Dominika Elmlund

Architecture of an RNA Polymerase II Transcription Pre-Initiation Complex

Introduction RNA polymerase II (pol II) is capable of RNA synthesis but is unable to recognize a promoter or to initiate transcription. For these essential functions, a set of general transcription factors (GTFs)—termed TFIIB, -D, -E, -F, and -H—is required. The GTFs escort promoter DNA through the stages of recruitment to pol II, unwinding to create a transcription bubble, descent into the pol II cleft, and RNA synthesis to a length of 25 residues and transition to a stable elongating complex. The structural basis for these transactions is largely unknown. Only TFIIB has been solved by means of x-ray diffraction, in a complex with pol II. We report on the structure of a complete set of GTFs, assembled with pol II and promoter DNA in a 32-protein, 1.5 megaDalton “pre-initiation complex” (PIC), as revealed with cryo-electron microscopy (cryo-EM) and chemical cross-linking. A section through the cryo-EM structure of the complete PIC. Cut surfaces are shown in gray. Locations of densities due to pol II and the GTFs (TFIIA, TFIIB C-terminal domain, TBP subunit of TFIID, TFIIE, and TFIIH, including its helicase subunit Ssl2 and its kinase module TFIIK) are indicated. Density due to DNA is indicated by the superimposed double helix model. TFIIF is not seen in this section. Methods Three technical advances enabled the structural analysis of the PIC. First, a procedure was established for the preparation of a stable, abundant PIC. Both the homogeneity and functional activity of the purified PIC were demonstrated. Second, an algorithm was developed for alignment of cryo-EM images that requires no prior information (no “search model”) and that can distinguish multiple conformational states. Last, a computational method was devised for determining the arrangement of protein subunits and domains within a cryo-EM density map from a pattern of chemical cross-linking. Results The density map of the PIC showed a pronounced division in two parts, one pol II and the other the GTFs. Promoter DNA followed a straight path, in contact with the GTFs but well separated from pol II, suspended above the active center cleft. Cross-linking and computational analysis led to a most probable arrangement of the GTFs, with IIB at the upstream end of the pol II cleft, followed by IIF, IIE, and IIH. The Ssl2 helicase subunit of IIH was located at the downstream end of the cleft. Discussion A principle of the PIC revealed by this work is the interaction of promoter DNA with the GTFs and not with pol II. The GTFs position the DNA above the pol II cleft, but interaction with pol II can only occur after melting of the DNA to enable bending for entry in the cleft. Contact of the DNA with the Ssl2 helicase in the PIC leads to melting (in the presence of adenosine triphosphatase). Cryo-EM by others, based on sequential assembly and analysis of partial complexes rather than of the complete PIC, did not show a separation between pol II and GTFs and revealed direct DNA–pol II interaction. The discrepancy calls attention to a role of the GTFs in preventing direct DNA-polymerase interaction. Pre-Initiation Complex in 3D The regulation of gene expression is critical for almost every aspect of biology. Transcription—generating an RNA copy of a gene—requires the assembly of a large pre-initiation complex (PIC) at every RNA polymerase II (pol II) promoter. Roughly 32 proteins—the subunits of pol II and the general transcription factors—form a PIC that can recognize a minimal TATA-box promoter, select a transcription start site, and synthesize a nascent transcript. Murakami et al. (p. 10.1126/science.1238724, published online 26 September; see the Perspective by Malik and Roeder) determined the three-dimensional map of the Saccharomyces cerevisiae 30-subunit PIC using cryo-electron microscopy. The saddle-shaped TATA binding protein, the boot-shaped transcription factor IIA (TFIIA), and promoter DNA ∼27 bp downstream of the TATA-box could all be seen. Cross-linking and mass spectrometry was used to determine the spatial proximity of the 30 subunits, revealing that the PIC forms two lobes with TFIIF forming a bridge between them. The yeast transcription pre-initiation complex has a bi-lobed structure that may reflect the assembly pathway of the complex. [Also see Perspective by Malik and Roeder] The protein density and arrangement of subunits of a complete, 32-protein, RNA polymerase II (pol II) transcription pre-initiation complex (PIC) were determined by means of cryogenic electron microscopy and a combination of chemical cross-linking and mass spectrometry. The PIC showed a marked division in two parts, one containing all the general transcription factors (GTFs) and the other pol II. Promoter DNA was associated only with the GTFs, suspended above the pol II cleft and not in contact with pol II. This structural principle of the PIC underlies its conversion to a transcriptionally active state; the PIC is poised for the formation of a transcription bubble and descent of the DNA into the pol II cleft.

SIMPLE: Software for ab initio reconstruction of heterogeneous single-particles.

The open source software suite SIMPLE: Single-particle IMage Processing Linux Engine provides data analysis methods for single-particle cryo-electron microscopy (cryo-EM). SIMPLE addresses the problem of obtaining 3D reconstructions from 2D projections only, without using an input reference volume for approximating orientations. The SIMPLE reconstruction algorithm is tailored to asymmetrical and structurally heterogeneous single-particles. Its basis is global optimization with the use of Fourier common lines. The advance that enables ab initio reconstruction and heterogeneity analysis is the separation of the tasks of in-plane alignment and projection direction determination via bijective orientation search - a new concept in common lines-based strategies. Bijective orientation search divides the configuration space into two groups of paired parameters that are optimized separately. The first group consists of the rotations and shifts in the plane of the projection; the second group consists of the projection directions and state assignments. In SIMPLE, ab initio reconstruction is feasible because the 3D in-plane alignment is approximated using reference-free 2D rotational alignment. The subsequent common lines-based search hence searches projection directions and states only. Thousands of class averages are analyzed simultaneously in a matter of hours. Novice SIMPLE users get a head start via the well documented front-end. The structured, object-oriented back-end invites advanced users to develop new alignment and reconstruction algorithms. An overview of the package is presented together with benchmarks on simulated data. Executable binaries, source code, and documentation are available at http://simple.stanford.edu.

ATP-Induced Conformational Dynamics in the AAA+ Motor Unit of Magnesium Chelatase

Mg-chelatase catalyzes the first committed step of the chlorophyll biosynthetic pathway, the ATP-dependent insertion of Mg(2+) into protoporphyrin IX (PPIX). Here we report the reconstruction using single-particle cryo-electron microscopy of the complex between subunits BchD and BchI of Rhodobacter capsulatus Mg-chelatase in the presence of ADP, the nonhydrolyzable ATP analog AMPPNP, and ATP at 7.5 A, 14 A, and 13 A resolution, respectively. We show that the two AAA+ modules of the subunits form a unique complex of 3 dimers related by a three-fold axis. The reconstructions demonstrate substantial differences between the conformations of the complex in the presence of ATP and ADP, and suggest that the C-terminal integrin-I domains of the BchD subunits play a central role in transmitting conformational changes of BchI to BchD. Based on these data a model for the function of magnesium chelatase is proposed.

Ab Initio Structure Determination from Electron Microscopic Images of Single Molecules Coexisting in Different Functional States

We have developed methods for ab initio three-dimensional (3D) structure determination from projection images of randomly oriented single molecules coexisting in multiple functional states, to aid the study of complex samples of macromolecules and nanoparticles by electron microscopy (EM). New algorithms for the determination of relative 3D orientations and conformational state assignment of single-molecule projection images are combined with well-established techniques for alignment and statistical image analysis. We describe how the methodology arrives at homogeneous groups of images aligned in 3D and discuss application to experimental EM data sets of the Escherichia coli ribosome and yeast RNA polymerase II.

Subunit arrangement in the dodecameric chloroplast small heat shock protein Hsp21

Unfolding proteins are prevented from irreversible aggregation by small heat shock proteins (sHsps) through interactions that depend on a dynamic equilibrium between sHsp subunits and sHsp oligomers. A chloroplast‐localized sHsp, Hsp21, provides protection to client proteins to increase plant stress resistance. Structural information is lacking concerning the oligomeric conformation of this sHsp. We here present a structure model of Arabidopsis thaliana Hsp21, obtained by homology modeling, single‐particle electron microscopy, and lysine‐specific chemical crosslinking. The model shows that the Hsp21 subunits are arranged in two hexameric discs, similar to a cytosolic plant sHsp homolog that has been structurally determined after crystallization. However, the two hexameric discs of Hsp21 are rotated by 25° in relation to each other, suggesting a role for global dynamics in dodecamer function.

Structural studies show energy transfer within stabilized phycobilisomes independent of the mode of rod-core assembly

The major light harvesting complex in cyanobacteria and red algae is the phycobilisome (PBS), comprised of hundreds of seemingly similar chromophores, which are protein bound and assembled in a fashion that enables highly efficient uni-directional energy transfer to reaction centers. The PBS is comprised of a core containing 2-5 cylinders surrounded by 6-8 rods, and a number of models have been proposed describing the PBS structure. One of the most critical steps in the functionality of the PBS is energy transfer from the rod substructures to the core substructure. In this study we compare the structural and functional characteristics of high-phosphate stabilized PBS (the standard fashion of stabilization of isolated complexes) with cross-linked PBS in low ionic strength buffer from two cyanobacterial species, Thermosynechococcus vulcanus and Acaryochloris marina. We show that chemical cross-linking preserves efficient energy transfer from the phycocyanin containing rods to the allophycocyanin containing cores with fluorescent emission from the terminal emitters. However, this energy transfer is shown to exist in PBS complexes of different structures as characterized by determination of a 2.4Å structure by X-ray crystallography, single crystal confocal microscopy, mass spectrometry and transmission electron microscopy of negatively stained and cryogenically preserved complexes. We conclude that the PBS has intrinsic structural properties that enable efficient energy transfer from rod substructures to the core substructures without requiring a single unique structure. We discuss the significance of our observations on the functionality of the PBS in vivo.

The activity of barley NADPH-dependent thioredoxin reductase C is independent of the oligomeric state of the protein: tetrameric structure determined by cryo-electron microscopy

Thioredoxin and thioredoxin reductase can regulate cell metabolism through redox regulation of disulfide bridges or through removal of H(2)O(2). These two enzymatic functions are combined in NADPH-dependent thioredoxin reductase C (NTRC), which contains an N-terminal thioredoxin reductase domain fused with a C-terminal thioredoxin domain. Rice NTRC exists in different oligomeric states, depending on the absence or presence of its NADPH cofactor. It has been suggested that the different oligomeric states may have diverse activity. Thus, the redox status of the chloroplast could influence the oligomeric state of NTRC and thereby its activity. We have characterized the oligomeric states of NTRC from barley (Hordeum vulgare L.). This also includes a structural model of the tetrameric NTRC derived from cryo-electron microscopy and single-particle reconstruction. We conclude that the tetrameric NTRC is a dimeric arrangement of two NTRC homodimers. Unlike that of rice NTRC, the quaternary structure of barley NTRC complexes is unaffected by addition of NADPH. The activity of NTRC was tested with two different enzyme assays. The N-terminal part of NTRC was tested in a thioredoxin reductase assay. A peroxide sensitive Mg-protoporphyrin IX monomethyl ester (MPE) cyclase enzyme system of the chlorophyll biosynthetic pathway was used to test the catalytic ability of both the N- and C-terminal parts of NTRC. The different oligomeric assembly states do not exhibit significantly different activities. Thus, it appears that the activities are independent of the oligomeric state of barley NTRC.

The AAA(+) motor complex of subunits CobS and CobT of cobaltochelatase visualized by single particle electron microscopy.

Cobalamins belong to the tetrapyrrole family of prosthetic groups. The presence of a metal ion is a key feature of these compounds. In the oxygen-dependent (aerobic) cobalamin biosynthetic pathway, cobalt is inserted into a ring-contracted tetrapyrrole called hydrogenobyrinic acid a,c-diamide (HBAD) by a cobaltochelatase that is constituted by three subunits, CobN, CobS and CobT, with molecular masses of 137, 37 and 71kDa, respectively. Based on the similarities with magnesium chelatase, cobaltochelatase has been suggested to belong to the AAA(+) superfamily of proteins. In this paper we present the cloning of the Brucella melitensis cobN, cobS and cobT, the purification of the encoded protein products, and a single-particle reconstruction of the macromolecular assembly formed between CobS and CobT from negatively stained electron microscopy images of the complex. The results show for the first time that subunits CobS and CobT form a chaperone-like complex, characteristic for the AAA(+) class of proteins. The molecules are arranged in a two-tiered ring structure with the six subunits in each ring organized as a trimer of dimers. The similarity between this structure and that of magnesium chelatase, as well as analysis of the amino acid sequences confirms the suggested evolutionary relationship between the two enzymes.

High-resolution single-particle orientation refinement based on spectrally self-adapting common lines.

Three-dimensional (3D) structure determination from electron microscopic images of single molecules can be difficult for particles with low or no internal symmetry, and for images with low signal-to-noise ratio (SNR), due to the existence of false maxima in the scoring function used for orientation search. In attempt to improve robustness of orientation parameter refinement towards noise and poor starting reconstruction quality, we have developed a method for common lines-based orientation search in Fourier space. The Fourier-space formulation enables inclusion of resolution (spatial frequency of the low-pass limit) as a variable that is adjusted in a particle-dependent, self-adaptive manner. The method allows for the underlying 3D structure to be estimated to high resolution, and requires only a crude, low-resolution reconstruction as starting-point for refinement. Benchmarking of the method is performed on experimental and synthetic data.