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Statistics of data collection and structure refinement.

Statistics of data collection and structure refinement.</p

Purification of QL-nanoKAZ from 800 mL of cultured <i>E</i>. <i>coli</i> cells using a Ni-chelate column.

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Inhibition of luminescence activity of CTZ-utilizing luciferases with deaza-coelenterazine (daCTZ) analogs as inhibitors.

Inhibition of luminescence activity of CTZ-utilizing luciferases with deaza-coelenterazine (daCTZ) analogs as inhibitors.</p

Expression of QL-nanoKAZ in the presence or absence of the secretory signal peptide sequence from <i>Gaussia</i> luciferase (GLsp) in CHO-K1 cells.

Expression of QL-nanoKAZ in the presence or absence of the secretory signal peptide sequence from Gaussia luciferase (GLsp) in CHO-K1 cells.</p

Comparison of the secondary structures between nanoKAZ and QL-nanoKAZ.

The amino acid sequences of nanoKAZ and QL-nanoKAZ are shown with their positions of the secondary structure, and the letters highlighted in orange indicate the substituted 16 amino acid residues in wild KAZ to prepare reverse mutations of nanoKAZ. The cylinders and arrows indicate the regions of α-helices (yellow, α1–α4) and β-strands (blue, β1–β11), respectively. The green in the cylinder (α3) and the arrows (β6 and β7) in QK-nanoKAZ indicate the structural differences compared to nanoKAZ. Tyr 109 is highlighted in red.</p

Luminescence properties of QL-nanoKAZ.

A. Luminescence kinetics of QL-nanoKAZ with CTZ and its analogs as substrates. B. Normalized luminescence spectra of QL-nanoKAZ with CTZ and its analogs, based on the luminescence intensity of QL-nanoKAZ with CTZ. C. Linearity of luminescence intensity (Imax) of QL-nanoKAZ with CTZ, in comparison with nanoKAZ, SNH-nanoKAZ, GLase, and aequorin at the protein concentrations of 0.3 pg to 3 ng (n = 6). Solid and dashed lines represent blank + 3 SD for aequorin and the CTZ-utilizing luciferases, respectively.</p

Time-Resolved Raman and Polyacrylamide Gel Electrophoresis Observations of Nucleotide Incorporation and Misincorporation in RNA within a Bacterial RNA Polymerase Crystal

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 <i>Thermus thermophilus</i> EC (TthEC) RNAP. When the cognate base GTP, labeled with ¹³C and ¹⁵N (*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, ¹³C- and ¹⁵N-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

Primer list used for site-directed mutagenesis to prepare reverse mutant genes for nanoKAZ by PCR.

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Luminescence reaction of coelenterazine (CTZ) catalyzed by the CTZ-utilizing luciferase and chemical structures of CTZ analogs and deaza-CTZ analogs.

A. Oxidation process of CTZ with O2 by CTZ-utilizing luciferases and the degradation product of coelenteramine (CTM), 4-hydroxyphenylacetic acid (4HPAA), and 4-hydroxyphenylpyruvic acid (4HPPA) through 2-peroxycoelenterazine (CTZ-OOH). B. Chemical structures of C2- and C6-modified CTZ analogs. The C6-group of CTZ analogs was colored in red, and the C2- and C8-groups of CTZ analogs were colored in blue. C. Chemical structures of deaza-analogs for CTZ and CTZ-OOH as inhibitors.</p