Mutations in RNAs of both ribosomal subunits cause defects in translation termination

The processes of association and dissociation of ribosomal subunits are of great importance for the protein biosynthesis. The mechanistic details of these processes, however, are not well known. In bacteria, upon translation termination, ribosome dissociates into subunits which is necessary for its further involvement into new initiation step. The dissociated state of ribosome is maintained by initiation factor 3 (IF3) which binds to free small subunits and prevents their premature association with the large subunits. In this work, we have exchanged IF3 in E.coli cells by its ortholog from Saccharomyces cerevisiae mitochondria (Aim23p) and showed that yeast protein cannot functionally substitute the bacterial one and is even slightly toxic for bacterial cells. Our in vitro experiments have demonstrated that Aim23p does not split E.coli ribosomes into subunits. Instead, it fixes an intermediate state of ribosomes dissociation characterized by sedimentation coefficient about 60S. Using molecular modeling, we show that such fixation is due to mitochondria-specific terminal extensions of Aim23p that stabilize the position of the protein on the bacterial ribosome.

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An intermediate state of bacterial ribosome dissociation is fixed by yeast mitochondrial initiation factor 3

10.7287/peerj.preprints.26997 ◽

2018 ◽

Author(s):

Sergey Levitskii ◽

Ksenia Derbikova ◽

Andrey V Golovin ◽

Anton Kuzmenko ◽

Maria V Baleva ◽

...

Keyword(s):

Intermediate State ◽

Translation Termination ◽

Protein Biosynthesis ◽

Sedimentation Coefficient ◽

Initiation Factor ◽

Bacterial Cells ◽

Ribosomal Subunits ◽

Bacterial Ribosome ◽

Dissociated State

The processes of association and dissociation of ribosomal subunits are of great importance for the protein biosynthesis. The mechanistic details of these processes, however, are not well known. In bacteria, upon translation termination, ribosome dissociates into subunits which is necessary for its further involvement into new initiation step. The dissociated state of ribosome is maintained by initiation factor 3 (IF3) which binds to free small subunits and prevents their premature association with the large subunits. In this work, we have exchanged IF3 in E.coli cells by its ortholog from Saccharomyces cerevisiae mitochondria (Aim23p) and showed that yeast protein cannot functionally substitute the bacterial one and is even slightly toxic for bacterial cells. Our in vitro experiments have demonstrated that Aim23p does not split E.coli ribosomes into subunits. Instead, it fixes an intermediate state of ribosomes dissociation characterized by sedimentation coefficient about 60S. Using molecular modeling, we show that such fixation is due to mitochondria-specific terminal extensions of Aim23p that stabilize the position of the protein on the bacterial ribosome.

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A Uniquely Complex Mitochondrial Proteome from Euglena gracilis

Molecular Biology and Evolution ◽

10.1093/molbev/msaa061 ◽

2020 ◽

Vol 37 (8) ◽

pp. 2173-2191 ◽

Cited By ~ 1

Author(s):

Michael J Hammond ◽

Anna Nenarokova ◽

Anzhelika Butenko ◽

Martin Zoltner ◽

Eva Lacová Dobáková ◽

...

Keyword(s):

Euglena Gracilis ◽

Translation Termination ◽

Mitochondrial Gene ◽

Label Free ◽

Free Living ◽

Ribosomal Subunits ◽

Mitochondrial Proteome ◽

Liquid Chromatography Tandem Mass ◽

Emergence Of Complexity ◽

Environmental Importance

Abstract Euglena gracilis is a metabolically flexible, photosynthetic, and adaptable free-living protist of considerable environmental importance and biotechnological value. By label-free liquid chromatography tandem mass spectrometry, a total of 1,786 proteins were identified from the E. gracilis purified mitochondria, representing one of the largest mitochondrial proteomes so far described. Despite this apparent complexity, protein machinery responsible for the extensive RNA editing, splicing, and processing in the sister clades diplonemids and kinetoplastids is absent. This strongly suggests that the complex mechanisms of mitochondrial gene expression in diplonemids and kinetoplastids occurred late in euglenozoan evolution, arising independently. By contrast, the alternative oxidase pathway and numerous ribosomal subunits presumed to be specific for parasitic trypanosomes are present in E. gracilis. We investigated the evolution of unexplored protein families, including import complexes, cristae formation proteins, and translation termination factors, as well as canonical and unique metabolic pathways. We additionally compare this mitoproteome with the transcriptome of Eutreptiella gymnastica, illuminating conserved features of Euglenida mitochondria as well as those exclusive to E. gracilis. This is the first mitochondrial proteome of a free-living protist from the Excavata and one of few available for protists as a whole. This study alters our views of the evolution of the mitochondrion and indicates early emergence of complexity within euglenozoan mitochondria, independent of parasitism.

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Structural Role of rRNAs on the Ribosomal Subunits from E. Coli by Scanning Transmission Electron Microscopy

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100098629 ◽

1981 ◽

Vol 39 ◽

pp. 424-425

Author(s):

M. Boublik ◽

N. Robakis ◽

J.S. Wall

Keyword(s):

Three Dimensional ◽

Uranyl Acetate ◽

Dimensional Structure ◽

Electron Microscopic ◽

Ribosomal Subunits ◽

E Coli ◽

Freeze Dried ◽

16S Rna ◽

Scanning Transmission ◽

And Function

The three-dimensional structure and function of biological supramolecular complexes are, in general, determined and stabilized by conformation and interactions of their macromolecular components. In the case of ribosomes, it has been suggested that one of the functions of ribosomal RNAs is to act as a scaffold maintaining the shape of the ribosomal subunits. In order to investigate this question, we have conducted a comparative TEM and STEM study of the structure of the small 30S subunit of E. coli and its 16S RNA.The conventional electron microscopic imaging of nucleic acids is performed by spreading them in the presence of protein or detergent; the particles are contrasted by electron dense solution (uranyl acetate) or by shadowing with metal (tungsten). By using the STEM on freeze-dried specimens we have avoided the shearing forces of the spreading, and minimized both the collapse of rRNA due to air drying and the loss of resolution due to staining or shadowing. Figure 1, is a conventional (TEM) electron micrograph of 30S E. coli subunits contrasted with uranyl acetate.

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Three-dimensional reconstruction of the 40S ribosomal subunit from rabbit reticulocytes

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100128961 ◽

1987 ◽

Vol 45 ◽

pp. 936-937

Author(s):

Neng-Yu Zhang ◽

Terence Wagenknecht ◽

Michael Radermacher ◽

Tom Obrig ◽

Joachim Frank

Keyword(s):

Three Dimensional ◽

Ribosomal Subunit ◽

Uranyl Acetate ◽

Reconstruction Method ◽

Single Exposure ◽

Electron Dose ◽

Ribosomal Subunits ◽

40S Ribosomal Subunit ◽

Dimensional Reconstruction ◽

Rabbit Reticulocytes

We have reconstructed the 40S ribosomal subunit at a resolution of 4 nm using the single-exposure pseudo-conical reconstruction method of Radermacher et al.Small (40S) ribosomal subunits were Isolated from rabbit reticulocytes, applied to grids and negatively stained (0.5% uranyl acetate) in a manner that “sandwiches” the specimen between two layers of carbon. Regions of the grid exhibiting uniform and thick staining were identified and photographed twice (magnification 49,000X). The first micrograph was always taken with the specimen tilted by 50° and the second was of the Identical area untilted (Fig. 1). For each of the micrographs the specimen was subjected to an electron dose of 2000-3000 el/nm2.Three hundred thirty particles appearing in the L view (defined in [4]) were selected from both tilted- and untilted-specimen micrographs. The untilted particles were aligned and their rotational alignment produced the azimuthal angles of the tilted particles in the conical tilt series.

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Angular Calibration of Ribosomal Subuntts in Factor Space

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100181063 ◽

1990 ◽

Vol 48 (1) ◽

pp. 462-463

Author(s):

Minakhi Pujari ◽

Joachim Frank

Keyword(s):

Three Dimensional ◽

Carbon Layer ◽

Uranyl Acetate ◽

Particle Analysis ◽

Factor Space ◽

Multivariate Statistical ◽

Ribosomal Subunits ◽

Layer Method ◽

Dimensional Reconstruction ◽

Number Of Particles

In single-particle analysis of macromolecule images with the electron microscope, variations of projections are often observed that can be attributed to the changes of the particle’s orientation on the specimen grid (“rocking”). In the multivariate statistical analysis (MSA) of such projections, a single factor is often found that expresses a large portion of these variations. Successful angle calibration of this “rocking factor” would mean that correct angles can be assigned to a large number of particles, thus facilitating three-dimensional reconstruction.In a study to explore angle calibration in factor space, we used 40S ribosomal subunits, which are known to rock around an axis approximately coincident with their long axis. We analyzed micrographs of a field of these particles, taken with 20° tilt and without tilt, using the standard methods of alignment and MSA. The specimen was prepared with the double carbon-layer method, using uranyl acetate for negative staining. In the MSA analysis, the untilted-particle projections were used as active, the tilted-particle projections as inactive objects. Upon tilting, those particles whose rocking axes are parallel to the tilt axis will change their appearance in the same way as under the influence of rocking. Therefore, each vector, in factor space, joining a tilted and untilted projection of the same particle can be regarded as a local 20-degree calibration bar.

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Influence of nascent polypeptide positive charges on translation dynamics

Biochemical Journal ◽

10.1042/bcj20200303 ◽

2020 ◽

Vol 477 (15) ◽

pp. 2921-2934

Author(s):

Rodrigo D. Requião ◽

Géssica C. Barros ◽

Tatiana Domitrovic ◽

Fernando L. Palhano

Keyword(s):

Mrna Decay ◽

Translation Termination ◽

Published Data ◽

Translation Efficiency ◽

Amino Acid Residues ◽

High Concentration ◽

Strong Argument ◽

Translation Arrest ◽

Exit Tunnel ◽

Positively Charged

Protein segments with a high concentration of positively charged amino acid residues are often used in reporter constructs designed to activate ribosomal mRNA/protein decay pathways, such as those involving nonstop mRNA decay (NSD), no-go mRNA decay (NGD) and the ribosome quality control (RQC) complex. It has been proposed that the electrostatic interaction of the positively charged nascent peptide with the negatively charged ribosomal exit tunnel leads to translation arrest. When stalled long enough, the translation process is terminated with the degradation of the transcript and an incomplete protein. Although early experiments made a strong argument for this mechanism, other features associated with positively charged reporters, such as codon bias and mRNA and protein structure, have emerged as potent inducers of ribosome stalling. We carefully reviewed the published data on the protein and mRNA expression of artificial constructs with diverse compositions as assessed in different organisms. We concluded that, although polybasic sequences generally lead to lower translation efficiency, it appears that an aggravating factor, such as a nonoptimal codon composition, is necessary to cause translation termination events.

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