Concerted Protein and Nucleic Acid Conformational
Changes Observed Prior to Nucleotide Incorporation in a Bacterial
RNA Polymerase: Raman Crystallographic Evidence
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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 <i>Thermus thermophilus</i> elongation complex (TthEC)
is reported. When [<sup>13</sup>C,<sup>15</sup>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, <i>Thermus thermophilus</i> (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) <i>Biochemistry
54</i>, 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