Assembling the archaeal ribosome: roles for translation-factor-related GTPases

2011 ◽  
Vol 39 (1) ◽  
pp. 45-50 ◽  
Author(s):  
Fabian Blombach ◽  
Stan J.J. Brouns ◽  
John van der Oost
Keyword(s):  
Conformational Changes ◽  
Ribosomal Proteins ◽  
Major Type ◽  
Ribosome Assembly ◽  
Ribosomal Subunits ◽  
Translation Factor ◽  
Associated Functions ◽  
Assembly Factor ◽  
Switch Mechanism ◽  
Stepwise Assembly

The assembly of ribosomal subunits from their individual components (rRNA and ribosomal proteins) requires the assistance of a multitude of factors in order to control and increase the efficiency of the assembly process. GTPases of the TRAFAC (translation-factor-related) class constitute a major type of ribosome-assembly factor in Eukaryota and Bacteria. They are thought to aid the stepwise assembly of ribosomal subunits through a ‘molecular switch’ mechanism that involves conformational changes in response to GTP hydrolysis. Most conserved TRAFAC GTPases are involved in ribosome assembly or other translation-associated processes. They typically interact with ribosomal subunits, but in many cases, the exact role that these GTPases play remains unclear. Previous studies almost exclusively focused on the systems of Bacteria and Eukaryota. Archaea possess several conserved TRAFAC GTPases as well, with some GTPase families being present only in the archaeo–eukaryotic lineage. In the present paper, we review the occurrence of TRAFAC GTPases with translation-associated functions in Archaea.

scholarly journals Ribosomal proteins produced in excess are degraded by the ubiquitin–proteasome system

2016 ◽  
Vol 27 (17) ◽  
pp. 2642-2652 ◽  
Author(s):  
Min-Kyung Sung ◽  
Justin M. Reitsma ◽  
Michael J. Sweredoski ◽  
Sonja Hess ◽  
Raymond J. Deshaies
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Quality Control ◽  
Ribosomal Proteins ◽  
Ubiquitin Proteasome System ◽  
Ribosome Assembly ◽  
Protein Overexpression ◽  
Ribosomal Subunits ◽  
Nuclear Compartment ◽  
Ubiquitin Proteasome ◽  
Autophagy Inhibition

Ribosome assembly is an essential process that consumes prodigious quantities of cellular resources. Ribosomal proteins cannot be overproduced in Saccharomyces cerevisiae because the excess proteins are rapidly degraded. However, the responsible quality control (QC) mechanisms remain poorly characterized. Here we demonstrate that overexpression of multiple proteins of the small and large yeast ribosomal subunits is suppressed. Rpl26 overexpressed from a plasmid can be detected in the nucleolus and nucleoplasm, but it largely fails to assemble into ribosomes and is rapidly degraded. However, if the endogenous RPL26 loci are deleted, plasmid-encoded Rpl26 assembles into ribosomes and localizes to the cytosol. Chemical and genetic perturbation studies indicate that overexpressed ribosomal proteins are degraded by the ubiquitin–proteasome system and not by autophagy. Inhibition of the proteasome led to accumulation of multiple endogenous ribosomal proteins in insoluble aggregates, consistent with the operation of this QC mechanism in the absence of ribosomal protein overexpression. Our studies reveal that ribosomal proteins that fail to assemble into ribosomes are rapidly distinguished from their assembled counterparts and ubiquitinated and degraded within the nuclear compartment.

scholarly journals Co-Assembly of 40S and 60S Ribosomal Proteins in Early Steps of Eukaryotic Ribosome Assembly

2019 ◽  
Vol 20 (11) ◽  
pp. 2806 ◽  
Author(s):  
Jesse M. Fox ◽  
Rebekah L. Rashford ◽  
Lasse Lindahl
Keyword(s):  
Ribosomal Rna ◽  
Ribosomal Proteins ◽  
Ribosome Assembly ◽  
Relative Timing ◽  
Ribosomal Subunits ◽  
Eukaryotic Ribosome ◽  
Rrna Molecule ◽  
Ribosomal Precursor ◽  
Its1 And Its2

In eukaryotes three of the four ribosomal RNA (rRNA) molecules are transcribed as a long precursor that is processed into mature rRNAs concurrently with the assembly of ribosomal subunits. However, the relative timing of association of ribosomal proteins with the ribosomal precursor particles and the cleavage of the precursor rRNA into the subunit-specific moieties is not known. To address this question, we searched for ribosomal precursors containing components from both subunits. Particles containing specific ribosomal proteins were targeted by inducing synthesis of epitope-tagged ribosomal proteins followed by pull-down with antibodies targeting the tagged protein. By identifying other ribosomal proteins and internal rRNA transcribed spacers (ITS1 and ITS2) in the immuno-purified ribosomal particles, we showed that eS7/S7 and uL4/L4 bind to nascent ribosomes prior to the separation of 40S and 60S specific segments, while uS4/S9, uL22, and eL13/L13 are bound after, or simultaneously with, the separation. Thus, the incorporation of ribosomal proteins from the two subunits begins as a co-assembly with a single rRNA molecule, but is finished as an assembly onto separate precursors for the two subunits.

The control of accuracy during protein synthesis in Escherichia coli and perturbations of this control by streptomycin, neomycin, or ribosomal mutations

1984 ◽  
Vol 62 (5) ◽  
pp. 231-244 ◽  
Author(s):  
Léa Brakier-Gingras ◽  
Pauline Phoenix
Keyword(s):  
Escherichia Coli ◽  
Protein Synthesis ◽  
Conformational Changes ◽  
Ribosomal Proteins ◽  
High Energy ◽  
Ribosomal Subunits ◽  
Reversible Binding ◽  
Step Model ◽  
Experimental Approaches

This review surveys the different experimental approaches which describe the binding of tRNA to mRNA-programmed ribosomes and the control of tRNA selection. This selection is best described by the two-step model proposed by Hopfield and demonstrated by Thompson and his collaborators. The model involves a first control at the initial reversible binding of tRNA to the ribosome and a second control, the proofreading control, which promotes rejection of the incorrect tRNA from a high-energy intermediate during the transition from the initial to the final binding state. Streptomycin, neomycin, and ribosomal fidelity mutations appear to affect both control steps. Their effect can be related to the location of the mutated ribosomal proteins and to the conformational changes induced in the ribosome by the misreading agents. An alteration of the first control probably results from a distortion of the codon–anticodon interaction, while an alteration of the second control may be caused by a change in the association between ribosomal subunits.

scholarly journals Mitochondrial ribosome assembly in Neurospora. Structural analysis of mature and partially assembled ribosomal subunits by equilibrium centrifugation in CsCl gradients.

1982 ◽  
Vol 95 (1) ◽  
pp. 267-277 ◽  
Author(s):  
R J Lapolla ◽  
A M Lambowitz
Keyword(s):  
Ribosomal Proteins ◽  
Mitochondrial Protein ◽  
Ribosomal Subunit ◽  
Small Subunit ◽  
Ribosome Assembly ◽  
Ribosomal Subunits ◽  
Mitochondrial Ribosome ◽  
Equilibrium Centrifugation ◽  
Insight Into

In Neurospora, one protein associated with the mitochondrial small ribosomal subunit (S-5, Mr 52,000) is synthesized intramitochondrially and is assumed to be encoded by mtDNA. When mitochondrial protein synthesis is inhibited, either by chloramphenicol or by mutation, cells accumulate incomplete mitochondrial small subunits (CAP-30S and INC-30S particles) that are deficient in S-5 and several other proteins. To gain additional insight into the role of S-5 in mitochondrial ribosome assembly, the structures of Neurospora mitochondrial ribosomal subunits, CAP-30S particles, and INC-30S particles were analyzed by equilibrium centrifugation in CsCl gradients containing different concentrations of Mg+2. The results show (a) that S-5 is tightly associated with small ribosomal subunits, as judged by the fact that it is among the last proteins to be dissociated in CsCl gradients as the Mg+2 concentration is decreased, and (b) that CAP-30S and INC-30S particles, which are deficient in S-5, contain at most 12 proteins that are bound as tightly as in mature small subunits. The CAP-30S particles isolated from sucrose gradients contain a number of proteins that appear to be loosely bound, as judged by dissociation of these proteins in CsCl gradients under conditions in which they remain associated with mature small subunits. The results suggest that S-5 is required for the stable binding of a subset of small subunit ribosomal proteins.

scholarly journals A combined quantitative mass spectrometry and electron microscopy analysis of ribosomal 30S subunit assembly in E. coli

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Dipali G Sashital ◽  
Candacia A Greeman ◽  
Dmitry Lyumkis ◽  
Clinton S Potter ◽  
Bridget Carragher ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Mass Spectrometry ◽  
Ribosomal Proteins ◽  
Hybrid Approach ◽  
Protein Composition ◽  
Ribosome Assembly ◽  
Quantitative Mass Spectrometry ◽  
Complex Process ◽  
Electron Microscopy Analysis ◽  
Assembly Factor

Ribosome assembly is a complex process involving the folding and processing of ribosomal RNAs (rRNAs), concomitant binding of ribosomal proteins (r-proteins), and participation of numerous accessory cofactors. Here, we use a quantitative mass spectrometry/electron microscopy hybrid approach to determine the r-protein composition and conformation of 30S ribosome assembly intermediates in Escherichia coli. The relative timing of assembly of the 3′ domain and the formation of the central pseudoknot (PK) structure depends on the presence of the assembly factor RimP. The central PK is unstable in the absence of RimP, resulting in the accumulation of intermediates in which the 3′-domain is unanchored and the 5′-domain is depleted for r-proteins S5 and S12 that contact the central PK. Our results reveal the importance of the cofactor RimP in central PK formation, and introduce a broadly applicable method for characterizing macromolecular assembly in cells.

scholarly journals Structural insights into assembly of the ribosomal nascent polypeptide exit tunnel

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Daniel M. Wilson ◽  
Yu Li ◽  
Amber LaPeruta ◽  
Michael Gamalinda ◽  
Ning Gao ◽  
...  
Keyword(s):  
Ribosomal Rna ◽  
Ribosome Assembly ◽  
Ribosomal Subunits ◽  
Nascent Polypeptide ◽  
The Core ◽  
Cryo Electron Microscopy ◽  
Associated Proteins ◽  
Assembly Factor ◽  
Exit Tunnel ◽  
Structural Insights

Abstract The nascent polypeptide exit tunnel (NPET) is a major functional center of 60S ribosomal subunits. However, little is known about how the NPET is constructed during ribosome assembly. We utilized molecular genetics, biochemistry, and cryo-electron microscopy (cryo-EM) to investigate the functions of two NPET-associated proteins, ribosomal protein uL4 and assembly factor Nog1, in NPET assembly. Structures of mutant pre-ribosomes lacking the tunnel domain of uL4 reveal a misassembled NPET, including an aberrantly flexible ribosomal RNA helix 74, resulting in at least three different blocks in 60S assembly. Structures of pre-ribosomes lacking the C-terminal extension of Nog1 demonstrate that this extension scaffolds the tunnel domain of uL4 in the NPET to help maintain stability in the core of pre-60S subunits. Our data reveal that uL4 and Nog1 work together in the maturation of ribosomal RNA helix 74, which is required to ensure proper construction of the NPET and 60S ribosomal subunits.

Protein-induced conformational changes in 16S rRNA during assembly of the Escherichia Coli 30S ribosomal subunit: A STEM study

1987 ◽  
Vol 45 ◽  
pp. 910-911
Author(s):  
M. Boublik ◽  
V. Mandiyan ◽  
J.F. Hainfeld ◽  
J.S. Wall
Keyword(s):  
16S Rrna ◽  
Conformational Changes ◽  
Ribosomal Proteins ◽  
Structural Changes ◽  
Ribosomal Subunit ◽  
Compact Structure ◽  
E Coli ◽  
Freeze Dried ◽  
16S Rna ◽  
30S Subunit

The aim of this study is to understand the mechanism of 16S rRNA folding into the compact structure of the small 30S subunit of E. coli ribosome. The assembly of the 30S E. coli ribosomal subunit is a sequence of specific interactions of 16S rRNA with 21 ribosomal proteins (S1-S21). Using dedicated high resolution STEM we have monitored structural changes induced in 16S rRNA by the proteins S4, S8, S15 and S20 which are involved in the initial steps of 30S subunit assembly. S4 is the first protein to bind directly and stoichiometrically to 16S rRNA. Direct binding also occurs individually between 16S RNA and S8 and S15. However, binding of S20 requires the presence of S4 and S8. The RNA-protein complexes are prepared by the standard reconstitution procedure, dialyzed against 60 mM KCl, 2 mM Mg(OAc)2, 10 mM-Hepes-KOH pH 7.5 (Buffer A), freeze-dried and observed unstained in dark field at -160°.

Sign in / Sign up

Export Citation Format

Share Document