Wei-Hau Chang

Electron Crystal Structure of the Transcription Factor and DNA Repair Complex, Core TFIIH

Core TFIIH from yeast, made up of five subunits required both for RNA polymerase II transcription and nucleotide excision DNA repair, formed 2D crystals on charged lipid layers. Diffraction from electron micrographs of the crystals in negative stain extended to about 13 angstrom resolution, and 3D reconstruction revealed several discrete densities whose volumes corresponded well with those of individual TFIIH subunits. The structure is based on a ring of three subunits, Tfb1, Tfb2, and Tfb3, to which are appended several functional moieties: Rad3, bridged to Tfb1 by SsI1; SsI2, known to interact with Tfb2; and Kin28, known to interact with Tfb3.

Dibenzo[f,h]thieno[3,4-b] quinoxaline-based small molecules for efficient bulk-heterojunction solar cells.

Two isomeric compounds (1 and 2) containing a dibenzo[f,h]thieno[3,4-b]quinoxaline core and two peripheral arylamines were synthesized. Solution-processed bulk heterojunction (BHJ) solar cells based on these sensitizers and [6,6]-phenyl-C61-butyric acid methyl ester (PCBM) are reported. The cell fabricated from 1/67 wt % of PCBM exhibited a high power conversion efficiency of 1.70% and an external quantum yield of 55%. The film of the cell was found to have balanced electron and hole mobility and good film morphology.

Mapping RNA exit channel on transcribing RNA polymerase II by FRET analysis

A simple genetic tag-based labeling method that permits specific attachment of a fluorescence probe near the C terminus of virtually any subunit of a protein complex is implemented. Its immediate application to yeast RNA polymerase II (pol II) enables us to test various hypotheses of RNA exit channel by using fluorescence resonance energy transfer (FRET) analysis. The donor dye is labeled on a site near subunit Rpb3 or Rpb4, and the acceptor dye is attached to the 5′ end of RNA transcript in the pol II elongation complex. Both in-gel and single-molecule FRET analysis show that the growing RNA is leading toward Rpb4, not Rpb3, supporting the notion that RNA exits through the proposed channel 1. Distance constraints derived from our FRET results, in conjunction with triangulation, reveal the exit track of RNA transcript on core pol II by identifying amino acids in the vicinity of the 5′ end of RNA and show that the extending RNA forms contacts with the Rpb7 subunit. The significance of RNA exit route in promoter escape and that in cotranscriptional mRNA processing is discussed.

Revised subunit structure of yeast transcription factor IIH (TFIIH) and reconciliation with human TFIIH

Tfb4 is identified as a subunit of the core complex of yeast RNA polymerase II general transcription factor IIH (TFIIH) by affinity purification, by peptide sequence analysis, and by expression of the entire complex in insect cells. Tfb3, previously identified as a component of the core complex, is shown instead to form a complex with cdk and cyclin subunits of TFIIH. This reassignment of subunits resolves a longstanding discrepancy between yeast and human TFIIH complexes.

Zernike Phase Plate Cryoelectron Microscopy Facilitates Single Particle Analysis of Unstained Asymmetric Protein Complexes

Single particle reconstruction from cryoelectron microscopy images, though emerging as a powerful means in structural biology, is faced with challenges as applied to asymmetric proteins smaller than megadaltons due to low contrast. Zernike phase plate can improve the contrast by restoring the microscope contrast transfer function. Here, by exploiting simulated Zernike and conventional defocused cryoelectron microscope images with noise characteristics comparable to those of experimental data, we quantified the efficiencies of the steps in single particle analysis of ice-embedded RNA polymerase II (500 kDa), transferrin receptor complex (290 kDa), and T7 RNA polymerase lysozyme (100 kDa). Our results show Zernike phase plate imaging is more effective as to particle identification and also sorting of orientations, conformations, and compositions. Moreover, our analysis on image alignment indicates that Zernike phase plate can, in principle, reduce the number of particles required to attain near atomic resolution by 10-100 fold for proteins between 100 kDa and 500 kDa.

Phase TEM for biological imaging utilizing a Boersch electrostatic phase plate: theory and practice

A Boersch electrostatic phase plate (BEPP) used in a transmission electron microscope (TEM) system can provide tuneable phase shifts and overcome the low contrast problem for biological imaging. Theoretically, a pure phase image with a high phase contrast can be obtained using a BEPP. However, a currently available TEM system utilizing a BEPP cannot achieve sufficiently high phase efficiency for biological imaging, owing to the practical conditions. The low phase efficiency is a result of the blocking of partial unscattered electrons by BEPP, and the contribution of absorption contrast. The fraction of blocked unscattered beam is related to BEPP dimensions and to divergence of the illumination system of the TEM. These practical issues are discussed in this paper. Phase images of biological samples (negatively stained ferritin) obtained by utilizing a BEPP are reported, and the phase contrast was found to be enhanced by a factor of approximately 1.5, based on the calculation using the Rose contrast criterion. The low gain in phase contrast is consistent with the expectation from the current TEM/BEPP system. A new generation of phase TEM utilizing BEPP and designed for biological imaging with a high phase efficiency is proposed.

Role of the DxxDxD motif in the assembly and stability of betanodavirus particles

Piscine betanodavirus possesses a bipartite genome of single-stranded (+)RNAs. RNA2 cDNA of dragon grouper nervous necrosis virus (DGNNV) has been expressed previously to form virus-like particles (VLPs), which are highly similar to the native virion. Experiments with calcium-chelating or reducing/oxidizing reagents showed that the DGNNV VLPs required only calcium for particle assembly. With the recombinant VLPs, site-directed mutagenesis can be employed to investigate the roles of calcium-binding ligands in particle formation. The results of mutational analysis of DxxDxD, which is putatively involved in the coordination of calcium ions, showed that the D133N mutation significantly disrupted the assembly of VLPs, while D130N and D135N mutants produced heterogeneous VLPs with broken shapes. The thermal stability of the VLP-forming fractions demonstrated that VLPs of the D135N mutant were stable at a temperature of 85°C, which is slightly higher than that for wild-type, whereas VLPs of the D130N mutant could not tolerate the thermal effects at a temperature higher than 60°C. It was deduced that the three aspartate residues of the motif DxxDxD are all important for the efficient formation of DGNNV VLPs and that, among them, the DxxD provides a more stable coordinate of calcium ligand than DxD.

Regulation of mammalian transcription by Gdown1 through a novel steric crosstalk revealed by cryo-EM

In mammals, a distinct RNA polymerase II form, RNAPII(G) contains a novel subunit Gdown1 (encoded by POLR2M), which represses gene activation, only to be reversed by the multisubunit Mediator co‐activator. Here, we employed single‐particle cryo‐electron microscopy (cryo‐EM) to disclose the architectures of RNAPII(G), RNAPII and RNAPII in complex with the transcription initiation factor TFIIF, all to ∼19 Å. Difference analysis mapped Gdown1 mostly to the RNAPII Rpb5 shelf‐Rpb1 jaw, supported by antibody labelling experiments. These structural features correlate with the moderate increase in the efficiency of RNA chain elongation by RNAP II(G). In addition, our updated RNAPII–TFIIF map showed that TFIIF tethers multiple regions surrounding the DNA‐binding cleft, in agreement with cross‐linking and biochemical mapping. Gdown1s binding sites overlap extensively with those of TFIIF, with Gdown1 sterically excluding TFIIF from RNAPII, herein demonstrated by competition assays using size exclusion chromatography. In summary, our work establishes a structural basis for Gdown1 impeding initiation at promoters, by obstruction of TFIIF, accounting for an additional dependent role of Mediator in activated transcription.

Fabrication of influenza virus-like particles using M2 fusion proteins for imaging single viruses and designing vaccines

Influenza virus-like particles (VLPs) are noninfectious and the assembly of influenza VLPs depends on the interactions of M1 proteins and/or other viral surface proteins, such as HA, NA, and M2, with the cellular lipid membranes. In this study we propose that M2 protein can be used as a molecular fabricator without disrupting the assembly of VLPs and while retaining the native structures of HA and NA envelope protein oligomers on the particle surfaces. First, we demonstrated that influenza VLPs can be fabricated by the M2 fusion of enhanced green fluorescent protein for imaging single virus entering A549 cells. Second, we engineered two molecular adjuvants (flagellin and profilin) fused to M2 protein to generate molecular adjuvanted VLPs. Theses molecular adjuvanted VLPs had stimulatory functions, including increasing TNF-α production and promoting the maturation of dendritic cells. Immunization of mice with molecular adjuvanted VLPs also enhanced the response of the neutralizing antibodies against homologous and heterologous H5N1 viruses. The results can provide useful information for imaging single viruses and designing novel vaccines against influenza virus infection.

Electron crystallography of yeast RNA polymerase II preserved in vitreous ice.

Two-dimensional (2-D) crystals of yeast RNA polymerase preserved in vitreous ice were studied by electron crystallographic and single-particle techniques. An electron density projection map of the enzyme was calculated from the data, which extended to a resolution of about 12 A, but was unexpectedly weak at resolutions higher than about 20 A. Multivariate statistics analysis revealed a large amount of variability in unit-cell structure in the polymerase crystals, partially related to high mobility of certain polymerase domains. Those same domains were previously identified as being involved in a conformational transition in the enzyme that controls DNA processivity and access to the active center cleft. Electron microscopic studies of other large multiprotein complexes are likely to require similar approaches to those described here.