Hydroxyl Radical Probing Research Articles

New Insights into the Phototoxicity of Anthracene-Based Chromophores: The Chloride Salt Effect†.

Unraveling the causes underlying polycyclic aromatic hydrocarbon phototoxicity is an essential step in understanding the harmful effects of these compounds in nature. Toward this end, we have studied the DNA interactions and photochemistry of N1-(anthracen-9-ylmethyl)ethane-1,2-diaminium dichloride in the presence and absence of NaF, KF, NaCl, KCl, NaBr, KBr, NaI, and KI (350 nm hν, pH 7.0). Exposing pUC19 plasmid to UV light in solutions containing 400 mM KCl formed significantly more direct strand breaks in DNA compared to no-salt control reactions. In contrast, NaCl increased DNA damage moderately, while the sodium(I) and potassium(I) fluoride, bromide, and iodide salts generally inhibited cleavage (I- > Br- > F-). A halide anion-induced heavy-atom effect was indicated by monitoring anthracene photodegradation and by employing the hydroxyl radical (•OH) probe hydroxyphenyl fluorescein (HPF). These studies revealed that among no-salt controls and the eight halide salts, only NaCl and KCl enabled the anthracene to photosensitize the production of high levels of DNA-damaging reactive oxygen species (ROS). Pre-irradiation of N1-(anthracen-9-ylmethyl)ethane-1,2-diaminium dichloride at 350 nm increased the amounts of chloride salt-induced •OH detected by HPF in subsequent anthracene photoactivation experiments. Taking into consideration that •OH and other highly reactive ROS are extremely short-lived, this result suggests that the pre-irradiation step might lead to the formation of oxidized anthracene photoproducts that are exceedingly redox-active. The fluorometric probes HPF and Singlet Oxygen Sensor Green revealed that KCl concentrations ranging from 150 to 400 mM and from 100 to 400 mM, respectively, enhanced N1-(anthracen-9-ylmethyl)ethane-1,2-diaminium dichloride photosensitized •OH and singlet oxygen (1O2) production over no-salt controls. Considering the relatively high levels of Na+, K+, and Cl- ions that exist in the environment and in living organisms, our findings may be relevant to the phototoxic effects exhibited by anthracenes and other polycyclic hydrocarbons in vivo.

A study on photodegradation of trichloroethylene using an optical fiber coated with different photocatalysts

In this research, optical fiber photocatalytic technology was used to treat trichloroethylene (TCE) in simulated groundwater. The study investigated the effects of various conditions on TCE treatment, including the type and modification method of photocatalyst, pH value of the solution, and pollutant concentration on the photocatalytic reaction rate and hydroxyl radical conversion rate (ROH,UV). The objective was to improve the transmission and extension of light energy using the high light-conducting properties of optical fibers. This technique is particularly useful in areas where light transmission is obstructed and can address the issues associated with traditional photocatalytic treatment methods, such as photocatalyst recovery. Soil reaction flasks were used as containers, and a LED as the experimental light source. The coated photocatalysts were analyzed for their basic characteristics using SEM/EDS, UV–Vis DRS, and XRD. In addition, p-chlorobenzoic acid (pCBA) as a hydroxyl radical (･OH) probe for kinetic study and calculation of ROH,UV. The results show that N-doped TiO2 (NT) with the addition of 10 mL NH4OH improved TCE removal efficiency at sampling point A from about 20% to 49.6%, while Ag-doped TiO2 (AT) was increased to 53.8% with the addition of 0.5 g AgNO3. These two dosages had the highest hydroxyl radical generation efficiency compared to other higher dosage conditions. However, excessive dosage can inhibit crystal formation. The study also conducted reaction rates under different pH solution environments, showing that NT had the highest reaction rate (kapp) at pH 7 and AT at pH 10. Overall, this research highlights the potential of optical fiber photocatalytic technology for TCE treatment in groundwater and provides insights into the factors that affect its efficiency.

Degradation of 1,2,3-trichloropropane by pyrite activating sodium percarbonate and the implications for groundwater remediation

1,2,3-trichloropropane (TCP) is a newly regulated groundwater contaminant, however, its remediation technology has not been fully investigated. In this research, pyrite (FeS2) was used as a novel activator of sodium percarbonate (SPC) for TCP degradation. TCP could be effectively removed by FeS2/SPC system with a much higher removal efficiency than the classic Fenton system. Free radical quenching test, hydroxyl radical probe reaction, EPR, XPS, XRD and SEM were used to elucidate the reaction mechanism. SPC was decomposed to produce H2O2, which was activated by Fe2+ dissolved from pyrite to produce HO· and O2-·. Both HO· and O2-· contributed to TCP degradation, but HO· played a more important role. The S elements of pyrite promoted the regeneration of Fe2+, thus enhancing the TCP degradation. The TCP removal efficiency significantly increased with increases in concentrations of pyrite and SPC and decreases in pyrite particle size and pH. Although HCO3-, Cl- and NOM inhibited TCP degradation by FeS2/SPC, the reaction system could still remove TCP in real groundwater. Overall, pyrite-activate SPC is a promising in-situ groundwater remediation technology.

Simulating micropollutant abatement during cobalt mediated peroxymonosulfate process by probe-based kinetic models

Prediction of micropollutant abatement during advanced oxidation processes by modelling can provide valuable information to optimize the process for efficient water treatment. In this study, a probe-based kinetic model was developed to simulate micropollutant abatement during water treatment by a cobalt-mediated peroxymonosulfate (Co(II)/PMS) process. Eight micropollutants (atrazine, primidone, sulfisoxazole, sulfamethoxypyridazine, gemfibrozil, chloramphenicol, p-chlorobenzoic acid, and perfluorooctanoic acid) were spiked in various water matrices (synthetic water and real surface water), which were then treated by the Co(II)/PMS process. By following the decay of PMS, atrazine, and primidone (as sulfate and hydroxyl radical (SO4·- and ·OH) probes) during the process, the exposures of PMS, SO4·- and ·OH were calculated using the model. Then, based on the determined exposures, the abatement efficiencies of the other micropollutants could be reasonably predicted with the model. The results indicate that by using a couple of probes to measure the SO4·- and ·OH exposures, the model can be used for the generalized prediction of the abatement of various micropollutants during water treatment by the Co(II)/PMS process. In addition, modelling results show that for a given water matrix, the ratios of SO4·- and ·OH concentrations to PMS concentrations (RSP and RHP) are almost constant during the Co(II)/PMS process. This finding allows the transient concentrations of SO4·- and ·OH to be quantified based on the constant RSP and RHP ratios and the PMS concentrations observed during the process. The results of this study suggest that the probe-based kinetic models can provide a useful tool to characterize the persulfate-based process and to predict the abatement efficiencies of various micropollutants during water treatment.

Are microbubbles magic or just small? a direct comparison of hydroxyl radical generation between microbubble and conventional bubble ozonation under typical operational conditions

The application of microbubbles for water treatment is an emerging technology which has been shown to significantly enhance gas–liquid contacting processes. When applied to ozonation, microbubble technology has been shown to enhance mass transfer and the speed and extent of compound removal compared with conventional bubbling techniques. One explanation as to why microbubble systems outperform conventional systems is that microbubbles shrink, collapse and spontaneously generate hydroxyl radicals which is thought to enhance the speed of compound removal. This study compared microbubble (mean diameter 37 μm) and conventional bubble (mean diameter 5.4 mm) ozonation systems under identical conditions. The experiments were normalised for effective ozone dose to determine whether microbubble ozonation generated significantly more hydroxyl radicals than conventional bubble ozonation. 4-chlorobenzoic was used as the hydroxyl radical probe and the proportion of hydroxyl radicals generated for a given effective ozone dose was quantified. The •OH-exposure to O3-exposure (the Rct) was used to compare the systems. The ratio of the mean RctMicrobubble to mean RctConventional was 0.73, 0.84 and 1.12 at pH 6, 7 and 8 respectively. Statistical assessment of the Rct showed that there was no significant difference between the bubble systems. No evidence was found to support the hypothesis that microbubble systems generate more •OH. Instead, the level of •OH-exposure is linked to the effective dose and pH of the system and future designs should focus on those factors to deliver •OH based benefits.

Linear Relationship between Temperature and the Apparent Reaction Rate Constant of Hydroxyl Radical with 4-chlorobenzoic Acid

ABSTRACT 4-Chlorobenzoic acid (p-CBA) is frequently used as a hydroxyl radical (HO·) probe substance in studies of ozonation and advanced oxidation processes. However, the temperature dependence of the reaction between HO· and p-CBA remains unclear. In this context, we identified the relationship between temperature ( T , K) and the apparent second-order reaction rate constant of HO· with p- CBA ( k H O ⋅ , p − C B A T , M−1 s−1): 10 10 . They were measured by a novel competitive method using 2-methylpropan-2-ol (tert-butyl alcohol) as a reference substance in the range of 1.0–40.0℃. The linear regression equation was more appropriate than the exponential regression equation to express this relationship. More generally, our simulation shows that the linear regression equation can be more accurate than the exponential regression equation to express the relationship between temperature and apparent reaction rate constants of HO.

Occurrence of unknown reactive species in UV/H2O2 system leading to false interpretation of hydroxyl radical probe reactions

The UV/H2O2 process is a benchmark advanced oxidation process (AOP) that in situ generates highly reactive and nonselective hydroxyl radical (•OH) to oxidatively destroy a wide range of organic compounds. Accurately quantifying the concentration of short-lived •OH is essential to predict process performance, optimize the operation parameters, and compare with other process options. The •OH concentration is typically measured using organic probe molecules that react with •OH but not with other oxidants. In the extremely well-characterized UV/H2O2 system in which •OH is proven to be the dominant oxidant, using photolysis-resistant probes such as benzoic acid and its derivatives is a widely agreed and practiced norm. We herein report that certain •OH probe compounds can be degraded in UV/H2O2 system by unknown reactive species that has not been reported in the past. Several common organic probes, particularly p-substituted benzoic acid compounds (i.e., p-hydroxybenzoic acid, p-chlorobenzoic acid, and p-phthalic acid), were found to be vulnerable to attack by the unknown reactive species, leading to false quantification of •OH concentration under high radical scavenging conditions. Lines of evidence obtained from a series of •OH scavenging experiments performed under various conditions (i.e., different concentrations of H2O2, •OH probe compounds, and dissolved oxygen) point toward excited state H2O2. The results from this study suggest the importance of using appropriate •OH probe compounds in mechanistic studies and needs for considering the unidentified role of excited state of H2O2 on the UV/H2O2 process and related AOPs.

Richard Jackson (1940–2020) ‐ A towering presence in translation

Richard Jackson (1940–2020) ‐ A towering presence in translation

Platinum Nanozyme-Enabled Colorimetric Determination of Total Antioxidant Level in Saliva.

Redox imbalance and oxidative stress-related biomarkers are raising increasing consensus in the scientific community for their significant role in a wide range of human disorders. In this framework, the total antioxidant capacity (TAC), namely, the overall pattern of both enzymatic and nonenzymatic antioxidant compounds within the body, represents an important bioanalytical parameter. To date, however, antioxidant assays require costly instrumentations, laboratory setups, and reagents, and they are invasive. Yet, their accuracy typically suffers from strong sensitivity to interfering matrices and inability to detect the complete pattern of physiological antioxidant molecules, due to the use of reaction schemes and probes/substrates that are not sensitive to the diverse range of relevant target species. Here, we exploit the enzyme-mimetic properties of platinum nanoparticles combined with hydroxyl radical probes produced at the particle surface to develop an effective detection scheme that is sensitive to both single electron transfer (SET) and hydrogen atom transfer (HAT) reactions, thus covering all the physiologically relevant antioxidant species. Importantly, the nanozyme-enabled method allows fast (5 min), accurate, and noninvasive evaluation of the body TAC through saliva via simple naked-eye or smartphone-based inspection.

Binding interface and impact on protease cleavage for an RNA aptamer to HIV-1 reverse transcriptase.

RNA aptamers that bind HIV-1 reverse transcriptase (RT) inhibit RT in enzymatic and viral replication assays. Some aptamers inhibit RT from only a few viral clades, while others show broad-spectrum inhibition. Biophysical determinants of recognition specificity are poorly understood. We investigated the interface between HIV-1 RT and a broad–spectrum UCAA-family aptamer. SAR and hydroxyl radical probing identified aptamer structural elements critical for inhibition and established the role of signature UCAA bulge motif in RT-aptamer interaction. HDX footprinting on RT ± aptamer shows strong contacts with both subunits, especially near the C-terminus of p51. Alanine scanning revealed decreased inhibition by the aptamer for mutants P420A, L422A and K424A. 2D proton nuclear magnetic resonance and SAXS data provided constraints on the solution structure of the aptamer and enable computational modeling of the docked complex with RT. Surprisingly, the aptamer enhanced proteolytic cleavage of precursor p66/p66 by HIV-1 protease, suggesting that it stabilizes the productive conformation to allow maturation. These results illuminate features at the RT-aptamer interface that govern recognition specificity by a broad-spectrum antiviral aptamer, and they open new possibilities for accelerating RT maturation and interfering with viral replication.

Assessment of p-nitroso dimethylaniline (pNDA) suitability as a hydroxyl radical probe: Investigating bleaching mechanism using immobilized zero-valent iron nanoparticles

Use of p-nitroso dimethylaniline (pNDA) as a selective hydroxyl radical (OH) probe compound has been extensively reported in the literature for evaluating the performance of the catalysts used in Advanced Oxidation Processes (AOPs). Some recent studies speculated that pNDA might be oxidized by other oxidizing species, and might also be directly reduced. This study, for the first time, investigates pNDA bleaching mechanism by an Fe0-containing composite, capable of causing both reduction and Fenton-like oxidation. The composite consisted of Santa Barbara-15 (SBA-15) mesoporous silica as the supporting medium for nZVI immobilization (nZVI/SBA-15). The quantity of iron in the composite was optimized by synthesizing four various nZVI/SBA-15 samples with different iron to silica weight ratios and comparing their pNDA bleaching efficiency. Kinetic studies showed a pseudo-second-order model for pNDA bleaching using all nZVI/SBA-15 samples. The effects of OH scavengers and dissolved oxygen concentration on pNDA bleaching rate were examined. FTIR analysis of pNDA solutions showed different molecular structures for pNDA bleached under different DO conditions, leading to the conclusion that pNDA is susceptible to reductive bleaching. Results demonstrated that when using pNDA as a probe for free radicals’ generation assessment, the reductive potential of the catalysts needs to be considered.

Treatment of Trichloroethylene with Photocatalyst-Coated Optical Fiber

In this present study, we investigated the effect of photocatalyzation on the degradation of trichloroethylene (TCE) in the aqueous phase by a photocatalyst-coated plastic optical fiber (POF). Two light-emitting diodes (LEDs) with low light intensity were used as the light source and TiO2 and ZnO were used as photocatalysts, which were characterized by scanning electron microscope (SEM) and UV-Vis diffuse reflectance spectroscopy (DRS). The para-chlorobenzoic acid (pCBA) was used as the hydroxyl radical probe for kinetic study and for the calculation of hydroxyl radical conversion rate (ROH,UV ). Experimental results show that POF coated with TiO2 exhibited higher degradation efficiency of TCE in basic solution, but POF coated with ZnO performed better in acidic solution. The increase of coating times resulted in the decrease in degradation efficiency of TCE due to increased thickness of the photocatalyst layer. The enhancement of light intensity contributed to the improvement of photocatalytic treatment efficiency. The ROH,UV for TiO2 and ZnO coated POF increased from 2 × 103 to 8 × 103 M s cm2 mJ−1 and from 8 × 102 to 2 × 103 M s cm2 mJ−1, respectively, as the pH increased from 4 to 10.

Confirming the Formation of Hydroxyl Radicals in the Catalytic Decomposition of H2O2 on Metal Oxides Using Coumarin as a Probe

AbstractHydrogen peroxide can be catalytically decomposed to O2 and H2O on metal oxide surfaces in contact with aqueous solutions containing H2O2. The initial step in this process has been proposed to be the formation of surface‐bound hydroxyl radicals which has recently been verified using tris as a radical scavenger. Here, we make use of the unique fluorescent product 7‐hydroxycoumarin formed in the reaction between hydroxyl radicals and coumarin to probe the formation of surface‐bound hydroxyl radicals. The experiments clearly show that 7‐hydroxycoumarin is formed upon catalytic decomposition of H2O2 in aqueous suspensions containing ZrO2‐particles and coumarin, thereby confirming the formation of surface‐bound hydroxyl radicals in this process. The results are quantitatively compared to results on the same system using tris as a probe for hydroxyl radicals. The effects of the two probes on the system under study are compared and it is concluded that coumarin has a significantly lower impact on the system.

Abatement of ozone-recalcitrant micropollutants during municipal wastewater ozonation: Kinetic modelling and surrogate-based control strategies

Although ozonation is nowadays recognized as one of the most efficient technologies for micropollutants abatement in municipal wastewater effluents, several of the compounds potentially present in those waters exhibit a strong resistance to direct ozone oxidation. In addition, the real-time control of the removal process is still challenging. In this work, the abatement of ozone-recalcitrant micropollutants during wastewater ozonation of six different wastewater effluents was explored using the pesticide acetamiprid as hydroxyl radical (OH) probe. By means of this data, the oxidation efficiency (i.e., hydroxyl radical exposure per consumed ozone) could be described by means of a two-stage model based on the ROHO3 concept. This was possible using a semi-continuous bubbling ozone contactor in all experiments, which permitted the inclusion of the ozone mass balance in the model. ROHO3 values of (1.53–7.60)·10−7 s for initial ozonation stage and (0.61–2.95)·10−6 s for the secondary stage were obtained allowing the characterization and comparison of the process performance in a wide range of effluent qualities, including water matrices with a high content of dissolved and particulate organic matter (total organic carbon (TOC), dissolved organic carbon (DOC) and turbidity tested ranges: 6.7–50 mg C L−1, 6.6–27.6 mg C L−1 and 0.3–28.6 NTU, respectively). Finally, a surrogate strategy involving OH exposure estimation by means of ultraviolet absorbance at 254 nm (UVA254) measurements was proposed based on the ROHO3 concept, and by means of its application the removal of atrazine and ibuprofen in six different wastewaters could be rightly predicted (R2 > 0.98).

Synergy of combined adsorption and electrochemical degradation of aqueous organics by granular activated carbon particulate electrodes

Abstract Electrochemical oxidation (EO) and activated carbon (AC) adsorption are, despite difference in maturation and market dissemination, both well-proven water treatment principles for the abatement of micropollutants. Both technologies suffer some drawbacks as mass transfer limitations (in case of EO) and poor adsorption affinity of some compounds (in case of AC). Granular active carbon may, when placed within an electric field, be polarized generating particulate reactive microelectrodes in bulk solution increasing the overall active electrode area, a concept known as 3D electrochemistry. In this paper, a potential synergy by combining EO and AC was studied in a potentiostatic batch setup at different applied electric field strengths (25–500 V/m), using boron-doped diamond (BDD) as active anode and the hydroxyl radical probe compound p-nitrosodimethylaniline (RNO) and the groundwater contaminants 2-methyl-4-chlorophenoxy acetic acid (MCPA), 2-methyl-4-chlorophenoxy propionic acid (MCPP or mecoprop) and 2,6-benzamide (BAM) as target model contaminants. Synergy was assessed based on comparison of the 3D process with removal kinetics in conventional 2D electrochemical process and pure AC adsorption. In demineralized water model solutions, synergies of 121–126% was found for RNO, MCPA and MCPA at 375 V/m electric field strength and w/w AC:organic ratio of 5:1. For BAM, the synergy was 192%, primarily due to stronger AC adsorption affinity. The study showed interesting perspectives of this treatment concept that needs to be pursued and studied in matrices of higher complexity.

Terephthalate Probe for Hydroxyl Radicals: Yield of 2-Hydroxyterephthalic Acid and Transition Metal Interference

ABSTRACTHydroxyl radicals (.OH) are key players in chemistry in surface waters, clouds, and aerosols. Additionally, .OH may contribute to the inflammation underlying adverse health outcomes associated with particulate matter exposure. Terephthalate is a particularly sensitive probe for hydroxyl radicals, with a detection limit as low as 2 nM. However, there is uncertainty in .OH quantification using this method, and potential for interferences from some transition metals. Terephthalate reacts with .OH to form a fluorescent product, 2-hydroxyterephthalic acid (hTA), with a moderate dependence on pH and temperature. However, there is disagreement in the literature on the yield of the fluorescent product (YhTA), which introduces a large uncertainty in the quantification of OH. Additionally, TA and similar organic probes are known to complex Cu(II) at high concentrations; thus, if this reaction is important at lower concentrations, Cu(II) could reduce apparent hTA formation, and reduce activity of Cu(II) in target samples. Using a pH 3.5 dark ferrous Fenton system to generate .OH radicals, we find that YhTA = 31.5 ± 7%. This is about double the recent literature value measured, but in excellent agreement with earlier measurements. Additionally, we find that interactions between Cu(II) and hTA are small enough to be ignored at Cu(II) concentrations below ∼50 µM.

Persulfate activation by glucose for in situ chemical oxidation

Sodium persulfate has become the most popular oxidant source for the in situ chemical oxidation (ISCO) treatment of organic contaminants in the subsurface. The most common persulfate activators, iron chelates and base, are often ineffective in initiating the generation of reactive oxygen species in field applications. In this study, glucose was investigated as a persulfate activator in systems containing varying concentrations of sodium hydroxide using nitrobenzene as a hydroxyl radical probe and hexachloroethane as a reductant + nucleophile probe. Glucose activation of persulfate increased as a function of sodium hydroxide addition, but was still effective at circumneutral pH regimes. Use of central composite rotatable experimental designs showed that hydroxyl radical and reductant + nucleophile generation rates increased as a function of persulfate at near-neutral pH regimes. Glucose activation of persulfate has the advantages over other activation pathways of more options and flexibility for effective process chemistry and of minimizing or eliminating the mass of sodium hydroxide added to the subsurface. The results of this research can be applied in the field by first evaluating glucose activation compared to base and iron chelate activation of persulfate in laboratory treatability studies.

Directed hydroxyl radical probing reveals Upf1 binding to the 80S ribosomal E site rRNA at the L1 stalk.

Upf1 is an SF1-family RNA helicase that is essential for the nonsense-mediated decay (NMD) process in eukaryotes. While Upf1 has been shown to interact with 80S ribosomes, the molecular details of this interaction were unknown. Using purified recombinant proteins and high-throughput sequencing combined with Fe-BABE directed hydroxyl radical probing (HTS-BABE) we have characterized the interaction between Upf1 and the yeast 80S ribosome. We identify the 1C domain of Upf1, an alpha-helical insertion in the RecA helicase core, to be essential for ribosome binding, and determine that the L1 stalk of 25S rRNA is the binding site for Upf1 on the ribosome. Using the cleavage sites identified by hydroxyl radical probing and high-resolution structures of both yeast Upf1 and the human 80S ribosome, we provide a model of a Upf1:80S structure. Our model requires that the L1 stalk adopt an open configuration as adopted by an un-rotated, or classical-state, ribosome. Our results shed light on the interaction between Upf1 and the ribosome, and suggest that Upf1 may specifically engage a classical-state ribosome during translation.

Low levels of iron enhance UV/H2O2 efficiency at neutral pH

While the presence of iron is generally not seen as favorable for UV-based treatment systems due to lamp fouling and decreased UV transmittance, we show that low levels of iron can lead to improvements in the abatement of chemicals in the UV-hydrogen peroxide advanced oxidation process. The oxidation potential of an iron-assisted UV/H2O2 (UV254 + H2O2 + iron) process was evaluated at neutral pH using iron levels below USEPA secondary drinking water standards (<0.3 mg/L). Para-chlorobenzoic acid (pCBA) was used as a hydroxyl radical (HO) probe to quantify HO steady state concentrations. Compounds degraded by different mechanisms including, carbamazepine (CBZ, HO oxidation) and N-nitrosodimethylamine (NDMA, direct photolysis), were used to investigate the effect of iron on compound degradation for UV/H2O2 systems. The effects of iron species (Fe2+ and Fe3+), iron concentration (0–0.3 mg/L), H2O2 concentration (0–10 mg/L) and background water matrix (low-carbon tap (LCT) and well water) on HO production and compound removal were examined. Iron-assisted UV/H2O2 efficiency was most influenced by the target chemical and the water matrix. Added iron to UV/H2O2 was shown to increase the steady-state HO concentration by approximately 25% in all well water scenarios. While CBZ removal was unchanged by iron addition, 0.3 mg/L iron improved NDMA removal rates in both LCT and well water matrices by 15.1% and 4.6% respectively. Furthermore, the combination of UV/Fe without H2O2 was also shown to enhance NDMA removal when compared to UV photolysis alone indicating the presence of degradation pathways other than HO oxidation.

The TFIIE-related Rpc82 subunit of RNA polymerase III interacts with the TFIIB-related transcription factor Brf1 and the polymerase cleft for transcription initiation.

Rpc82 is a TFIIE-related subunit of the eukaryotic RNA polymerase III (pol III) complex. Rpc82 contains four winged-helix (WH) domains and a C-terminal coiled-coil domain. Structural resolution of the pol III complex indicated that Rpc82 anchors on the clamp domain of the pol III cleft to interact with the duplex DNA downstream of the transcription bubble. However, whether Rpc82 interacts with a transcription factor is still not known. Here, we report that a structurally disordered insertion in the third WH domain of Rpc82 is important for cell growth and in vitro transcription activity. Site-specific photo-crosslinking analysis indicated that the WH3 insertion interacts with the TFIIB-related transcription factor Brf1 within the pre-initiation complex (PIC). Moreover, crosslinking and hydroxyl radical probing analyses revealed Rpc82 interactions with the upstream DNA and the protrusion and wall domains of the pol III cleft. Our genetic and biochemical analyses thus provide new molecular insights into the function of Rpc82 in pol III transcription.