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. 2013 Feb;1829(2):187-98.
doi: 10.1016/j.bbagrm.2012.11.005. Epub 2012 Nov 30.

The RNA polymerase bridge helix YFI motif in catalysis, fidelity and translocation

Yuri A Nedialkov  1 , Kristopher Opron, Fadi Assaf, Irina Artsimovitch, Maria L Kireeva, Mikhail Kashlev, Robert I Cukier, Evgeny Nudler, Zachary F Burton
Affiliations

The RNA polymerase bridge helix YFI motif in catalysis, fidelity and translocation

Yuri A Nedialkov et al. Biochim Biophys Acta. 2013 Feb.

Abstract

The bridge α-helix in the β' subunit of RNA polymerase (RNAP) borders the active site and may have roles in catalysis and translocation. In Escherichia coli RNAP, a bulky hydrophobic segment near the N-terminal end of the bridge helix is identified (β' 772-YFI-774; the YFI motif). YFI is located at a distance from the active center and adjacent to a glycine hinge (β' 778-GARKG-782) involved in dynamic bending of the bridge helix. Remarkably, amino acid substitutions in YFI significantly alter intrinsic termination, pausing, fidelity and translocation of RNAP. F773V RNAP largely ignores the λ tR2 terminator at 200μM NTPs and is strongly reduced in λ tR2 recognition at 1μM NTPs. F773V alters RNAP pausing and backtracking and favors misincorporation. By contrast, the adjacent Y772A substitution increases fidelity and exhibits other transcriptional defects generally opposite to those of F773V. All atom molecular dynamics simulation revealed two separate functional connections emanating from YFI explaining the distinct effects of substitutions: Y772 communicates with the active site through the link domain in the β subunit, whereas F773 communicates through the fork domain in the β subunit. I774 interacts with the F-loop, which also contacts the glycine hinge of the bridge helix. These results identified negative and positive circuits coupled at YFI and employed for regulation of catalysis, elongation, termination and translocation.

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Figures

Fig. 1
Fig. 1
The bridge helix YFI motif. On the right, an N-terminal end on view of the bridge helix is shown. Two images on the left indicate the RNAP regions surrounding YFI, the F-loop (β′ 736 to 770; Ec RNAP numbering), the fork (β 550 to 570) and the link domain approaching the active site (β 670 to 680). Blue ovals circumscribe YFI. The image is derived from PDB 2O5J [3] after molecular dynamics simulation. Secondary structures of RNAP chains are indicated: purple is α-helix; blue is 310 helix; yellow is β-sheet; white is coil; and cyan is turn. The image was drawn using Visual Molecular Dynamics [40]. A multiple sequence alignment of bridge helix sequences is shown to indicate the relationship of YFI and the adjacent, more N-terminal glycine hinge. Abbreviations: Tt, Thermus thermophilus; Ec, Escherichia coli; Mj, Methanocaldococcus jannaschii; Sc, Saccharomyces cerevisiae.
Fig. 2
Fig. 2
Elongation on a long DNA template comparing F773V and other RNAP mutants. The DNA template is indicated at the top of the figure. Elongation times are 10, 20, 30, 60, 120, 240, and 480 s. Mutants other than F773V are shown for comparison and are not discussed in detail.
Fig. 3
Fig. 3
Bridge helix YFI substitutions affect λ tR2 intrinsic terminator recognition. A) 32P-labeled A32 RNA TECs were initiated from the bacteriophage T7 A1 promoter, walked to A32 and extended up to or through the λ tR2 terminator at 200 and 1 µM NTPs. B) λ tR2 termination efficiency. Triplicate samples at 30 s (200 µM NTPs; black bars) or 40 min (1 µM NTPs; gray bars) were done. Error bars indicate standard deviation.
Fig. 4
Fig. 4
Millisecond phase kinetic analysis of YFI substitutions. 200 µM GTP and ATP were added to G9 RNAP TECs assembled in vitro. Reactions were quenched in a KinTek RQF-3 instrument with EDTA or HCl. A) Wild type RNAP. B) Y772A RNAP. C) F773V RNAP. Data were fit with double or single phase exponential curves (Fig. 6B).
Fig. 5
Fig. 5
Elongation of G9 RNAP TECs at 1 µM GTP and ATP (as in Fig. 4). A) Wild type RNAP. B) Y772A RNAP. C) F773V RNAP.
Fig. 6
Fig. 6
Reproducibility and model-independent analysis of the fast kinetic experiment. A) HCl quench data at 200 µM GTP + ATP (as in Fig. 4) was obtained in triplicate for wild type and Y772A RNAPs and compared to F773V RNAP. Error bars indicate standard deviation of three replicates. B) Model-independent analysis. Apparent rates (s−1) and amplitudes (% of TECs) based on double or single phase exponential curve fitting. Error bars indicate standard error.
Fig. 7
Fig. 7
YFI substitutions Y772A and F773V strongly and conversely affect transcriptional fidelity. A) Normalized AMP (from ATP) misincorporation for GMP (from GTP) done as an ATP versus GTP competition experiment in the presence of high ATP and low GTP. A*10A11 indicates misincorporation of AMP for GMP at A*10 followed by accurate incorporation at A11. Phosphorimager quantification is shown at the right. Error bars indicate standard deviation. B) Misincorporation rates for UMP (from UTP) in place of CMP (with no CTP added). Phosphorimager quantification is shown at the bottom of the figure.
Fig. 8
Fig. 8
RNAP C14 TEC downstream border Exo III mapping indicating the slow post → pre and pre → backtracked transitions. A) Protocol and schematic. In vitro assembled Ec RNAP G9 TECs were extended to G10 and washed. After releasing TECs from beads, ATP and CTP were added together with Exo III to extend the TEC to C14 and to map the downstream RNAP border, as indicated. Filled asterisk indicates the position of the 5′ DNA radiolabel. Open asterisk indicates that the template DNA strand is blocked from Exo III digestion using a thio linkage. B) Exo III mapping (20–90 s incubation at 25 °C) and RNA gel data. 500 µM GMPcPP was added to the indicated reactions as a non-incorporatable GTP analog to stabilize the hyper-, post- and pre-translocated TEC Exo III gel bands. Rectangles and ovals highlight apparent differences in the stability of DNA A51 for wild type and Y772A RNAPs.
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