Including Protons in Solid-State NMR Resonance Assignment and Secondary Structure Analysis: The Example of RNA Polymerase II Subunits Rpo4/7

Anahit Torosyan,Peter Güntert,Maarten Schledorn,Daniel Klose,Beat H Meier,Thomas Wiegand,Anja Böckmann

doi:10.3389/fmolb.2019.00100

Abstract

1H-detected solid-state NMR experiments feasible at fast magic-angle spinning (MAS) frequencies allow accessing 1H chemical shifts of proteins in solids, which enables their interpretation in terms of secondary structure. Here we present 1H and 13C-detected NMR spectra of the RNA polymerase subunit Rpo7 in complex with unlabeled Rpo4 and use the 13C, 15N, and 1H chemical-shift values deduced from them to study the secondary structure of the protein in comparison to a known crystal structure. We applied the automated resonance assignment approach FLYA including 1H-detected solid-state NMR spectra and show its success in comparison to manual spectral assignment. Our results show that reasonably reliable secondary-structure information can be obtained from 1H secondary chemical shifts (SCS) alone by using the sum of 1Hα and 1HN SCS rather than by TALOS. The confidence, especially at the boundaries of the observed secondary structure elements, is found to increase when evaluating 13C chemical shifts, here either by using TALOS or in terms of 13C SCS.

Highlights

Solid-state NMR and, in particular, proton-detected spectroscopy under fast magic-angle spinning (MAS) allows to characterize larger and larger proteins and protein complexes (Linser et al, 2011; Andreas et al, 2015; Struppe et al, 2017; Schubeis et al, 2018; Bougault et al, 2019)
How these functions are achieved in molecular detail remains elusive, and conformational changes of Rpo4/7 in response to RNA binding have not been detected when probed by labeling techniques, such as fluorescence and electron paramagnetic resonance spectroscopy (Grohmann et al, 2010)
The side chains were mainly assigned by analyzing NCACX and CCC spectra [employing Dipolar Recoupling Enhanced by Amplitude Modulation (DREAM) (Verel et al, 2001; Westfeld et al, 2012) and Dipolar Assisted Rotational Resonance (DARR) (Takegoshi et al, 2003) transfer steps]

Summary

Introduction

Solid-state NMR and, in particular, proton-detected spectroscopy under fast MAS allows to characterize larger and larger proteins and protein complexes (Linser et al, 2011; Andreas et al, 2015; Struppe et al, 2017; Schubeis et al, 2018; Bougault et al, 2019). The archaeal RNAP has served as a model system for dissecting the functions of the individual subunits of the human RNAP II (Werner, 2007, 2008) Two of these subunits, Rpb4/Rpb, that form a stalk-like protrusion in RNAP II, or rather their archaeal homologs Rpo4/Rpo (or Rpo4/7), are known to bind the nascent single-stranded RNA, contribute to transcription initiation as well as termination efficiency and increase processivity during elongation (Meka, 2005; Újvári and Luse, 2006; Grohmann and Werner, 2010, 2011). Rpb4/Rpb, that form a stalk-like protrusion in RNAP II, or rather their archaeal homologs Rpo4/Rpo (or Rpo4/7), are known to bind the nascent single-stranded RNA, contribute to transcription initiation as well as termination efficiency and increase processivity during elongation (Meka, 2005; Újvári and Luse, 2006; Grohmann and Werner, 2010, 2011) How these functions are achieved in molecular detail remains elusive, and conformational changes of Rpo4/7 in response to RNA binding have not been detected when probed by labeling techniques, such as fluorescence and electron paramagnetic resonance spectroscopy (Grohmann et al, 2010). NMR spectroscopy could provide further information at the atomic level

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Results

Conclusion