Julio O. Ortiz

The Native 3D Organization of Bacterial Polysomes

Recent advances have led to insights into the structure of the bacterial ribosome, but little is known about the 3D organization of ribosomes in the context of translating polysomes. We employed cryoelectron tomography and a template-matching approach to map 70S ribosomes in vitrified bacterial translation extracts and in lysates of active E. coli spheroplasts. In these preparations, polysomal arrangements were observed in which neighboring ribosomes are densely packed and exhibit preferred orientations. Analysis of characteristic examples of polysomes reveals a staggered or pseudohelical organization of ribosomes along the mRNA trace, with the transcript being sequestered on the inside, the tRNA entrance sites being accessible, and the polypeptide exit sites facing the cytosol. Modeling of elongating nascent polypeptide chains suggests that this arrangement maximizes the distance between nascent chains on adjacent ribosomes, thereby reducing the probability of intermolecular interactions that would give rise to aggregation and limit productive folding.

A movie of the RNA polymerase nucleotide addition cycle.

During gene transcription, RNA polymerase (Pol) passes through repetitive cycles of adding a nucleotide to the growing mRNA chain. Here we obtained a movie of the nucleotide addition cycle by combining structural information on different functional states of the Pol II elongation complex (EC). The movie illustrates the two-step loading of the nucleoside triphosphate (NTP) substrate, closure of the active site for catalytic nucleotide incorporation, and the presumed two-step translocation of DNA and RNA, which is accompanied by coordinated conformational changes in the polymerase bridge helix and trigger loop. The movie facilitates teaching and a mechanistic analysis of transcription and can be downloaded from http://www.lmb.uni-muenchen.de/cramer/pr-materials.

Structure of hibernating ribosomes studied by cryoelectron tomography in vitro and in situ

CryoET shows the configuration of the ephemeral translationally inactive 100S ribosomal dimer.

Noise contributions in an inducible genetic switch: a whole-cell simulation study.

Stochastic expression of genes produces heterogeneity in clonal populations of bacteria under identical conditions. We analyze and compare the behavior of the inducible lac genetic switch using well-stirred and spatially resolved simulations for Escherichia coli cells modeled under fast and slow-growth conditions. Our new kinetic model describing the switching of the lac operon from one phenotype to the other incorporates parameters obtained from recently published in vivo single-molecule fluorescence experiments along with in vitro rate constants. For the well-stirred system, investigation of the intrinsic noise in the circuit as a function of the inducer concentration and in the presence/absence of the feedback mechanism reveals that the noise peaks near the switching threshold. Applying maximum likelihood estimation, we show that the analytic two-state model of gene expression can be used to extract stochastic rates from the simulation data. The simulations also provide mRNA–protein probability landscapes, which demonstrate that switching is the result of crossing both mRNA and protein thresholds. Using cryoelectron tomography of an E. coli cell and data from proteomics studies, we construct spatial in vivo models of cells and quantify the noise contributions and effects on repressor rebinding due to cell structure and crowding in the cytoplasm. Compared to systems without spatial heterogeneity, the model for the fast-growth cells predicts a slight decrease in the overall noise and an increase in the repressors rebinding rate due to anomalous subdiffusion. The tomograms for E. coli grown under slow-growth conditions identify the positions of the ribosomes and the condensed nucleoid. The smaller slow-growth cells have increased mRNA localization and a larger internal inducer concentration, leading to a significant decrease in the lifetime of the repressor–operator complex and an increase in the frequency of transcriptional bursts.

Identification and Characterization of Transducin Functional Cysteines, Lysines, and Acidic Residues by Group-Specific Labeling and Chemical Cross-Linking

Guanine nucleotide binding proteins or G-proteins function as molecular switches in a diverse set of signaling pathways by coupling seven-helix transmembrane receptors to specific intracellular effectors (Kaziro et al., 1991; Dohlman et al., 1991). G-proteins are heterotrimers composed of a-, s-, and y-subunits. Activation of the appropiate receptor causes a GDP molecule bound to the resting form of a G-protein to be exchanged for GTP. As a consequence, the G-protein dissociates to form the a-subunit complexed to GTP, and the sy-dimer. The GTP-bound conformation of the a-subunit is capable of activating or inhibiting a variety of downstream effectors including enzymes as well as ion channels (Birnbaumer, 1992; Hepler & Gilman, 1992; Simon et al., 1991). The released sy-complex can itself activate or modulate some effectors (Logothetis et al., 1987; Tang et al., 1991; Katz et al., 1992). A GTPase-controlled timing mechanism inherent in all a-subunits and, in some cases, modulated by other proteins (Berstein et al., 1992; Arshaysky & Bownds, 1992), returns the GTP-activated a-subunit to the inactive GDP-bound conformation. The a-subunit complexed to GDP reassociates with the sy-complex and forms again the hetero-trimer in its resting state. Conklin & Bourne (1993) proposed a structural model for a general G-protein a-subunit, on the basis of biochemical, immunologic, and molecular genetic observations. This model provided a blurred but revealing view of the orientation of membrane-bound Ga with regard to Gpy, receptors, and effectors.

Combination of Different Techniques in Cryo-Electron Tomography with a Volta Phase Plate

Due to the sensitivity of the biological samples to beam irradiation resulting from induced changes to their structure, the limited allowed dose to frozen-hydrated biological specimens leads cryo images typically noisy and low in contrast. One way to improve the contrast is by applying few microns of defocus which is a well-known conventional technique since many years ago. The drawback of this technique is resolution deterioration caused by applied defocus that suppresses high-frequency information due to its sine type CTF. As opposed to the convectional technique, another way of increasing contrast is using a phase plate. Phase plate is located on the back-focal plane of the TEM. It changes the sine type CTF to a cosine type and the object information in the low spatial frequency domain is transferred well into the image information. There are various types of phase plates such as electrostatic, magnetic, anamorphotic, tulip type and etc. Nevertheless, none of them is as applicable as the thin film phase plate in terms of difficulties in fabrication and usage [1]. Among all different types of thin film phase plate, Zenrike phase plate has been the most successful one until the proposed novel phase plate. In the last two years, Volta phase plate (VPP) was developed and it improves on the performance of the Zernike phase plate by having a longer life time, overall better performance, simplicity in use and no fringe artefacts. The performance of the VPP is based on volta potential resulting from the incident electron beam and physical and chemical changes on the surface of the carbon [2].

70 Three-dimensional arrangements of ribosomes inside fast growing E. coli cells

and title adapted from T. Gerling, K. Wagenbauer, A. Neuner, & H. Dietz, Science, accepted manuscript.