scholarly journals The structural basis for release factor activation during translation termination revealed by time-resolved cryogenic electron microscopy

2018 ◽  
Author(s):  
Ziao Fu ◽  
Gabriele Indrisiunaite ◽  
Sandip Kaledhonkar ◽  
Binita Shah ◽  
Ming Sun ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
High Speed ◽  
Stop Codon ◽  
Structural Basis ◽  
Ester Bond ◽  
Dependent Manner ◽  
Release Factor ◽  
Accuracy Class ◽  
Time Resolved ◽  
Class 1

AbstractWhen the mRNA translating ribosome encounters a stop codon in its aminoacyl site (A site), it recruits a class-1 release factor (RF) to induce hydrolysis of the ester bond between peptide chain and peptidyl-site (P-site) tRNA. This process, called termination of translation, is under strong selection pressure for high speed and accuracy. Class-1 RFs (RF1, RF2 in bacteria, eRF1 in eukarya and aRF1 in archaea), have structural motifs that recognize stop codons in the decoding center (DC) and a universal GGQ motif for induction of ester bond hydrolysis in the peptidyl transfer center (PTC) 70 Å away from the DC. The finding that free RF2 is compact with only 20 Å between its codon reading and GGQ motifs came therefore as a surprise1. Cryo-electron microscopy (cryo-EM) then showed that ribosome-bound RF1 and RF2 have extended structures2,3, suggesting that bacterial RFs are compact when entering the ribosome and switch to the extended form in a stop signal-dependent manner3. FRET4, cryo-EM5,6 and X-ray crystallography7, along with a rapid kinetics study suggesting a pre-termination conformational change on the millisecond time-scale of ribosome-bound RF1 and RF28, have lent indirect support to this proposal. However, direct experimental evidence for such a short-lived compact conformation on the native pathway to RF-dependent termination is missing due to its transient nature. Here we use time-resolved cryo-EM9,10,11,12,13 to visualize compact and extended forms of RF1 and RF2 at 3.5 and 4 Å resolution, respectively, in the codon-recognizing complex on the pathway to termination. About 25% of ribosomal complexes have RFs in the compact state at 24 ms reaction time after mixing RF and ribosomes, and within 60 ms virtually all ribosome-bound RFs are transformed to their extended forms.

scholarly journals The Structural Basis for Release Factor Activation during Translation Termination Revealed by Time-Resolved Cryogenic Electron Microscopy

2019 ◽  
Vol 116 (3) ◽  
pp. 574a-575a ◽  
Author(s):  
Ziao Fu ◽  
Gabriele Indrisiunaite ◽  
Sandip Kaledhonkar ◽  
Binita Shah ◽  
Ming Sun ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Translation Termination ◽  
Structural Basis ◽  
Release Factor ◽  
Time Resolved

Identification of amino acids responsible for stop codon recognition for polypeptide chain release factor

2013 ◽  
Vol 91 (3) ◽  
pp. 155-164 ◽  
Author(s):  
Lijun Xu ◽  
Yanrong Hao ◽  
Cui Li ◽  
Quan Shen ◽  
Baofeng Chai ◽  
...  
Keyword(s):  
Stop Codon ◽  
Translation Termination ◽  
Release Factor ◽  
Codon Reassignment ◽  
Eukaryotic Translation ◽  
Codon Recognition ◽  
Terminal Domain ◽  
Class 1 ◽  
Stop Codon Recognition ◽  
Stop Codon Reassignment

One factor involved in eukaryotic translation termination is class 1 release factor in eukaryotes (eRF1), which functions to decode stop codons. Variant code species, such as ciliates, frequently exhibit altered stop codon recognition. Studies revealed that some class-specific residues in the eRF1 N-terminal domain are responsible for stop codon reassignment in ciliates. Here, we investigated the effects on stop codon recognition of chimeric eRF1s containing the N-terminal domain of Euplotes octocarinatus and Blepharisma japonicum eRF1 fused to Saccharomyces cerevisiae M and C domains using dual luciferase read-through assays. Mutation of class-specific residues in different eRF1 classes was also studied to identify key residues and motifs involved in stop codon decoding. As expected, our results demonstrate that 3 pockets within the eRF1 N-terminal domain were involved in decoding stop codon nucleotides. However, allocation of residues to each pocket was revalued. Our data suggest that hydrophobic and class-specific surface residues participate in different functions: modulation of pocket conformation and interaction with stop codon nucleotides, respectively. Residues conserved across all eRF1s determine the relative orientation of the 3 pockets according to stop codon nucleotides. However, quantitative analysis of variant ciliate and yeast eRF1 point mutants did not reveal any correlation between evolutionary conservation of class-specific residues and termination-related functional specificity and was limited in elucidating a detailed mechanism for ciliate stop codon reassignment. Thus, based on isolation of suppressor tRNAs from Euplotes and Tetrahymena, we propose that stop codon reassignment in ciliates may be controlled by cooperation between eRF1 and suppressor tRNAs.

scholarly journals Structural basis of nucleosome assembly by the Abo1 AAA+ ATPase histone chaperone

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Carol Cho ◽  
Juwon Jang ◽  
Yujin Kang ◽  
Hiroki Watanabe ◽  
Takayuki Uchihashi ◽  
...  
Keyword(s):  
High Speed ◽  
Atp Hydrolysis ◽  
Structural Features ◽  
Structural Basis ◽  
Dependent Manner ◽  
Histone Chaperones ◽  
Nucleosome Assembly ◽  
Chromatin Dynamics ◽  
Aaa Atpase

AbstractThe fundamental unit of chromatin, the nucleosome, is an intricate structure that requires histone chaperones for assembly. ATAD2 AAA+ ATPases are a family of histone chaperones that regulate nucleosome density and chromatin dynamics. Here, we demonstrate that the fission yeast ATAD2 homolog, Abo1, deposits histone H3–H4 onto DNA in an ATP-hydrolysis-dependent manner by in vitro reconstitution and single-tethered DNA curtain assays. We present cryo-EM structures of an ATAD2 family ATPase to atomic resolution in three different nucleotide states, revealing unique structural features required for histone loading on DNA, and directly visualize the transitions of Abo1 from an asymmetric spiral (ATP-state) to a symmetric ring (ADP- and apo-states) using high-speed atomic force microscopy (HS-AFM). Furthermore, we find that the acidic pore of ATP-Abo1 binds a peptide substrate which is suggestive of a histone tail. Based on these results, we propose a model whereby Abo1 facilitates H3–H4 loading by utilizing ATP.

Microtubule nucleation sequence studied by time-resolved x-ray scattering and electron microscopy

1983 ◽  
Vol 41 ◽  
pp. 766-767
Author(s):  
Eva-Maria Mandelkow ◽  
Eckhard Mandelkow ◽  
Joan Bordas
Keyword(s):  
Electron Microscopy ◽  
Dynamic Equilibrium ◽  
Time Resolved ◽  
X Ray ◽  
Additional Information ◽  
X Ray Scattering ◽  
Low Concentrations ◽  
Microtubule Growth ◽  
Ray Patterns ◽  
Ray Scattering

When a solution of microtubule protein is changed from non-polymerising to polymerising conditions (e.g. by temperature jump or mixing with GTP) there is a series of structural transitions preceding microtubule growth. These have been detected by time-resolved X-ray scattering using synchrotron radiation, and they may be classified into pre-nucleation and nucleation events. X-ray patterns are good indicators for the average behavior of the particles in solution, but they are difficult to interpret unless additional information on their structure is available. We therefore studied the assembly process by electron microscopy under conditions approaching those of the X-ray experiment. There are two difficulties in the EM approach: One is that the particles important for assembly are usually small and not very regular and therefore tend to be overlooked. Secondly EM specimens require low concentrations which favor disassembly of the particles one wants to observe since there is a dynamic equilibrium between polymers and subunits.

Electron Microscopy and image reconstruction reveal the structural basis for spectrin's elastic properties

1992 ◽  
Vol 50 (1) ◽  
pp. 510-511
Author(s):  
Amy M. McGough ◽  
Robert Josephs
Keyword(s):  
Electron Microscopy ◽  
Elastic Properties ◽  
Conformational Changes ◽  
Quaternary Structure ◽  
Three Dimensional ◽  
Membrane Skeleton ◽  
Projection Algorithm ◽  
Structural Basis ◽  
Iterative Approximation ◽  
Membrane Skeletons

The remarkable deformability of the erythrocyte derives in large part from the elastic properties of spectrin, the major component of the membrane skeleton. It is generally accepted that spectrin's elasticity arises from marked conformational changes which include variations in its overall length (1). In this work the structure of spectrin in partially expanded membrane skeletons was studied by electron microscopy to determine the molecular basis for spectrin's elastic properties. Spectrin molecules were analysed with respect to three features: length, conformation, and quaternary structure. The results of these studies lead to a model of how spectrin mediates the elastic deformation of the erythrocyte.Membrane skeletons were isolated from erythrocyte membrane ghosts, negatively stained, and examined by transmission electron microscopy (2). Particle lengths and end-to-end distances were measured from enlarged prints using the computer program MACMEASURE. Spectrin conformation (straightness) was assessed by calculating the particles’ correlation length by iterative approximation (3). Digitised spectrin images were correlation averaged or Fourier filtered to improve their signal-to-noise ratios. Three-dimensional reconstructions were performed using a suite of programs which were based on the filtered back-projection algorithm and executed on a cluster of Microvax 3200 workstations (4).

Image quality vs data obtainment in biological electron microscopy

1986 ◽  
Vol 44 ◽  
pp. 42-43
Author(s):  
J. E. Johnson
Keyword(s):  
Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Electron Beam ◽  
Image Quality ◽  
High Speed ◽  
Early Period ◽  
Early Years ◽  
Fluorescent Screen ◽  
Embedding Medium

In the early years of biological electron microscopy, scientists had their hands full attempting to describe the cellular microcosm that was suddenly before them on the fluorescent screen. Mitochondria, Golgi, endoplasmic reticulum, and other myriad organelles were being examined, micrographed, and documented in the literature. A major problem of that early period was the development of methods to cut sections thin enough to study under the electron beam. A microtome designed in 1943 moved the specimen toward a rotary “Cyclone” knife revolving at 12,500 RPM, or 1000 times as fast as an ordinary microtome. It was claimed that no embedding medium was necessary or that soft embedding media could be used. Collecting the sections thus cut sounded a little precarious: “The 0.1 micron sections cut with the high speed knife fly out at a tangent and are dispersed in the air. They may be collected... on... screens held near the knife“.

Time-resolved high-resolution electron microscopy of structural stability in mgo clusters

1996 ◽  
Vol 54 ◽  
pp. 672-673
Author(s):  
T. Kizuka ◽  
N. Tanaka
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Magnesium Oxide ◽  
Structural Stability ◽  
High Resolution Electron Microscopy ◽  
Video Tape ◽  
Solid State Physics ◽  
Time Resolved ◽  
Resolution Electron ◽  
Structural And Functional Properties

Structure and stability of atomic clusters have been studied by time-resolved high-resolution electron microscopy (TRHREM). Typical examples are observations of structural fluctuation in gold (Au) clusters supported on silicon oxide films, graphtized carbon films and magnesium oxide (MgO) films. All the observations have been performed on the clusters consisted of single metal element. Structural stability of ceramics clusters, such as metal-oxide, metal-nitride and metal-carbide clusters, has not been observed by TRHREM although the clusters show anomalous structural and functional properties concerning to solid state physics and materials science.In the present study, the behavior of ceramic, magnesium oxide (MgO) clusters is for the first time observed by TRHREM at 1/60 s time resolution and at atomic resolution down to 0.2 nm.MgO and gold were subsequently deposited on sodium chloride (001) substrates. The specimens, single crystalline MgO films on which Au particles were dispersed were separated in distilled water and observed by using a 200-kV high-resolution electron microscope (JEOL, JEM2010) equipped with a high sensitive TV camera and a video tape recorder system.

The bright future of digital imaging in scanning electron microscopy

1993 ◽  
Vol 51 ◽  
pp. 768-769
Author(s):  
M. T. Postek ◽  
A. E. Vladar
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Digital Imaging ◽  
High Speed ◽  
High Performance ◽  
Mass Storage ◽  
Analog To Digital ◽  
Central Processing ◽  
Digital Imaging Technology ◽  
Scanning Electron

One of the major advancements applied to scanning electron microscopy (SEM) during the past 10 years has been the development and application of digital imaging technology. Advancements in technology, notably the availability of less expensive, high-density memory chips and the development of high speed analog-to-digital converters, mass storage and high performance central processing units have fostered this revolution. Today, most modern SEM instruments have digital electronics as a standard feature. These instruments, generally have 8 bit or 256 gray levels with, at least, 512 × 512 pixel density operating at TV rate. In addition, current slow-scan commercial frame-grabber cards, directly applicable to the SEM, can have upwards of 12-14 bit lateral resolution permitting image acquisition at 4096 × 4096 resolution or greater. The two major categories of SEM systems to which digital technology have been applied are:In the analog SEM system the scan generator is normally operated in an analog manner and the image is displayed in an analog or "slow scan" mode.

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