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

• Published in: Biochimica et biophysica acta 2013-02, Vol.1829 (2), p.187-198
• Main Authors: Nedialkov, Yuri A., Opron, Kristopher, Assaf, Fadi, Artsimovitch, Irina, Kireeva, Maria L., Kashlev, Mikhail, Cukier, Robert I., Nudler, Evgeny, Burton, Zachary F.
• Format: Article
• Language: English
• Subjects: Amino Acid Motifs; Amino Acid Sequence; Binding Sites; Bridge helix; Catalysis; Catalytic Domain; DNA-Directed RNA Polymerases - chemistry; DNA-Directed RNA Polymerases - metabolism; Escherichia coli; Escherichia coli - enzymology; Kinetics; Molecular Dynamics Simulation; Molecular Sequence Data; Mutagenesis; Pausing; Protein Conformation; Protein Structure, Secondary; RNA polymerase; Termination; Transcriptional fidelity; Translocation
• ISSN: 1874-9399; 0006-3002; 1876-4320
• DOI: 10.1016/j.bbagrm.2012.11.005

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. ► The bridge helix contains a bendable glycine hinge. ► Adjacent bulky hydrophobic residues and surrounding protein regulate the hinge. ► Mutations in these bulky residues modify transcription elongation. ► Pausing, catalysis, fidelity, termination and translocation are affected.