Thomas Becker

Structure of the signal recognition particle interacting with the elongation-arrested ribosome

Cotranslational translocation of proteins across or into membranes is a vital process in all kingdoms of life. It requires that the translating ribosome be targeted to the membrane by the signal recognition particle (SRP), an evolutionarily conserved ribonucleoprotein particle. SRP recognizes signal sequences of nascent protein chains emerging from the ribosome. Subsequent binding of SRP leads to a pause in peptide elongation and to the ribosome docking to the membrane-bound SRP receptor. Here we present the structure of a targeting complex consisting of mammalian SRP bound to an active 80S ribosome carrying a signal sequence. This structure, solved to 12u2009Å by cryo-electron microscopy, enables us to generate a molecular model of SRP in its functional conformation. The model shows how the S domain of SRP contacts the large ribosomal subunit at the nascent chain exit site to bind the signal sequence, and that the Alu domain reaches into the elongation-factor-binding site of the ribosome, explaining its elongation arrest activity.

Cavity quantum electrodynamics

This paper reviews the work on cavity quantum electrodynamics of free atoms. In recent years, cavity experiments have also been conducted on a variety of solid-state systems resulting in many interesting applications, of which microlasers, photon bandgap structures and quantum dot structures in cavities are outstanding examples. Although these phenomena and systems are very interesting, discussion is limited here to free atoms and mostly single atoms because these systems exhibit clean quantum phenomena and are not disturbed by a variety of other effects. At the centre of our review is the work on the one-atom maser, but we also give a survey of the entire field, using free atoms in order to show the large variety of problems dealt with. The cavity interaction can be separated into two main regimes: the weak coupling in cavity or cavity-like structures with low quality factors Q and the strong coupling when high-Q cavities are involved. The weak coupling leads to modification of spontaneous transitions and level shifts, whereas the strong coupling enables one to observe a periodic exchange of photons between atoms and the radiation field. In this case, atoms and photons are entangled, this being the basis for a variety of phenomena observed, some of them leading to interesting applications in quantum information processing. The cavity experiments with free atoms reached a new domain with the advent of experiments in the visible spectral region. A review on recent achievements in this area is also given.

PMAS: The Potsdam Multi-Aperture Spectrophotometer. I. Design, Manufacture, and Performance

We describe the design, manufacture, commissioning, and performance of PMAS, the Potsdam Multi-Aperture Spectrophotometer. PMAS is a dedicated integral field spectrophotometer optimized to cover the optical wavelength regime of 0.35-1 μm. It is based on the lens array-fiber bundle principle of operation. The instrument employs an all-refractive fiber spectrograph, built with CaF2 optics, to provide good transmission and high image quality over the entire nominal wavelength range. A set of user-selectable reflective gratings provides low to medium spectral resolution of approximately 1.5, 3.2, and 7 A in first order, depending on the groove density (1200, 600, 300 grooves mm–1). While the standard integral field unit (IFU) uses a 16 × 16 element lens array, which provides seeing-limited sampling in a relatively small field of view (FOV) in one of three magnifications (8 × 8, 12 × 12, or 16 × 16), a recently retrofitted bare fiber bundle IFU (PPak: PMAS fiber pack) expands the FOV to a hexagonal area with a footprint of 65 × 74. Other special features include a cryogenic CCD camera for field acquisition and guiding, a nod-shuffle mode for beam switching and improved sky background subtraction, and a scanning Fabry-Perot etalon in combination with the standard IFU (PYTHEAS mode). PMAS was initially designed and built as an experimental traveling instrument with optical interfaces to various telescopes (Calar Alto 3.5 m, ESO VLT, LBT). It is offered as a common-user instrument at Calar Alto under contract to MPIA Heidelberg since 2002.

PMAS: The Potsdam Multi-Aperture Spectrophotometer. II. The Wide Integral Field Unit PPak

PPak is a new fiber-based integral field unit (IFU) developed at the Astrophysical Institute of Potsdam and implemented as a module into the existing Potsdam Multi-Aperture Spectrophotometer (PMAS) spectrograph. The purpose of PPak is to provide an extended field of view with a large light-collecting power for each spatial element, as well as an adequate spectral resolution. The PPak system consists of a fiber bundle with 331 object fibers, 36 sky fibers, and 15 calibration fibers. The object and sky fibers collect the light from the focal plane behind a focal reducer lens. The object fibers of PPak, each 2.7 in diameter, provide a contiguous hexagonal field of view of 74 × 64 on the sky, with a filling factor of 60%. The operational wavelength range is from 400 to 900 nm. The PPak IFU, together with the PMAS spectrograph, are intended for the study of extended, low surface brightness objects, offering an optimization of total light-collecting power and spectral resolution. This paper describes the instrument design, the assembly, integration, and tests, the commissioning and operational procedures, and presents the measured performance at the telescope.

Structure of the no-go mRNA decay complex Dom34–Hbs1 bound to a stalled 80S ribosome

No-go decay (NGD) is a mRNA quality-control mechanism in eukaryotic cells that leads to degradation of mRNAs stalled during translational elongation. The key factors triggering NGD are Dom34 and Hbs1. We used cryo-EM to visualize NGD intermediates resulting from binding of the Dom34–Hbs1 complex to stalled ribosomes. At subnanometer resolution, all domains of Dom34 and Hbs1 were identified, allowing the docking of crystal structures and homology models. Moreover, the close structural similarity of Dom34 and Hbs1 to eukaryotic release factors (eRFs) enabled us to propose a model for the ribosome-bound eRF1–eRF3 complex. Collectively, our data provide structural insights into how stalled mRNA is recognized on the ribosome and how the eRF complex can simultaneously recognize stop codons and catalyze peptide release.

Structure of Eef3 and the Mechanism of Transfer RNA Release from the E-Site.

Elongation factor eEF3 is an ATPase that, in addition to the two canonical factors eEF1A and eEF2, serves an essential function in the translation cycle of fungi. eEF3 is required for the binding of the aminoacyl-tRNA–eEF1A–GTP ternary complex to the ribosomal A-site and has been suggested to facilitate the clearance of deacyl-tRNA from the E-site. Here we present the crystal structure of Saccharomyces cerevisiae eEF3, showing that it consists of an amino-terminal HEAT repeat domain, followed by a four-helix bundle and two ABC-type ATPase domains, with a chromodomain inserted in ABC2. Moreover, we present the cryo-electron microscopy structure of the ATP-bound form of eEF3 in complex with the post-translocational-state 80S ribosome from yeast. eEF3 uses an entirely new factor binding site near the ribosomal E-site, with the chromodomain likely to stabilize the ribosomal L1 stalk in an open conformation, thus allowing tRNA release.

Structural Basis for Translational Stalling by Human Cytomegalovirus and Fungal Arginine Attenuator Peptide.

Specific regulatory nascent chains establish direct interactions with the ribosomal tunnel, leading to translational stalling. Despite a wealth of biochemical data, structural insight into the mechanism of translational stalling in eukaryotes is still lacking. Here we use cryo-electron microscopy to visualize eukaryotic ribosomes stalled during the translation of two diverse regulatory peptides: the fungal arginine attenuator peptide (AAP) and the human cytomegalovirus (hCMV) gp48 upstream open reading frame 2 (uORF2). The C terminus of the AAP appears to be compacted adjacent to the peptidyl transferase center (PTC). Both nascent chains interact with ribosomal proteins L4 and L17 at tunnel constriction in a distinct fashion. Significant changes at the PTC were observed: the eukaryotic-specific loop of ribosomal protein L10e establishes direct contact with the CCA end of the peptidyl-tRNA (P-tRNA), which may be critical for silencing of the PTC during translational stalling. Our findings provide direct structuralxa0insight into two distinct eukaryotic stalling processes.

p3d: a general data-reduction tool for fiber-fed integral-field spectrographs

The reduction of integral-field spectrograph (IFS) data is demanding work. Many repetitive operations are required to convert raw data into, typically, a large number of spectra. This effort can be markedly simplified through the use of a tool or pipeline, which is designed to complete many of the repetitive operations without human interaction. Here we present our semi-automatic data-reduction tool p3d, which is designed to be used with fiber-fed IFSs. Important components of p3d include a novel algorithm for automatic finding and tracing of spectra on the detector and two methods of optimal spectrum extraction in addition to standard aperture extraction. p3d also provides tools to combine several images, perform wavelength calibration and flat field data. p3d is at the moment configured for four IFSs. To evaluate its performance, we tested the different components of the tool. For these tests we used both simulated and observational data. We demonstrate that a correction for so-called cross-talk due to overlapping spectra on the detector is required for three of the IFSs. Without such a correction, spectra will be inaccurate, in particular if there is a significant intensity gradient across the object. Our tests showed that p3d is able to produce accurate results. p3d is a highly general and freely available tool. It is easily extended to include improved algorithms, new visualization tools, and support for additional instruments. The program code can be downloaded from the p3d-project web site http://p3d.sourceforge.net.

ERj1p uses a universal ribosomal adaptor site to coordinate the 80S ribosome at the membrane

Ribosomes translating secretory and membrane proteins are targeted to the endoplasmic reticulum membrane and attach to the protein-conducting channel and ribosome-associated membrane proteins (RAMPs). Recently, a new RAMP, ERj1p, has been identified that recruits BiP to ribosomes and regulates translational activity. Here we present the cryo-EM structure of a ribosome–ERj1p complex, revealing how ERj1p coordinates the ribosome at the membrane and how allosteric effects may mediate ERj1ps regulatory activity.

Structure of a human translation termination complex.

In contrast to bacteria that have two release factors, RF1 and RF2, eukaryotes only possess one unrelated release factor eRF1, which recognizes all three stop codons of the mRNA and hydrolyses the peptidyl-tRNA bond. While the molecular basis for bacterial termination has been elucidated, high-resolution structures of eukaryotic termination complexes have been lacking. Here we present a 3.8 Å structure of a human translation termination complex with eRF1 decoding a UAA(A) stop codon. The complex was formed using the human cytomegalovirus (hCMV) stalling peptide, which perturbs the peptidyltransferase center (PTC) to silence the hydrolysis activity of eRF1. Moreover, unlike sense codons or bacterial stop codons, the UAA stop codon adopts a U-turn-like conformation within a pocket formed by eRF1 and the ribosome. Inducing the U-turn conformation for stop codon recognition rationalizes how decoding by eRF1 includes monitoring geometry in order to discriminate against sense codons.