scholarly journals Pol5 is required for recycling of small subunit biogenesis factors and for formation of the peptide exit tunnel of the large ribosomal subunit

2019 ◽  
Author(s):  
Christina M Braun ◽  
Philipp Hackert ◽  
Catharina E Schmid ◽  
Markus T Bohnsack ◽  
Katherine E Bohnsack ◽  
...  
Keyword(s):  
Binding Sites ◽  
Ribosomal Proteins ◽  
Ribosomal Subunit ◽  
Small Subunit ◽  
Large Ribosomal Subunit ◽  
Rrna Sequence ◽  
Ribosomal Subunits ◽  
External Transcribed Spacer ◽  
Exit Tunnel ◽  
Domain Iii

Abstract More than 200 assembly factors (AFs) are required for the production of ribosomes in yeast. The stepwise association and dissociation of these AFs with the pre-ribosomal subunits occurs in a hierarchical manner to ensure correct maturation of the pre-rRNAs and assembly of the ribosomal proteins. Although decades of research have provided a wealth of insights into the functions of many AFs, others remain poorly characterized. Pol5 was initially classified with B-type DNA polymerases, however, several lines of evidence indicate the involvement of this protein in ribosome assembly. Here, we show that depletion of Pol5 affects the processing of pre-rRNAs destined for the both the large and small subunits. Furthermore, we identify binding sites for Pol5 in the 5′ external transcribed spacer and within domain III of the 25S rRNA sequence. Consistent with this, we reveal that Pol5 is required for recruitment of ribosomal proteins that form the polypeptide exit tunnel in the LSU and that depletion of Pol5 impairs the release of 5′ ETS fragments from early pre-40S particles. The dual functions of Pol5 in 60S assembly and recycling of pre-40S AFs suggest that this factor could contribute to ensuring the stoichiometric production of 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 Cryo-EM structure of an early precursor of large ribosomal subunit reveals a half assembled intermediate

2018 ◽  
Author(s):  
Dejian Zhou ◽  
Xing Zhu ◽  
Sanduo Zheng ◽  
Dan Tan ◽  
Meng-Qiu Dong ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Ribosomal Proteins ◽  
Large Subunit ◽  
Ribosomal Subunit ◽  
Large Ribosomal Subunit ◽  
Ribosomal Subunits ◽  
Cryo Electron Microscopy ◽  
5.8S Rrna ◽  
Early Processing ◽  
Large Subunit Rrna

AbstractAssembly of eukaryotic ribosome is a complicated and dynamic process that involves a series of intermediates. How the highly intertwined structure of 60S large ribosomal subunits is established is unknown. Here, we report the structure of an early nucleolar pre-60S ribosome determined by cryo-electron microscopy at 3.7 Å resolution, revealing a half assembled subunit. Domains I, II and VI of 25S/5.8S rRNA tightly pack into a native-like substructure, but domains III, IV and V are not assembled. The structure contains 12 assembly factors and 19 ribosomal proteins, many of which are required for early processing of large subunit rRNA. The Brx1-Ebp2 complex would interfere with the assembly of domains IV and V. Rpf1, Mak16, Nsa1 and Rrp1 form a cluster that consolidates the joining of domains I and II. Our structure reveals a key intermediate on the path to the establishment of the global architecture of 60S subunits.

A Possible Role for 5 S rRNA as a Bridge Between Ribosomal Subunits

1973 ◽  
Vol 51 (12) ◽  
pp. 1669-1672 ◽  
Author(s):  
A. A. Azad ◽  
B. G. Lane
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Large Subunit ◽  
Ribosomal Subunit ◽  
Small Subunit ◽  
Low Molecular Weight ◽  
Large Ribosomal Subunit ◽  
Ribosomal Subunits ◽  
Wheat Embryo ◽  
Present Context

18 S rRNA is a high molecular weight polyribonucleotide found in the small subunit, and 26 S rRNA is a high molecular weight polyribonucleotide found in the large subunit, whereas 5 S rRNA and 5.8 S rRNA are low molecular weight ("satellite") polyribonucleotides confined to the large subunit of wheat-embryo ribosomes. Under the same conditions in which 5.8 S rRNA is known to complex efficiently and preferentially with 26 S rRNA, it has been observed that 5 S rRNA complexes efficiently and preferentially with 18 S rRNA. Since 5 S rRNA is a component of the large ribosomal subunit, but it complexes preferentially with 18 S rRNA, which is a component of the small ribosomal subunit, it has been proposed that 5 S rRNA may serve as a "bridge" to mediate reversible association between the small and large ribosomal subunits. The possible role that a polycistronic precursor of rRNA might be visualized to play in the biogenesis and assembly of reversibly associating ribosomal subunits is alluded to in the present context.

scholarly journals Cryo-EM structure of the highly atypical cytoplasmic ribosome of Euglena gracilis

2020 ◽  
Vol 48 (20) ◽  
pp. 11750-11761
Author(s):  
Donna Matzov ◽  
Masato Taoka ◽  
Yuko Nobe ◽  
Yoshio Yamauchi ◽  
Yehuda Halfon ◽  
...  
Keyword(s):  
Ribosomal Rna ◽  
Euglena Gracilis ◽  
Ribosomal Proteins ◽  
Ribosomal Subunit ◽  
Catalytic Rna ◽  
Central Component ◽  
Large Ribosomal Subunit ◽  
Eukaryotic Alga ◽  
Cytoplasmic Ribosome ◽  
Exit Tunnel

Abstract Ribosomal RNA is the central component of the ribosome, mediating its functional and architectural properties. Here, we report the cryo-EM structure of a highly divergent cytoplasmic ribosome from the single-celled eukaryotic alga Euglena gracilis. The Euglena large ribosomal subunit is distinct in that it contains 14 discrete rRNA fragments that are assembled non-covalently into the canonical ribosome structure. The rRNA is substantially enriched in post-transcriptional modifications that are spread far beyond the catalytic RNA core, contributing to the stabilization of this highly fragmented ribosome species. A unique cluster of five adenosine base methylations is found in an expansion segment adjacent to the protein exit tunnel, such that it is positioned for interaction with the nascent peptide. As well as featuring distinctive rRNA expansion segments, the Euglena ribosome contains four novel ribosomal proteins, localized to the ribosome surface, three of which do not have orthologs in other eukaryotes.

Structural organization of escherichia coli ribosomes as determined by immuno electron microscopy

1978 ◽  
Vol 36 (2) ◽  
pp. 166-167 ◽  
Author(s):  
G. Stöffler ◽  
R.W. Bald ◽  
J. Dieckhoff ◽  
H. Eckhard ◽  
R. Lührmann ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Escherichia Coli ◽  
Ribosomal Proteins ◽  
Structural Organization ◽  
Three Dimensional ◽  
Ribosomal Subunit ◽  
Spatial Arrangement ◽  
Small Subunit ◽  
Antibody Binding ◽  
Immuno Electron Microscopy

A central step towards an understanding of the structure and function of the Escherichia coli ribosome, a large multicomponent assembly, is the elucidation of the spatial arrangement of its 54 proteins and its three rRNA molecules. The structural organization of ribosomal components has been investigated by a number of experimental approaches. Specific antibodies directed against each of the 54 ribosomal proteins of Escherichia coli have been performed to examine antibody-subunit complexes by electron microscopy. The position of the bound antibody, specific for a particular protein, can be determined; it indicates the location of the corresponding protein on the ribosomal surface.The three-dimensional distribution of each of the 21 small subunit proteins on the ribosomal surface has been determined by immuno electron microscopy: the 21 proteins have been found exposed with altogether 43 antibody binding sites. Each one of 12 proteins showed antibody binding at remote positions on the subunit surface, indicating highly extended conformations of the proteins concerned within the 30S ribosomal subunit; the remaining proteins are, however, not necessarily globular in shape (Fig. 1).

Ribosomal protein S14 is altered by two-step emetine resistance mutations in Chinese hamster cells

1983 ◽  
Vol 3 (2) ◽  
pp. 190-197
Author(s):  
J J Madjar ◽  
M Frahm ◽  
S McGill ◽  
D J Roufa
Keyword(s):  
Ribosomal Protein ◽  
Ribosomal Proteins ◽  
Ribosomal Subunit ◽  
Chinese Hamster ◽  
Complementation Group ◽  
Resistance Mutations ◽  
Cho Cell ◽  
Ribosomal Subunits ◽  
40S Ribosomal Subunit ◽  
One Step

Four two-dimensional polyacrylamide gel electrophoresis systems were used to identify 78 Chinese hamster cell ribosomal proteins by the uniform nomenclature based on rat liver ribosomal proteins. The 40S ribosomal subunit protein affected by Chinese hamster ovary (CHO) cell one-step emetine resistance mutations is designated S14 in the standard nomenclature. To seek unambiguous genetic evidence for a cause and effect relationship between CHO cell emetine resistance and mutations in the S14 gene, we mutagenized a one-step CHO cell mutant and isolated second-step mutant clones resistant to 10-fold-higher concentrations of emetine. All of the highly resistant, two-step CHO cell mutants obtained displayed additional alterations in ribosomal protein S14. Hybridization complementation tests revealed that the two-step CHO cell emetine resistance mutants were members of the same complementation group defined by one-step CHO cell mutants, EmtB. Two-step mutants obtained from a Chinese hamster lung cell emetine-resistant clone belong to the EmtA complementation group. The two-step and EmtB mutants elaborated 40S ribosomal subunits, which dissociated to 32S and 40S core particles in buffers containing 0.5 M KCl at 4 degrees C. In contrast, 40S ribosomal subunits purified from all EmtA, one-step EmtB EmtC mutants, and wild-type CHO and lung cells were stable at this temperature in buffers containing substantially higher concentrations of salt. Thus, two-step emtB mutations affect the structure of S14 protein directly and the stability of the 40S ribosomal subunit indirectly.

scholarly journals Analysis of subunit folding contribution of three yeast large ribosomal subunit proteins required for stabilisation and processing of intermediate nuclear rRNA precursors.

2021 ◽  
Author(s):  
Philipp Milkereit ◽  
Gisela Pöll ◽  
Michael Pilsl ◽  
Joachim Griesenbeck ◽  
Herbert Tschochner
Keyword(s):  
Electron Microscopy ◽  
Ribosomal Proteins ◽  
Ribosomal Subunit ◽  
Protein Assembly ◽  
Functional Coupling ◽  
Large Ribosomal Subunit ◽  
Cryo Electron Microscopy ◽  
Intrinsic Quality ◽  
Domain Arrangement ◽  
The Stability

In yeast and human cells many of the ribosomal proteins (r-proteins) are required for the stabilisation and productive processing of rRNA precursors. Functional coupling of r-protein assembly with the stabilisation and maturation of subunit precursors potentially promotes the production of ribosomes with defined composition. To further decipher mechanisms of such an intrinsic quality control pathway we analysed here the contribution of three yeast large ribosomal subunit r-proteins for intermediate nuclear subunit folding steps. Structure models obtained from single particle cryo-electron microscopy analyses provided evidence for specific and hierarchic effects on the stable positioning and remodelling of large ribosomal subunit domains. Based on these structural and previous biochemical data we discuss possible mechanisms of r-protein dependent hierarchic domain arrangement and the resulting impact on the stability of misassembled subunits.

scholarly journals The proteins of Xenopus ovary ribosomes

1971 ◽  
Vol 125 (4) ◽  
pp. 1091-1107 ◽  
Author(s):  
P J Ford
Keyword(s):  
Potassium Chloride ◽  
Ribosomal Proteins ◽  
Large Subunit ◽  
Buoyant Density ◽  
Small Subunit ◽  
Chemical Methods ◽  
Ribosomal Subunits ◽  
Molecular Weights ◽  
Caesium Chloride ◽  
Chloride Treatment

1. The preparation of ribosomes and ribosomal subunits from Xenopus ovary is described. 2. The yield of once-washed ribosomes (buoyant density in caesium chloride 1.601g·cm-3; 44% RNA, 56% protein by chemical methods) was 10.1mg/g wet wt. of tissue. 3. Buoyant density in caesium chloride and RNA/protein ratios by chemical methods have been determined for ribosome subunits produced by 1.0mm-EDTA or 0.5m-potassium chloride treatment and also for EDTA subunits extracted with 0.5m-, 1.0m- or 1.5m-potassium chloride, 4. Analysis of ribosomal protein on acrylamide gels at pH4.5 in 6m-urea reveals 24 and 26 bands from small and large EDTA subunits respectively. The actual numbers of proteins are greater than this, as many bands are obviously doublets. 5. Analysis of the proteins in the potassium chloride extract and particle fractions showed that some bands are completely and some partially extracted. Taking partial extraction as an indication of possible doublet bands it was found that there were 12 and 20 such bands in the small and large subunits respectively, making totals of 36 and 46 proteins. 6. From the measured protein contents and assuming weight-average molecular weights for the proteins of large and small subunits close to those observed for eukaryote ribosomal proteins it is possible to compute the total numbers of protein molecules per particle. It appears that too few protein bands have been identified on acrylamide gels to account for all the protein in the large subunit, but probably enough for the small subunit.

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