Recent studies demonstrated the existence of gene loops that juxtapose the promoter and terminator regions of genes with exceptionally long ORFs in yeast. Here we report that looping is not idiosyncratic to long genes but occurs between the distal ends of genes with ORFs as short as 1 kb. Moreover, looping is dependent upon the general transcription factor TFIIB: the E62K (glutamic acid 62 --> lysine) form of TFIIB adversely affects looping at every gene tested, including BLM10, SAC3, GAL10, SEN1, and HEM3. TFIIB crosslinks to both the promoter and terminator regions of the PMA1 and BLM10 genes, and its association with the terminator, but not the promoter, is adversely affected by E62K and by depletion of the Ssu72 component of the CPF 3' end processing complex, and is independent of TBP. We propose a model suggesting that TFIIB binds RNAP II at the terminator, which in turn associates with the promoter scaffold.
The thermodynamics of small-molecule (H(2), arene, alkane, and CO) addition to pincer-ligated iridium complexes of several different configurations (three-coordinate d(8), four-coordinate d(8), and five-coordinate d(6)) have been investigated by computational and experimental means. The substituent para to the iridium (Y) has been varied in complexes containing the (Y-PCP)Ir unit (Y-PCP = eta(3)-1,3,5-C(6)H(2)[CH(2)PR(2)](2)Y; R = methyl for computations; R = tert-butyl for experiments); substituent effects have been studied for the addition of H(2), C-H, and CO to the complexes (Y-PCP)Ir, (Y-PCP)Ir(CO), and (Y-PCP)Ir(H)(2). Para substituents on arenes undergoing C-H bond addition to (PCP)Ir or to (PCP)Ir(CO) have also been varied computationally and experimentally. In general, increasing electron donation by the substituent Y in the 16-electron complexes, (Y-PCP)Ir(CO) or (Y-PCP)Ir(H)(2), disfavors addition of H-H or C-H bonds, in contradiction to the idea of such additions being oxidative. Addition of CO to the same 16-electron complexes is also disfavored by increased electron donation from Y. By contrast, addition of H-H and C-H bonds or CO to the three-coordinate parent species (Y-PCP)Ir is favored by increased electron donation. In general, the effects of varying Y are markedly similar for H(2), C-H, and CO addition. The trends can be fully rationalized in terms of simple molecular orbital interactions but not in terms of concepts related to oxidation, such as charge-transfer or electronegativity differences.
DNA loops that juxtapose the promoter and terminator regions of RNA polymerase II-transcribed genes have been identified in yeast and mammalian cells. Loop formation is transcription-dependent and requires components of the pre-mRNA 39-end processing machinery. Here we report that looping at the yeast GAL10 gene persists following a cycle of transcriptional activation and repression. Moreover, GAL10 and a GAL1p-SEN1 reporter undergo rapid reactivation kinetics following a cycle of activation and repression-a phenomenon defined as ''transcriptional memory''-and this effect correlates with the persistence of looping. We propose that gene loops facilitate transcriptional memory in yeast.Supplemental material is available at http://www.genesdev.org.
The present study explores the nature and reactivity of iron-and zinc-containing species generated in hydrocarbon-oxidizing Gif IV -type solutions (Fe catalyst/Zn/O 2 in pyridine/acetic acid (10:1 v/v). The ultimate goal of this investigation is to unravel the role of metal sites in mediating dioxygen-dependent C-H activation, which in the case of Gif chemistry demonstrates an enhanced selectivity for the ketonization of secondary carbons. Reaction of [Fe 3 O(O 2 CCH 3 ) 6 (py) 3 ]‚py (1) with zinc powder in CH 3 CN/CH 3 COOH or CH 2 Cl 2 /CH 3 COOH affords the trinuclear compound [Zn 2 Fe II (O 2 CCH 3 ) 6 (py) 2 ] (2). Single-crystal X-ray analysis confirms that one monodentate and two bidentate acetate groups bridge adjacent pairs of metals with the iron atom occupying a centrosymmetric position. The analogous reduction of 1 in py/CH 3 COOH (10:1, 5:1, 2:1 v/v) yields [Fe II (O 2 CCH 3 ) 2 (py) 4 ] (3), [Fe II 2 (O 2 CCH 3 ) 4 (py) 3 ] n (4), and [Zn(O 2 CCH 3 ) 2 (py) 2 ] (5) depending on the isolation procedure employed. Compound 3 possesses a distorted octahedral geometry, featuring a C 2 axis bisecting the equatorial, pyridine-occupied plane, whereas the two acetate groups reside along the perpendicular axis. Compound 4 is a one-dimensional solid constructed by asymmetric diferrous units. Two bidentate and one monodentate acetate groups bridge the two iron sites, with the monodentate bridge also acting as a chelator to one ferrous center. The two iron centers exhibit weak antiferromagnetic coupling. Compounds 3 and 4 are also accessible from the reduction of 1 with iron powder or treatment with H 2 /Pd. Solutions of 3 and 4 in pyridine or py/CH 3 COOH react with pure dioxygen or air to eventually regenerate 1 in a concentrationdependent manner. Oxidation of 2 in py/CH 3 COOH with pure dioxygen or air yields [Fe 2.22(2) Zn 0.78(2) O(O 2 CCH 3 ) 6 -(py) 3 ]‚py (1′) and [Zn 2 (O 2 CCH 3 ) 4 (py) 2 ] (6). Compound 1′ is isostructural to 1, exhibiting rhombohedral symmetry at 223 K. The filtrate of the reduction of 1 with zinc in neat pyridine, when exposed to dioxygen, affords dichroic red-green crystals of monoclinic [Fe 2 ZnO(O 2 CCH 3 ) 6 (py) 3 ]‚py (1′′). Species 1′′ yields products identical with those provided by 1 under reducing conditions. Compounds 2-6 are related by pyridine-dependent equilibria, as demonstrated by mutual interconversions and electronic absorption data in pyridine and py/CH 3 COOH solutions. In nonpyridine solutions, Zn-containing species 5 and 6 rearrange to the crystallographically characterized species [Zn-(O 2 CCH 3 ) 2 (py)] n (7) and [Zn 3 (O 2 CCH 3 ) 6 (py) 2 ] (8). Compound 7 is a one-dimensional solid featuring a chain of Zn sites linked by a bidentate acetate group while additionally coordinated by a chelating acetate. Compound 8 is isostructural to 2. Further perturbations of the described structures are apparent in ionic iron-containing species, such as the pseudo-seven-coordinate iron in [Ph 3 PdNdPPh 3 ][Fe II (O 2 CCH 3 ) 3 (py)] (9), which is obtained from the react...
Saccharomyces cerevisiae Pta1 is a component of the cleavage/polyadenylation factor (CPF) 3-end processing complex and functions in pre-mRNA cleavage, poly(A) addition, and transcription termination. In this study, we investigated the role of the N-terminal region of Pta1 in transcription and processing. We report that a deletion of the first 75 amino acids (pta1-⌬75) causes thermosensitive growth, while the deletion of an additional 25 amino acids is lethal. The pta1-⌬75 mutant is defective for snoRNA termination, RNA polymerase II C-terminal domain Ser5-P dephosphorylation, and gene looping but is fully functional for mRNA 3-end processing. Furthermore, different regions of Pta1 interact with the CPF subunits Ssu72, Pti1, and Ysh1, supporting the idea that Pta1 acts as a scaffold to organize CPF. The first 300 amino acids of Pta1 are sufficient for interactions with Ssu72, which is needed for pre-mRNA cleavage. By the degron-mediated depletion of Pta1, we show that the removal of this essential region leads to a loss of Ssu72, yet surprisingly, in vitro cleavage and polyadenylation remain efficient. In addition, a fragment containing amino acids 1 to 300 suppresses 3-end processing in wild-type extracts. These findings suggest that the amino terminus of Pta1 has an inhibitory effect and that this effect can be neutralized through the interaction with Ssu72.
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