expressing wild-type Rpb1 [pJS757 RPB1] or Rpb1-E1103G [pJS781 /pb1-E1103G]. The numbers of the clones with trp1/HIS3 along with the total number of HIS3 clones are shown in parentheses.

Figure 2.. The E1103G Substitution in Rpb1 Promotes Misincorporation and Mismatch Extension In Vitro

TECs were assembled with fully complementary 45 nt template and nontemplate DNA strands and 7 nt or 8 nt RNA oligonucleotides. The assembled TECs were walked to position +9 with 1 μM each of ATP and GTP or only GTP (45A), washed, and incubated with purified NTPs for 5 min. The resulting 10 nt products are named according to the base at the 3’ end (A10 incorporated AMP at the 3′ end, etc.). Positions of the RNAs are indicated by arrows.

Figure 3.. The E1103G Mutation Promotes Misincorporation of Various Substrates

TEC8 obtained on the template 45C (Figure 2) was incubated with 10 μM ATP for 30 s, after which 100 μM of CTP (A), 10 μM dCTP (B), 100 μM UTP (C), or 1 μM ATP (D) was added. The reaction with CTP was stopped with 1 M HCl. Black symbols and lines show the data set for WT Pol II, and red symbols and lines represent E1103G Pol II. The data shown are an average of three parallels, and the error bars show the standard deviation. The curves represent single-exponential fits of the data, and the apparent rates (k) are shown.

Figure 4.. Translocation Properties of the WT and E1103G Pol II

(A) Experimental setup. (B) Dynamics of DNA degradation by Exo III reveals equilibrium between pre- and posttranslocated states of the TEC. TEC9 variants were obtained by 5 min incubation of TEC8 with 10 μM ATP or 3′dATP. The RNAs are shown in the lower panel. Note the aberrantly high mobility of the 9 nt RNA containing 3′dAMP. (C) The E1103G mutation confers an equilibrium shift toward the pretranslocated state in the stalled TEC. TEC10 variants were obtained by 5 min incubation of TEC9 with 10 μM of CTP or 3′dCTP. (D) Forward translocation of Pol II in TEC9 containing terminating 3′dAMP is induced by the incoming substrate. CTP was added at the final concentrations indicated on top of the gel. (E) NTP-stabilized forward translocation of Pol II in TEC9 containing terminating 3′dAMP requires complementary substrate. CTP, UTP, GTP, and ATP were added at 1 μM.

Figure 5.. The E1103G Mutation in Rpb1 Enhances Sequestration of the Incoming NTP and Suppresses Isomerization Reversal of the Elongation Complex

(A) E1103G Pol II sequesters the incoming NTP more efficiently than the WT Pol II. TEC9 was chased with α-[P³²] CTP for 0.002 or 5 s, and the reaction was quenched with 1 M HCl or 0.25 M EDTA or diluted with 3 mM unlabeled CTP and quenched with HCl after 5 s incubation. The longer RNA products (lanes 11 and 12) result from misincorporation of the unlabeled CMP in place of AMP and extension of the mismatch with the next CTP. The plot shows an average of the results of three experiments, and the error bars indicate standard deviation. (B) EDTA quench reveals the equilibrium between the open and closed conformations of the active center. The experiment was done as in Figure 3A, but the reaction was stopped with HCl (open symbols) or EDTA (solid symbols). The solid lines represent kinetic simulation according to the reaction scheme showed on top. The details of kinetic simulation are described in the Supplemental Experimental Procedures and Figure S8. (C) The reverse isomerization rate defines the efficiency of misincorporation. UTP concentrations in the reactions were 200 μM (blue), 400 μM (green), 600 μM (orange), 800 μM (cyan), 1200 μM (purple), and 2000 μM (red). The solid lines show kinetic simulation of the data according to the mechanism for misincorporation.

Figure 6.. Glu1103 May Stabilize an Open Conformation of the Trigger Loop by Interaction with Lys1112 and Thr1095

(A) TEC structures of S. cerevisiae Pol II with the closed trigger loop (cyan) and open trigger loop (yellow) (PDB 2E2H [Wang et al., 2006] and 1Y1V [Kettenberger et al., 2004]) aligned using the VMD software (Humphrey et al., 1996). Positions of Glu1103, Lys1112, and Thr1095 are shown for the open state (solid) and closed state (semitransparent) on the right panel. A hydrogen bond between the side chain of Glu1103 and the backbone amide of Lys1112 is shown. (B) Alignment of T. thermophilus RNA polymerase TEC structures (PDB 2O5J [Vassylyev et al., 2007b] and 2CW0 [Tuske et al., 2005]). (C) Sequence alignment of the regions undergoing a major shift during the trigger loop closing in yeast (S.c., Saccharomyces cerevisiae) Pol II and bacterial (E.c., E. coli; T. th., T. thermophilus) RNA polymerases.