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R D Kornberg

Publications and source records attributed to R D Kornberg.

At least 19 recordsLinked to original sources
PubMed2000

Architecture of RNA polymerase II and implications for the transcription mechanism.

A backbone model of a 10-subunit yeast RNA polymerase II has been derived from x-ray diffraction data extending to 3 angstroms resolution. All 10 subunits exhibit a high degree of identity with the corresponding human proteins, and 9 of the 10 subunits are conserved among the three eukaryotic RNA polymerases I, II, and III. Notable features of the model include a pair of jaws, formed by subunits Rpb1, Rpb5, and Rpb9, that appear to grip DNA downstream of the active center. A clamp on the DNA nearer the active center, formed by Rpb1, Rpb2, and Rpb6, may be locked in the closed position by RNA, accounting for the great stability of transcribing complexes. A pore in the protein complex beneath the active center may allow entry of substrates for polymerization and exit of the transcript during proofreading and passage through pause sites in the DNA.

Amino Acid Motifs↗
PubMed2000

Subunit interactions in yeast transcription/repair factor TFIIH. Requirement for Tfb3 subunit in nucleotide excision repair.

A yeast strain harboring a temperature-sensitive allele of TFB3 (tfb3(ts)), the 38-kDa subunit of the RNA polymerase II transcription/nucleotide excision repair factor TFIIH, was found to be sensitive to ultraviolet (UV) radiation and defective for nucleotide excision repair in vitro. Interestingly, tfb3(ts) failed to grow on medium containing caffeine. A comprehensive pairwise two-hybrid analysis between yeast TFIIH subunits identified novel interactions between Rad3 and Tfb3, Tfb4 and Ssl1, as well as Ssl2 and Tfb2. These interactions have facilitated a more complete model of the structure of TFIIH and the nucleotide excision repairosome.

Adenosine Triphosphatases↗
PubMed2000

Defocus-gradient corrected back-projection.

Three-dimensional reconstructions of icosahedral viruses from cryoelectron microscope images have reached resolutions where the microscope depth of field is a significant resolution-limiting factor. An analytical treatment presented here shows how the depth of field limitation can be understood as an envelope function which gradually attenuates the signal, starting well before the numerical depth of field is actually reached. A simple modification to the well-known back-projection reconstruction algorithm is described, called the defocus-gradient corrected back-projection, which computationally corrects for the contrast transfer function along a defocus gradient. Computer simulations demonstrate how the algorithm effectively eliminates the depth of field limitation.

Cryoelectron Microscopy↗
PubMed2000

Mediator-nucleosome interaction.

Mediator, a multiprotein complex involved in the regulation of RNA polymerase II transcription, binds to nucleosomes and acetylates histones. Three lines of evidence identify the Nut1 subunit of Mediator as responsible for the histone acetyltransferase (HAT) activity. An "in-gel" HAT assay reveals a single band of the appropriate size. Sequence alignment shows significant similarity of Nut1 to the GCN5-related N-acetyltransferase superfamily. Finally, recombinant Nut1 exhibits HAT activity in an in-gel assay.

Acetyltransferases↗
PubMed2000

Mediator of transcriptional regulation.

Three lines of evidence have converged on a multiprotein Mediator complex as a conserved interface between gene-specific regulatory proteins and the general transcription apparatus of eukaryotes. Mediator was discovered as an activity required for transcriptional activation in a reconstituted system from yeast. Upon resolution to homogeneity, the activity proved to reside in a 20-protein complex, which could exist in a free state or in a complex with RNA polymerase II, termed holoenzyme. A second line of evidence came from screens in yeast for mutations affecting transcription. Two-thirds of Mediator subunits are encoded by genes revealed by these screens. Five of the genetically defined subunits, termed Srbs, were characterized as interacting with the C-terminal domain of RNA polymerase II in vivo, and were shown to bind polymerase in vitro. A third line of evidence has come recently from studies in mammalian transcription systems. Mammalian counterparts of yeast Mediator were shown to interact with transcriptional activator proteins and to play an essential role in transcriptional regulation. Mediator evidently integrates and transduces positive and negative regulatory information from enhancers and operators to promoters. It functions directly through RNA polymerase II, modulating its activity in promoter-dependent transcription. Details of the Mediator mechanism remain obscure. Additional outstanding questions include the patterns of promoter-specificity of the various Mediator subunits, the possible cell-type-specificity of Mediator subunit composition, and the full structures of both free Mediator and RNA polymerase II holoenzyme.

Animals↗
PubMed1999

Electron crystal structure of an RNA polymerase II transcription elongation complex.

The structure of an actively transcribing complex, containing yeast RNA polymerase II with associated template DNA and product RNA, was determined by electron crystallography. Nucleic acid, in all likelihood the "transcription bubble" at the active center of the enzyme, occupies a previously noted 25 A channel in the protein structure. Details are indicative of a roughly 90 degrees bend of the DNA between upstream and downstream regions. The DNA apparently lies entirely on one face of the polymerase, rather than passing through a hole to the opposite side, as previously suggested.

Crystallography↗
PubMed1999

Yeast RNA polymerase II at 5 A resolution.

Appropriate treatment of X-ray diffraction from an unoriented 18-heavy atom cluster derivative of a yeast RNA polymerase II crystal gave significant phase information to 5 A resolution. The validity of the phases was shown by close similarity of a 6 A electron density map to a 16 A molecular envelope of the polymerase from electron crystallography. Comparison of the 6 A X-ray map with results of electron crystallography of a paused transcription elongation complex suggests functional roles for two mobile protein domains: the tip of a flexible arm forms a downstream DNA clamp; and a hinged domain may serve as an RNA clamp, enclosing the transcript from about 8-18 residues upstream of the 3'-end in a tunnel.

Crystallography, X-Ray↗
PubMed1999

Structure of DNA-dependent protein kinase: implications for its regulation by DNA.

DNA double-strand breaks are created by ionizing radiation or during V(D)J recombination, the process that generates immunological diversity. Breaks are repaired by an end-joining reaction that requires DNA-PKCS, the catalytic subunit of DNA-dependent protein kinase. DNA-PKCS is a 460 kDa serine-threonine kinase that is activated by direct interaction with DNA. Here we report its structure at 22 A resolution, as determined by electron crystallography. The structure contains an open channel, similar to those seen in other double-stranded DNA-binding proteins, and an enclosed cavity with three openings large enough to accommodate single-stranded DNA, with one opening adjacent to the open channel. Based on these structural features, we performed biochemical experiments to examine the interactions of DNA-PKCS with different DNA molecules. Efficient kinase activation required DNA longer than 12 bp, the minimal length of the open channel. Competition experiments demonstrated that DNA-PKCS binds to double- and single-stranded DNA via separate but interacting sites. Addition of unpaired single strands to a double-stranded DNA fragment stimulated kinase activation. These results suggest that activation of the kinase involves interactions with both double- and single-stranded DNA, as suggested by the structure. A model for how the kinase is regulated by DNA is described.

Binding Sites↗
PubMed1999

Conserved structures of mediator and RNA polymerase II holoenzyme.

Single particles of the mediator of transcriptional regulation (Mediator) and of RNA polymerase II holoenzyme were revealed by electron microscopy and image processing. Mediator alone appeared compact, but at high pH or in the presence of RNA polymerase II it displayed an extended conformation. Holoenzyme contained Mediator in a fully extended state, partially enveloping the globular polymerase, with points of apparent contact in the vicinity of the polymerase carboxyl-terminal domain and the DNA-binding channel. A similarity in appearance and conformational behavior of yeast and murine complexes indicates a conservation of Mediator structure among eukaryotes.

Animals↗
PubMed1999

Histone octamer transfer by a chromatin-remodeling complex.

RSC, an abundant, essential chromatin-remodeling complex related to SWI/SNF complex, catalyzes the transfer of a histone octamer from a nucleosome core particle to naked DNA. The newly formed octamer-DNA complex is identical with a nucleosome in all respects. The reaction requires ATP and involves an activated RSC-nucleosome intermediate. The mechanism may entail formation of a duplex displacement loop on the nucleosome, facilitating the entry of exogeneous DNA and the release of the endogenous molecule.

Animals↗
PubMed1999

Mediator protein mutations that selectively abolish activated transcription.

Deletion of any one of three subunits of the yeast Mediator of transcriptional regulation, Med2, Pgd1 (Hrs1), and Sin4, abolished activation by Gal4-VP16 in vitro. By contrast, other Mediator functions, stimulation of basal transcription and of TFIIH kinase activity, were unaffected. A different but overlapping Mediator subunit dependence was found for activation by Gcn4. The genetic requirements for activation in vivo were closely coincident with those in vitro. A whole genome expression profile of a Deltamed2 strain showed diminished transcription of a subset of inducible genes but only minor effects on "basal" transcription. These findings make an important connection between transcriptional activation in vitro and in vivo, and identify Mediator as a "global" transcriptional coactivator.

Cell-Free System↗
PubMed1999

Chromatin-modifying and -remodeling complexes.

Nucleosomes have long been known to inhibit DNA transactions on chromosomes and a remarkable abundance of multiprotein complexes that either enhance or relieve this inhibition have been described. Most is known about chromatin-remodeling complexes that perturb nucleosome structure.

Chromatin↗
PubMed1999

Two functionally distinct forms of the RSC nucleosome-remodeling complex, containing essential AT hook, BAH, and bromodomains.

RSC is an essential 15 protein nucleosome-remodeling complex from S. cerevisiae. We have identified two closely related RSC members, Rsc1 and Rsc2. Biochemical analysis revealed Rsc1 and Rsc2 in distinct complexes, defining two forms of RSC. Genetic analysis has shown that Rsc1 and Rsc2 possess shared and unique functions. Rsc1 and Rsc2 each contain two bromodomains, a bromo-adjacent homology (BAH) domain, and an AT hook. One of the bromodomains, the BAH domain, and the AT hook are each essential for Rsc1 and Rsc2 functions, although they are not required for assembly into RSC complexes. Therefore, these domains are required for RSC function. Additional genetic analysis provides further evidence that RSC function is related to transcriptional control.

Adenosine Triphosphatases↗
PubMed1999

Eukaryotic transcriptional control.

Some 30 years ago, following the elucidation of transcriptional control in prokaryotes, attention turned to the corresponding problem in eukaryotes: how are so many genes transcribed in a cell-type-specific, developmentally regulated manner? The answer has been found in two modes of regulation, one involving chromatin and the other the chief transcribing enzyme, RNA polymerase II. Although basic features of the prokaryotic mechanism have been preserved, the demands of eukaryotic transcription control are met by a huge increase in complexity and by the addition of new layers to the transcription apparatus. Discovering the components of this apparatus has been a major theme of research over the past three decades; unravelling the mechanisms is a challenge for the future.

Animals↗
PubMed1998

Identification of new mediator subunits in the RNA polymerase II holoenzyme from Saccharomyces cerevisiae.

Mediator was isolated from yeast on the basis of its requirement for transcriptional activation in a fully defined system. We have now identified three new members of mediator in the low molecular mass range by peptide sequence determination. These are the products of the NUT2, CSE2, and MED11 genes. The product of the NUT1 gene is evidently a component of mediator as well. NUT1 and NUT2 were earlier identified as negative regulators of the HO promoter, whereas mutations in CSE2 affect chromosome segregation. MED11 is a previously uncharacterized gene. The existence of these proteins in the mediator complex was verified by copurification and co-immunoprecipitation with RNA polymerase II holoenzyme.

Amino Acid Sequence↗
PubMed1998

Single-particle selection and alignment with heavy atom cluster-antibody conjugates.

A method is proposed for selecting and aligning images of single biological particles to obtain high-resolution structural information by cryoelectron microscopy. The particles will be labeled with multiple heavy atom clusters to permit the precise determination of particle locations and relative orientations even when imaged close to focus with a low electron dose, conditions optimal for recording high-resolution detail. Heavy atom clusters should also allow selection of images free from many kinds of defects, including specimen movement and particle inhomogeneity. Heavy atom clusters may be introduced in a general way by the construction of "adaptor" molecules based on single-chain Fv antibody fragments, consisting of a constant framework region engineered for optimal cluster binding and a variable antigen binding region selected for a specific target. The success of the method depends on the mobility of the heavy atom cluster on the particle, on the precision to which clusters can be located in an image, and on the sufficiency of cluster projections alone to orient and select particles for averaging. The necessary computational algorithms were developed and implemented in simulations that address the feasibility of the method.

Antibodies↗