Concerted Protein and Nucleic Acid Conformational Changes Observed Prior to Nucleotide Incorporation in a Bacterial RNA Polymerase: Raman Crystallographic Evidence

Abstract

Transcription elongation requires the continuous incorporation of ribonucleotide triphosphates into a growing transcript. RNA polymerases (RNAPs) are able to processively synthesize a growing RNA chain via translocation of the RNAP enzyme along its nucleic acid template strand after each nucleotide addition cycle. In this work, a time-resolved Raman spectroscopic analysis of nucleotide addition in single crystals of the Thermus thermophilus elongation complex (TthEC) is reported. When [(13)C,(15)N]GTP (*GTP) is soaked into crystals of the TthEC, large reversible changes in the Raman spectrum that are assigned to protein and nucleic acid conformational events during a single-nucleotide incorporation are observed. The *GTP population in the TthEC crystal reaches a stable population at 37 min, while substantial and reversible protein conformational changes (mainly ascribed to changes in α-helical Raman features) maximize at approximately 50 min. At the same time, changes in nucleic acid bases and phosphodiester backbone Raman marker bands occur. Catalysis begins at approximately 65-70 min, soon after the maximal protein and DNA changes, and is monitored via the decline in a triphosphate vibrational Raman mode from *GTP. The Raman data indicate that approximately 40% of the total triphosphate population, present as *GTP, reacts in the crystal. This may suggest that a second population of noncovalently bound *GTP resides in a site distinct from the catalytic site. The data reported here are an extension of our recent work on the elongation complex (EC) of a bacterial RNAP, Thermus thermophilus (Tth), where Raman spectroscopy and polyacrylamide gel electrophoresis were employed to monitor incorporation and misincorporation in single TthEC crystals [Antonopoulos, I. H., et al. (2015) Biochemistry 54, 652-665]. Therefore, the initial study establishes the groundwork for this study. In contrast to our previous study, in which incorporation takes place very rapidly inside the crystals, the data on this single crystal exhibit a slower time regime, which allows the dissection of the structural dynamics associated with GMP incorporation within the TthEC crystal.

Probing the Hydrogen-Bonding Environment of Individual Bases in DNA Duplexes with Isotope-Edited Infrared Spectroscopy

“…In nucleic acids, previous studies examined the conformation of adenine bases within DNA duplexes and DNA/protein complexes using deuterium-labeled adenine bases. More recent studies employed isotope editing experiments using commercially available, fully 15 N- and 13 C-labeled RNA bases to track incorporation of a single labeled base using Raman microscopy during an RNA polymerase reaction. − However, to the best of our knowledge, to date, no isotope editing studies have been performed that label an individual CO in nucleic acids.…”

Large α‐helical movements observed in ternary crystals of RB69 DNA polymerase following nucleotide incorporation

“…the broad band centered around 1160 cm −1 and the bands near 1072 and 1021 cm −1 . We have seen similar bands in the RNA polymerase T. thermophilus crystals during translocation, and we have tentatively assigned them to strained noncanonical phosphodiester bonds present as the backbone makes a bend in the active site. In addition, bands in the 1000–1100 cm −1 region may have contributions from pyrophosphate .…”