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Roger D Kornberg

Publications and source records attributed to Roger D Kornberg.

At least 19 recordsLinked to original sources
PubMed2006

Structural basis of transcription: role of the trigger loop in substrate specificity and catalysis.

New structures of RNA polymerase II (pol II) transcribing complexes reveal a likely key to transcription. The trigger loop swings beneath a correct nucleoside triphosphate (NTP) in the nucleotide addition site, closing off the active center and forming an extensive network of interactions with the NTP base, sugar, phosphates, and additional pol II residues. A histidine side chain in the trigger loop, precisely positioned by these interactions, may literally "trigger" phosphodiester bond formation. Recognition and catalysis are thus coupled, ensuring the fidelity of transcription.

Amanitins↗
PubMed2006

Head module control of mediator interactions.

Yeast Mediator proteins interacting with Med17(Srb4) have been expressed at a high level with the use of recombinant baculoviruses and recovered in homogeneous form as a seven subunit, 223 kDa complex. Electron microscopy and single-particle analysis identify this complex as the Mediator head module. The recombinant head module complements "headless" Mediator for the initiation of transcription in vitro. The module interacts with an RNA polymerase II-TFIIF complex, but not with the polymerase or TFIIF alone. This interaction is lost in the presence of a DNA template and associated RNA transcript, recapitulating the release of Mediator that occurs upon the initiation of transcription. Disruption of the head module in a temperature-sensitive mutant in vivo leads to the release of middle and tail modules from a transcriptionally active promoter. The head module evidently controls Mediator-RNA polymerase II and Mediator-promoter interactions.

Baculoviridae↗
PubMed2006

Rigid, specific, and discrete gold nanoparticle/antibody conjugates.

A general method of rigid, specific labeling of proteins with gold clusters has been devised. The method relies on the conjugation of a glutathione monolayer-protected gold cluster (MPC) with a single chain Fv antibody fragment (scFv), mutated to present an exposed cysteine residue. Efficient formation of a gold-thiolate bond between the MPC and scFv depends on activation of the gold cluster by chemical oxidation. Once formed, the MPC-scFv conjugate is treated with a reductant to quench cluster reactivity. The procedure has been performed with an MPC with an average Au(71) core and an scFv directed against a tetrameric protein, the influenza neuraminidase. A complex of the MPC-scFv conjugate with the neuraminidase was isolated, and the presence of four gold clusters was verified by cryoelectron microscopy.

Chromatography, Ion Exchange↗
PubMed2006

Chromatin remodeling by nucleosome disassembly in vitro.

The RSC chromatin-remodeling complex completely disassembles a nucleosome in the presence of the histone chaperone Nap1 and ATP. Disassembly occurs in a stepwise manner, with the removal of H2A/H2B dimers, followed by the rest of the histones and the release of naked DNA. RSC and related chromatin-remodeling complexes may be responsible for the removal of promoter nucleosomes during transcriptional activation in vivo.

Animals↗
PubMed2005

Mediator as a general transcription factor.

Others have shown that yeast strains bearing a ts mutation in the Srb4 subunit of Mediator cease transcription of all mRNA at the restrictive temperature, in a manner virtually indistinguishable from a strain bearing a ts mutation in the largest subunit of RNA polymerase II. We find that srb4ts Mediator is defective for the stimulation of basal RNA polymerase II transcription at the restrictive temperature in vitro. Taken together, these findings lead to the suggestion that Mediator is required for basal RNA polymerase II transcription in vivo. On this basis, Mediator is identified as a general transcription factor, comparable in importance to RNA polymerase II and other general factors for the initiation of transcription. The possibility that Mediator serves as an anti-inhibitor, opposing the effects of global negative regulators, is largely excluded.

Gene Expression Regulation, Fungal↗
PubMed2005

Defined DNA/nanoparticle conjugates.

Glutathione monolayer-protected gold clusters were reacted by place exchange with 19- or 20-residue thiolated oligonucleotides. The resulting DNA/nanoparticle conjugates could be separated on the basis of the number of bound oligonucleotides by gel electrophoresis and assembled with one another by DNA-DNA hybridization. This approach overcomes previous limitations of DNA/nanoparticle synthesis and yields conjugates that are precisely defined with respect to both gold and nucleic acid content.

Electrophoresis, Polyacrylamide Gel↗
PubMed2005

Thiolate ligands for synthesis of water-soluble gold clusters.

Water-soluble monolayer-protected gold clusters (MPCs) have been an object of investigation by many research groups since their first syntheses were reported in 1998 and 1999. The basic requirements for a ligand to form a monolayer protecting a gold cluster were established some time ago for alkanethiolate MPCs, but there has been no such information published for water-soluble MPCs. We identify 6 new ligands capable of forming water-soluble MPCs, as well as 22 water-soluble ligands that fail to form MPCs. Our findings contribute not only to the definition of the requirements for MPC formation but also to the variety of MPCs available for applications in chemistry and biology.

Gold↗
PubMed2005

Ubiquitin ligase activity of TFIIH and the transcriptional response to DNA damage.

Core transcription factor (TF) IIH purified from yeast possesses an E3 ubiquitin (Ub) ligase activity, which resides, at least in part, in a RING finger (RNF) domain of the Ssl1 subunit. Yeast strains mutated in the Ssl1 RNF domain are sensitive to ultraviolet (UV) light and to methyl methanesulfonate (MMS). This increased sensitivity to DNA-damaging agents does not reflect a deficiency in nucleotide excision repair. Rather, it correlates with reduced transcriptional induction of genes involved in DNA repair, suggesting that the E3 Ub ligase activity of TFIIH mediates the transcriptional response to DNA damage.

Amino Acid Sequence↗
PubMed2005

Structural basis of eukaryotic gene transcription.

An RNA polymerase II promoter has been isolated in transcriptionally activated and repressed states. Topological and nuclease digestion analyses have revealed a dynamic equilibrium between nucleosome removal and reassembly upon transcriptional activation, and have further shown that nucleosomes are removed by eviction of histone octamers rather than by sliding. The promoter, once exposed, assembles with RNA polymerase II, general transcription factors, and Mediator in a approximately 3 MDa transcription initiation complex. X-ray crystallography has revealed the structure of RNA polymerase II, in the act of transcription, at atomic resolution. Extension of this analysis has shown how nucleotides undergo selection, polymerization, and eventual release from the transcribing complex. X-ray and electron crystallography have led to a picture of the entire transcription initiation complex, elucidating the mechanisms of promoter recognition, DNA unwinding, abortive initiation, and promoter escape.

Crystallography, X-Ray↗
PubMed2005

Chromatin remodeling by DNA bending, not twisting.

Single-stranded regions (gaps) in nucleosomal DNA interfere with action of the RSC chromatin-remodeling complex, monitored by exposure of restriction endonuclease cutting sites. Single-strand breaks (nicks) in the DNA, by contrast, have no effect. Gaps on one side of the cutting site are inhibitory, but gaps on the other side are not. A gap >100 bp from the cutting site is as effective as a gap <20 bp from the site. These findings suggest a remodeling process involving bending, but not twisting, of the DNA and further point to the propagation of a bent region (loop or bulge) from one end of the nucleosome to the other.

Alu Elements↗
PubMed2005

Mediator and the mechanism of transcriptional activation.

Mediator was discovered because of its activity in a yeast RNA polymerase II (pol II) transcription system - it is needed for the system to respond to a transcriptional activator. Mediator is the central link in the enhancer-->activator-->Mediator-->pol II-->promoter pathway. The transduction of regulatory signals through this pathway is crucial for transcription of almost all pol II promoters in all eukaryote organisms.

Animals↗
PubMed2004

Diffusion of nucleoside triphosphates and role of the entry site to the RNA polymerase II active center.

Nucleoside triphosphates (NTPs) diffuse to the active center of RNA polymerase II through a funnel-shaped opening that narrows to a negatively charged pore. Computer simulation shows that the funnel and pore reduce the rate of diffusion by a factor of approximately 2 x 10(-7). The resulting limitation on the rate of RNA synthesis under conditions of low NTP concentration may be overcome by NTP binding to an entry site adjacent to the active center. Binding to the entry site greatly enhances the lifetime of an NTP in the active center region, and it prevents "backtracking" and the consequent occlusion of the active site.

Binding Sites↗
PubMed2004

Structural basis of transcription: nucleotide selection by rotation in the RNA polymerase II active center.

Binding of a ribonucleoside triphosphate to an RNA polymerase II transcribing complex, with base pairing to the template DNA, was revealed by X-ray crystallography. Binding of a mismatched nucleoside triphosphate was also detected, but in an adjacent site, inverted with respect to the correctly paired nucleotide. The results are consistent with a two-step mechanism of nucleotide selection, with initial binding to an entry (E) site beneath the active center in an inverted orientation, followed by rotation into the nucleotide addition (A) site for pairing with the template DNA. This mechanism is unrelated to that of single subunit RNA polymerases and so defines a new paradigm for the large, multisubunit enzymes. Additional findings from these studies include a third nucleotide binding site that may define the length of backtracked RNA; DNA double helix unwinding in advance of the polymerase active center; and extension of the diffraction limit of RNA polymerase II crystals to 2.3 A.

Base Pairing↗
PubMed2004

Removal of promoter nucleosomes by disassembly rather than sliding in vivo.

Previous work demonstrated the removal of nucleosomes from the PHO5 promoter upon transcriptional activation in yeast. Removal could occur by nucleosome disassembly or by sliding of nucleosomes away from the promoter. We have now activated the PHO5 promoter on chromatin circles following excision from the chromosomal locus. Whereas sliding would conserve the number of nucleosomes on the circle, we found that the number was diminished, demonstrating chromatin remodeling by nucleosome disassembly.

Acid Phosphatase↗
PubMed2004

Structural basis of transcription: an RNA polymerase II-TFIIB cocrystal at 4.5 Angstroms.

The structure of the general transcription factor IIB (TFIIB) in a complex with RNA polymerase II reveals three features crucial for transcription initiation: an N-terminal zinc ribbon domain of TFIIB that contacts the "dock" domain of the polymerase, near the path of RNA exit from a transcribing enzyme; a "finger" domain of TFIIB that is inserted into the polymerase active center; and a C-terminal domain, whose interaction with both the polymerase and with a TATA box-binding protein (TBP)-promoter DNA complex orients the DNA for unwinding and transcription. TFIIB stabilizes an early initiation complex, containing an incomplete RNA-DNA hybrid region. It may interact with the template strand, which sets the location of the transcription start site, and may interfere with RNA exit, which leads to abortive initiation or promoter escape. The trajectory of promoter DNA determined by the C-terminal domain of TFIIB traverses sites of interaction with TFIIE, TFIIF, and TFIIH, serving to define their roles in the transcription initiation process.

Amino Acid Sequence↗
PubMed2004

Structural basis of transcription: separation of RNA from DNA by RNA polymerase II.

The structure of an RNA polymerase II-transcribing complex has been determined in the posttranslocation state, with a vacancy at the growing end of the RNA-DNA hybrid helix. At the opposite end of the hybrid helix, the RNA separates from the template DNA. This separation of nucleic acid strands is brought about by interaction with a set of proteins loops in a strand/loop network. Formation of the network must occur in the transition from abortive initiation to promoter escape.

Base Pairing↗