Coactivator complexes regulate chromatin accessibility and transcription. SAGA (Spt-Ada-Gcn5 Acetyltransferase) is an evolutionary conserved coactivator complex. The core module scaffolds the entire SAGA complex and adopts a histone octamer-like structure, which consists of six histone-fold domain (HFD)-containing proteins forming three histone-fold (HF) pairs, to which the double HFD-containing SUPT3H adds one HF pair. Spt3, the yeast ortholog of SUPT3H, interacts genetically and biochemically with the TATA binding protein (TBP) and contributes to global RNA polymerase II (Pol II) transcription. Here we demonstrate that (i) SAGA purified from human U2OS or mouse embryonic stem cells (mESC) can assemble without SUPT3H, (ii) SUPT3H is not essential for mESC survival, but required for their growth and self-renewal, and (iii) the loss of SUPT3H from mammalian cells affects the transcription of only a specific subset of genes. Accordingly, in the absence of SUPT3H no major change in TBP accumulation at gene promoters was observed. Thus, SUPT3H is not required for the assembly of SAGA, TBP recruitment, or overall Pol II transcription, but plays a role in mESC growth and self-renewal. Our data further suggest that yeast and mammalian SAGA complexes contribute to transcription regulation by distinct mechanisms.
RNA polymerase III (Pol III) transcription initiation requires the action of the transcription factor IIIB (TFIIIB) and is highly regulated. Here, we determine the structures of Pol III pre-initiation complexes (PICs) using single particle cryo-electron microscopy (cryo-EM). We observe stable Pol III–TFIIIB complexes using nucleic acid scaffolds mimicking various functional states, in which TFIIIB tightly encircles the upstream promoter DNA. There is an intricate interaction between TFIIIB and Pol III, which stabilizes the winged-helix domains of the C34 subunit of Pol III over the active site cleft. The architecture of Pol III PIC more resembles that of the Pol II PIC than the Pol I PIC. In addition, we also obtain a 3D reconstruction of Pol III in complex with TFIIIB using the elongation complex (EC) scaffold, shedding light on the mechanism of facilitated recycling of Pol III prior to transcription re-initiation.
more » « less- NSF-PAR ID:
- 10150359
- Publisher / Repository:
- Nature Publishing Group
- Date Published:
- Journal Name:
- Cell Discovery
- Volume:
- 4
- Issue:
- 1
- ISSN:
- 2056-5968
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract Proofreading by replicative DNA polymerases is a fundamental mechanism ensuring DNA replication fidelity. In proofreading, mis-incorporated nucleotides are excised through the 3′-5′ exonuclease activity of the DNA polymerase holoenzyme. The exonuclease site is distal from the polymerization site, imposing stringent structural and kinetic requirements for efficient primer strand transfer. Yet, the molecular mechanism of this transfer is not known. Here we employ molecular simulations using recent cryo-EM structures and biochemical analyses to delineate an optimal free energy path connecting the polymerization and exonuclease states of
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Silhavy, Thomas J. (Ed.)ABSTRACT Expression of the Escherichia coli dnaN -encoded β clamp at ≥10-fold higher than chromosomally expressed levels impedes growth by interfering with DNA replication. A mutant clamp (β E202K bearing a glutamic acid-to-lysine substitution at residue 202) binds to DNA polymerase III (Pol III) with higher affinity than the wild-type clamp, suggesting that its failure to impede growth is independent of its ability to sequester Pol III away from the replication fork. Our results demonstrate that the dnaN E202K strain underinitiates DNA replication due to insufficient levels of DnaA-ATP and expresses several DnaA-regulated genes at altered levels, including nrdAB , that encode the class 1a ribonucleotide reductase (RNR). Elevated expression of nrdAB was dependent on hda function. As the β clamp-Hda complex regulates the activity of DnaA by stimulating its intrinsic ATPase activity, this finding suggests that the dnaN E202K allele supports an elevated level of Hda activity in vivo compared with the wild-type strain. In contrast, using an in vitro assay reconstituted with purified components the β E202K and wild-type clamp proteins supported comparable levels of Hda activity. Nevertheless, co-overexpression of the nrdAB -encoded RNR relieved the growth defect caused by elevated levels of the β clamp. These results support a model in which increased cellular levels of DNA precursors relieve the ability of elevated β clamp levels to impede growth and suggest either that multiple effects stemming from the dnaN E202K mutation contribute to elevated nrdAB levels or that Hda plays a noncatalytic role in regulating DnaA-ATP by sequestering it to reduce its availability. IMPORTANCE DnaA bound to ATP acts in initiation of DNA replication and regulates the expression of several genes whose products act in DNA metabolism. The state of the ATP bound to DnaA is regulated in part by the β clamp-Hda complex. The dnaN E202K allele was identified by virtue of its inability to impede growth when expressed ≥10-fold higher than chromosomally expressed levels. While the dnaN E202K strain exhibits several phenotypes consistent with heightened Hda activity, the wild-type and β E202K clamp proteins support equivalent levels of Hda activity in vitro . Taken together, these results suggest that β E202K -Hda plays a noncatalytic role in regulating DnaA-ATP. This, as well as alternative models, is discussed.more » « less
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Abstract Sorption of
myo ‐inositol hexakisphosphate (IHP), a common type of organic phosphorus in soils, largely controls its mobility and bioavailability. Research on the interaction between IHP and phyllosilicate minerals such as kaolinite, which is commonly present in highly weathered soils, has often been neglected, probably due to the common assumption that negatively charged phyllosilicate minerals have low sorption capacity and binding affinity to IHP and thus do not play any significant role in its fate. Here, the interaction between IHP and poorly crystallized kaolinite (KGa‐2) was investigated in batch experiments using Zeta (ζ ) potential measurement and31P nuclear magnetic resonance (NMR) spectroscopy. The results showed that dissolved Al(III) concentration at the adsorption initiation stage increased with increasing IHP concentration at pH 4.0. From pH 2.5 to 9.0, IHP presented a maximum sorption capacity (50 μmol g−1) at pH 4.0 at 24 hr. With IHP sorption, theζ potential of kaolinite first decreased sharply to a negative value, then gradually increased with resorption of Al(III) released from kaolinite dissolution at acidic pH, and finally approached the original value of the pure kaolinite.31P NMR spectroscopy andζ potential analyses revealed that IHP formed inner‐sphere surface complexes and aluminium phytate precipitated on kaolinite at low pH (2.5 and 4.0), whereas the formation of inner‐sphere surface complexes was the dominant sorption mechanism at pH ≥ 5.5. This study implies that various mechanisms, depending on ambient pH condition, can dominate the IHP sorption onto kaolinite, which impacts the mobility and bioavailability of phosphorus in highly weathered soils.Highlights IHP promotes the dissolution of kaolinite mainly through the formation of aluminium phytate complex.
IHP sorption presents a sharp maximum at pH 4.0.
IHP forms inner‐sphere complexes at the surface of kaolinite.
Formation of aluminium phytate surface precipitates is favourable at relatively low pH.
