David A. Bushnell

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.

Different forms of TFIIH for transcription and DNA repair: Holo-TFIIH and a nucleotide excision repairosome

Yeast TFIIH that is active in transcription can be dissociated into three components: a 5-subunit core, the SSL2 gene product, and a complex of 47 kDa, 45 kDa, and 33 kDa polypeptides that possesses protein kinase activity directed towards the C-terminal repeat domain of RNA polymerase II. These three components can reconstitute fully functional TFIIH, and all three are required for transcription in vitro. By contrast, TFIIH that is highly active in nucleotide excision repair (NER) lacks the kinase complex and instead contains the products of all other genes known to be required for NER in yeast: RAD1, RAD2, RAD4, RAD10, and RAD14. This repairosome is not active in reconstituted transcription in vitro and is significantly more active than any of the constituent polypeptides in correcting defective repair in extracts from strains mutated in NER genes.

Complete, 12-subunit RNA polymerase II at 4.1-A resolution: implications for the initiation of transcription.

The x-ray structure of complete RNA polymerase II from Saccharomyces cerevisiae has been determined, including a heterodimer of subunits Rpb4 and Rpb7 not present in previous “core” polymerase II structures. The heterodimer maintains the polymerase in the conformation of a transcribing complex, may bind RNA as it emerges from the enzyme, and is in a position to interact with general transcription factors and the Mediator of transcriptional regulation.

Structural basis of transcription: α-Amanitin–RNA polymerase II cocrystal at 2.8 Å resolution

The structure of RNA polymerase II in a complex with the inhibitor α-amanitin has been determined by x-ray crystallography. The structure of the complex indicates the likely basis of inhibition and gives unexpected insight into the transcription mechanism.

Structure of an RNA Polymerase II―TFIIB Complex and the Transcription Initiation Mechanism

Dissecting TFIIB Mechanics Eukaryotic RNA polymerase II (Pol II) requires five protein cofactors for promoter recognition and initiation of transcription. The factor TFIIB is implicated in start site selection and stabilization of the initial transcript. The co-crystal structure of Pol II and TFIIB showed an N-terminal “finger” region located in the RNA exit channel, but the core C-terminal region of TFIIB was disordered. Now Liu et al. (p. 206; published online 12 November) present a structure of the same complex determined under different conditions in which the C-terminal structure is well localized but the finger is disordered. Docking DNA into the structure suggests that the C-terminal region stabilizes initial promoter melting. After transcription of a few bases, TFIIB probably switches to the alternate conformation where the C-terminal region is released and the finger region stabilizes the initial transcript. X-ray structures provide more details on the initiation of transcription. Previous x-ray crystal structures have given insight into the mechanism of transcription and the role of general transcription factors in the initiation of the process. A structure of an RNA polymerase II–general transcription factor TFIIB complex at 4.5 angstrom resolution revealed the amino-terminal region of TFIIB, including a loop termed the “B finger,” reaching into the active center of the polymerase where it may interact with both DNA and RNA, but this structure showed little of the carboxyl-terminal region. A new crystal structure of the same complex at 3.8 angstrom resolution obtained under different solution conditions is complementary with the previous one, revealing the carboxyl-terminal region of TFIIB, located above the polymerase active center cleft, but showing none of the B finger. In the new structure, the linker between the amino- and carboxyl-terminal regions can also be seen, snaking down from above the cleft toward the active center. The two structures, taken together with others previously obtained, dispel long-standing mysteries of the transcription initiation process.

Structural basis of transcription: backtracked RNA polymerase II at 3.4 angstrom resolution.

Stepping Back to Go Forward Insight into the mechanism of transcription has come from crystal structures of actively transcribing RNA polymerase II complexes in the pre- and posttranslocation states. RNA polymerase also backtracks on the DNA template. Backtracking by only a few residues is reversible, but longer backtracking leads to arrest that is relieved by cleavage of the transcript by the transcription elongation factor SII (TFIIS). Now Wang et al. (p. 1203) report x-ray structures of backtracked ternary complexes and of a backtracked complex bound to a noncleaving mutant of TFIIS. The structures show a defined one-residue, backtracked state supporting the idea that RNA polymerase oscillates between backward and forward motion during active transcription. Mismatched residues disfavor forward translocation, increasing the lifetime of the backtracked state and facilitating cleavage by TFIIS. Thus, TFIIS-induced cleavage is likely to provide an important proofreading function during transcription. A backtracked RNA polymerase II reveals how the enzyme proofreads the RNA transcript. Transcribing RNA polymerases oscillate between three stable states, two of which, pre- and posttranslocated, were previously subjected to x-ray crystal structure determination. We report here the crystal structure of RNA polymerase II in the third state, the reverse translocated, or “backtracked” state. The defining feature of the backtracked structure is a binding site for the first backtracked nucleotide. This binding site is occupied in case of nucleotide misincorporation in the RNA or damage to the DNA, and is termed the “P” site because it supports proofreading. The predominant mechanism of proofreading is the excision of a dinucleotide in the presence of the elongation factor SII (TFIIS). Structure determination of a cocrystal with TFIIS reveals a rearrangement whereby cleavage of the RNA may take place.

KIR2DS4 is a product of gene conversion with KIR3DL2 that introduced specificity for HLA-A*11 while diminishing avidity for HLA-C

Human killer cell immunoglobulin-like receptors (KIRs) are distinguished by expansion of activating KIR2DS, whose ligands and functions remain poorly understood. The oldest, most prevalent KIR2DS is KIR2DS4, which is represented by a variable balance between “full-length” and “deleted” forms. We find that full-length 2DS4 is a human histocompatibility leukocyte antigen (HLA) class I receptor that binds specifically to subsets of C1+ and C2+ HLA-C and to HLA-A*11, whereas deleted 2DS4 is nonfunctional. Activation of 2DS4+ NKL cells was achieved with A*1102 as ligand, which differs from A*1101 by unique substitution of lysine 19 for glutamate, but not with A*1101 or HLA-C. Distinguishing KIR2DS4 from other KIR2DS is the proline–valine motif at positions 71–72, which is shared with KIR3DL2 and was introduced by gene conversion before separation of the human and chimpanzee lineages. Site-directed swap mutagenesis shows that these two residues are largely responsible for the unique HLA class I specificity of KIR2DS4. Determination of the crystallographic structure of KIR2DS4 shows two major differences from KIR2DL: displacement of contact loop L2 and altered bonding potential because of the substitutions at positions 71 and 72. Correlation between the worldwide distributions of functional KIR2DS4 and HLA-A*11 points to the physiological importance of their mutual interaction.

Synthesis and Characterization of Au102(p-MBA)44 Nanoparticles

The synthesis of Au(102)(p-MBA)(44) nanoparticles on a preparative scale in high yield is described. Various analytical methods are shown to give results consistent with the composition and known structure of the particles, showing the preparation is essentially homogeneous, and attesting to the validity of the methods as well. Derivatization of the particles with proteins and DNA is demonstrated, and conditions are described for imaging individual particles by cryo-EM at low electron dose, close to focus, conditions optimal for recording high-resolution details.

Electron microscopy of gold nanoparticles at atomic resolution

Detailed structure of a gold nanoparticle Adding only a few atoms or changing the capping ligand can dramatically change the structure of individual metal nanoparticles. Azubel et al. used aberration-corrected transmission electron microscopy to derive a three-dimensional reconstruction of water-soluble gold nanoparticles. Small-angle x-ray scattering and other techniques have also corroborated this model. They used this to determine the atomic structure, which compared favorably with density functional theory calculations, without assuming any a priori structural knowledge or the use of model fitting. Science, this issue p. 909 The atomic structure of a 68–gold atom nanoparticle is determined without prior structural knowledge or model fitting. Structure determination of gold nanoparticles (AuNPs) is necessary for understanding their physical and chemical properties, but only one AuNP larger than 1 nanometer in diameter [a 102–gold atom NP (Au102NP)] has been solved to atomic resolution. Whereas the Au102NP structure was determined by x-ray crystallography, other large AuNPs have proved refractory to this approach. Here, we report the structure determination of a Au68NP at atomic resolution by aberration-corrected transmission electron microscopy, performed with the use of a minimal electron dose, an approach that should prove applicable to metal NPs in general. The structure of the Au68NP was supported by small-angle x-ray scattering and by comparison of observed infrared absorption spectra with calculations by density functional theory.

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 ∼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.