Various complexes that contain the core subunits of RNA polymerase II associated with different transcription factors have been isolated from eukaryotes; their precise molecular constitution depends on the purification procedure. We estimated the numbers of various components of such complexes in an HeLa cell by quantitative immunoblotting. The cells were lysed with saponin in a physiological buffer; ϳ140,000 unengaged polymerases (mainly of form IIA) were released. Only ϳ4,000 of these soluble molecules sedimented in glycerol gradients as holoenzyme-sized complexes. About 180,000 molecules of polymerases (ϳ110,000 molecules of form IIO) and 10,000 to 30,000 molecules of each of TFIIB, TFIIE␣, TFIIE␤, TFIIF-RAP74, TFIIF-RAP30, and TFIIH-MAT1 remained tightly associated with the nuclear substructure. Most proteins and run-on activity were retained when ϳ50% of the chromatin was detached with a nuclease, but ϳ45,000 molecules of bound TATA binding protein (TBP) were detached. Similar results were obtained after cross-linking living cells with formaldehyde. The results provide little support for the existence of a large pool of soluble holoenzyme; they are consistent with TBP-promoter complexes in nuclease-sensitive chromatin being assembled into preinitiation complexes attached to the underlying structure.

Sulfur oxides (SO x ) are important atmospheric trace species in both gas and particulate phases, and sulfate is a major component of atmospheric aerosol. One potentially important source of particulate sulfate formation is the oxidation of dissolved SO2 by organic peroxides, which comprises a major fraction of secondary organic aerosol (SOA). In this study, we investigated the reaction kinetics and mechanisms between SO2 and condensed-phase peroxides. pH-dependent aqueous phase reaction rate constants between S­(IV) and organic peroxide standards were measured. Highly oxygenated organic peroxides with O/C > 0.6 in α-pinene SOA react rapidly with S­(IV) species in the aqueous phase. The reactions between organic peroxides and S­(IV) yield both inorganic sulfate and organosulfates (OS), as observed by electrospray ionization ion mobility mass spectrometry. For the first time, 34S-labeling experiments in this study revealed that dissolved SO2 forms OS via direct reactions without forming inorganic sulfate as a reactive intermediate. Kinetics of OS formation was estimated semiquantitatively, and such reaction was found to account for 30–60% of sulfur reacted. The photochemical box model GAMMA was applied to assess the implications of the measured SO2 consumption and OS formation rates. Our findings indicate that this novel pathway of SO2–peroxide reaction is important for sulfate formation in submicron aerosol.

Replication of HIV-1 requires the viral Tat protein, which increases the extent of transcription elongation by RNA polymerase II after activation at the single viral long terminal repeat (LTR) promoter. This effect of Tat on transcription requires Tat interactions with a 5 region (TAR) in nascent transcripts as well as Tat-specific cofactors. The present study identifies a cellular protein, TIP30, that interacts with Tat and with an SRB-containing RNA polymerase II complex both in vivo and in vitro. Coexpression of TIP30 specifically enhances transactivation by Tat in transfected cells, and immunodepletion of TIP30 from nuclear extracts abolishes Tat-activated transcription without affecting Tatindependent transcription. These results implicate TIP30 as a specific coactivator that may enhance formation of a Tat-RNA polymerase II holoenzyme complex.

In contrast to previous findings in cell-free systems reconstituted with partially purified metazoan factors, we demonstrate dramatic activation of transcription in a TBP-dependent but TAFII-independent manner in HeLa nuclear extracts immunodepleted of TBP and major TAFIIs. Single-round transcription assays reveal that TAFII-independent activation is manifested at the level of productive preinitiation complex formation and that TAFIIs actually impair functional preinitiation complex assembly in a core promoter-specific manner. Furthermore, TAFIIs appear to elevate absolute levels of transcription under multiple-round transcription conditions, presumably by facilitating secondary initiation events. Finally, human coactivator activities related to those in yeast RNA polymerase II/mediator complexes appear to function in unfractionated HeLa nuclear extracts.

No abstract

TATA-binding protein-associated factors (TAF II s) within TFIID control differential gene transcription through interactions with both activators and core promoter elements. In particular, TAF II 150 contributes to initiator-dependent transcription through an unknown mechanism. Here, we address whether TAF II s within TFIID are sufficient, in conjunction with highly purified general transcription factors (GTFs), for differential core promoter-dependent transcription by RNA polymerase II and whether additional cofactors are required. We identify the human homologue of Drosophila TAF II 150 through cognate cDNA cloning and show that it is a tightly associated component of human TFIID. More importantly, we demonstrate that the human TAF II 150-containing TFIID complex is not sufficient, in the context of all purified GTFs and RNA polymerase II, to mediate transcription synergism between TATA and initiator elements and initiator-directed transcription from a TAF II -dependent TATA-less promoter. Therefore, TAF II -promoter interactions are not sufficient for the productive core promoter-selective functions of TFIID. Consistent with this finding, we have partially purified novel cofactor activities (TICs) that potentiate the TAF II -mediated synergism between TATA and initiator elements (TIC-1) and TAF II -dependent transcription from TATA-less promoters (TIC-2 and -3). Furthermore, we demonstrate an essential function for TFIIA in TIC-and TAF II -dependent basal transcription from a TATA-less promoter. Our results reveal a parallel between the basal transcription activity of TAF II s through core promoter elements and TAF II -dependent activator function.The structure of the core promoter region (i.e., DNA sequences flanking the transcription start site and including the TATA, initiator, and downstream elements that interact with the general transcription machinery) of protein-coding genes has an important influence both on the efficiency of basal transcription and on the ability of the core promoter to respond to upstream promoter-bound activators in vivo and in vitro (reviewed in references 33, 38, and 46). Although the general transcription machinery has been well characterized, little is known about the factors and mechanisms that control its activity in a core promoter-specific manner. Whereas various factors like E2F, YY1, TFII-I, and USF may regulate transcription not only through upstream promoter elements but also through interactions with the core promoter regions of certain genes (for reviews, see references 38 and 50), several components of the basal transcription machinery have intrinsic and more general core promoter-selective functions. For instance, the differential affinity of TATA-binding protein (TBP) for different TATA box DNA sequences and conformations can influence the efficiency of transcription initiation (28, 37, 39; for a review, see reference 3). Previous studies have also demonstrated differential requirements for the general transcription factors (GTFs) TFIIE, TFIIF, and TFIIH for basal...

Nitrogen oxides (NO x ) play a key role in regulating the oxidizing capacity of the atmosphere through controlling the abundance of O3, OH, and other important gas and particle species. Some recent studies have suggested that particulate nitrate, which is conventionally considered as the ultimate oxidation product of NO x , can undergo “renoxification” via photolysis, recycling NO x and HONO back to the gas phase. However, there are large discrepancies in estimates of the importance of this channel, with reported renoxification rate constants spanning three orders of magnitude. In addition, previous laboratory studies derived the rate constant using bulk particle samples collected on substrates instead of suspended particles. In this work, we study renoxification of suspended submicron particulate sodium and ammonium nitrate through controlled laboratory photolysis experiments using an environmental chamber. We find that, under atmospherically relevant wavelengths and relative humidities, particulate inorganic nitrate releases NO x and HONO less than 10 times as rapidly as gaseous nitric acid, putting our measurements on the low end of recently reported renoxification rate constants. To the extent that our laboratory conditions are representative of the real atmosphere, renoxification from the photolysis of inorganic particulate nitrate appears to play a limited role in contributing to the NO x and OH budgets in remote environments. These results are based on simplified model systems; future studies should investigate renoxification of more complex aerosol mixtures that represent a broader spectrum of aerosol properties to better constrain the photolysis of ambient aerosols.

Particle size distributions in the range of 0.01–10 µm were measured in urban Shanghai in the summer of 2013 using a Wide‐range Particle Spectrometer (WPS). Size‐segregated aerosol samples were collected concurrently using a Micro‐Orifice Uniform Deposit Impactor (MOUDI), which aided our in‐depth understanding of the new particle formation (NPF) mechanism in the polluted Yangtze River Delta area. During the observations, 16 NPF events occurred at high temperatures (~34.7°C) on clear and sunny days. In the ammonium‐poor PM1.0 (particulate matter less than 1.0 µm), sulfate and ammonium accounted for 92% of the total water‐soluble inorganic species. Six aminiums were detected in these MOUDI samples, among which the group of diethylaminium and trimethylaminium (DEAH+ + TMAH+) was the most abundant. The very high level of aminiums (average concentration up to 86.4 ng m−3 in PM1.8), together with highly acidic aerosols, provided insight into the frequent NPF events. The high mass ratio of total aminiums to NH4+ (>0.2 for PM0.056) further highlighted the important role of amines in promoting NPF. The concentration of DEAH+ + TMAH+ in new particles below 180 nm was strongly correlated with aerosol phase acidity, indicating that acid‐base reactions dominated the aminium formation in NPF events. The unexpected enhancement of DEAH+ + TMAH+ on a nonevent day was attributed to the transportation of an SO2 plume. Our results reveal that the heterogeneous uptake of amines is dominated by the acid‐base reaction mechanism, which can effectively contribute to particle growth in NPF events.