The bacterial RNA polymerase (RNAP) elongation complex (EC) is highly stable and is able to extend an RNA chain for thousands of nucleotides. Understanding the processive mechanism of nucleotide addition requires detailed structural and temporal data for the EC reaction. Here, a time-resolved Raman spectroscopic analysis is combined with polyacrylamide gel electrophoresis (PAGE) to monitor nucleotide addition in single crystals of the Thermus thermophilus EC (TthEC) RNAP. When the cognate base GTP, labeled with (13)C and (15)N (*GTP), is soaked into crystals of the TthEC, changes in the Raman spectra show evidence of nucleotide incorporation and product formation. The major change is the reduction of *GTP's triphosphate intensity. Nucleotide incorporation is confirmed by PAGE assays. Both Raman and PAGE methods have a time resolution of minutes. There is also Raman spectroscopic evidence of a second population of *GTP in the crystal that does not become covalently linked to the nascent RNA chain. When this population is removed by "soaking out" (placing the crystal in a solution that contains no NTP), there are no perturbations to the Raman difference spectra, indicating that conformational changes are not detected in the EC. In contrast, the misincorporation of the noncognate base, (13)C- and (15)N-labeled UTP (*UTP), gives rise to large spectroscopic changes. As in the GTP experiment, reduction of the triphosphate relative intensity in the Raman soak-in data shows that the incorporation reaction occurs during the first few minutes of our instrumental dead time. This is also confirmed by PAGE analysis. Whereas PAGE data show *GTP converts 100% of the nascent RNA 14mer to 15mer, the noncognate *UTP converts only ∼50%. During *UTP soak-in, there is a slow, reversible formation of an α-helical amide I band in the Raman difference spectra peaking at 40 min. Similar to *GTP soak-in, *UTP soak-in shows Raman spectoscopic evidence of a second noncovalently bound *UTP population in the crystal. Moreover, the second population has a marked effect on the complex's conformational states because removing it by "soaking-out" unreacted *UTP causes large changes in protein and nucleic acid Raman marker bands in the time range of 10-100 min. The conformational changes observed for noncognate *UTP may indicate that the enzyme is preparing for proofreading to excise the misincorporated base. This idea is supported by the PAGE results for *UTP soak-out that show endonuclease activity is occurring.

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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.

Raman difference spectroscopy is used to monitor the conformational changes associated with dNMP incorporation in single crystals of a RB69 DNA polymerase (RB69pol) ternary complex. An inactive crystal of L415G RB69pol exo‐ with a primer/template duplex and a cognate dATP was produced in the presence of Ca2+. The Raman spectrum of the crystallized complex was obtained using a Raman microscope. Phosphodiester bond formation is triggered by soaking in Mg2+. The Raman difference spectra [ie, (ternary complex + Mg2+)–(ternary complex with no Mg2+)] show that conformational changes are complete prior to the acquisition of the first Raman spectrum following magnesium incorporation into the crystal. Polyacrylamide gel electrophoresis experiments carried in parallel show that dNMP incorporation occurs rapidly in the crystal. The Raman data indicate that significant changes in protein amide features, tyrosine ring modes, and nucleic acid base backbone modes have occurred. Overall, they are dominated by the dichroic effect with the molecular groups changing orientation with respect to the laser beam following nucleotide incorporation. The amide I and amide III α‐helix features are unusually narrow and are assigned to movement of the P and N helices. These results demonstrate the ability of Raman spectroscopy to monitor conformational changes in proteins via dichroism. The Raman spectra and the gel electrophoresis support the hypothesis that the major spectroscopic changes are because of structural changes in the ternary complex crystal following correct dNMP incorporation. Copyright © 2015 John Wiley & Sons, Ltd.