in-vitro-reassociation-assay-to-measure-formation-80s-ribosomal

<em>In Vitro</em> Reassociation Assay to Measure the Formation of 80S Ribosomal Particles Using Salt-washed Ribosomal Subunits

Ribosomes are molecular machines that are responsible for protein synthesis in all living cells. All ribosomes consist of two subunits. In eukaryotes, the ...

University of Tartu

A single-step PCR-based epitope tagging enables fast and efficient gene targeting with various epitope tags. This report presents a series of plasmids for the E2 epitope tagging of proteins in Saccharomyces cerevisiae and Schizosaccharomyces pombe. E2Tags are 10-amino acids (epitope E2a: SSTSSDFRDR)- and 12 amino acids (epitope E2b: GVSSTSSDFRDR)-long peptides derived from the E2 protein of bovine papillomavirus type 1. The modules for C-terminal tagging with E2a and E2b epitopes were constructed by the modification of the pYM-series plasmid. The N-terminal E2a and E2b tagging modules were based on pOM-series plasmid. The pOM-series plasmids were selected for this study because of their use of the Cre-loxP recombination system. The latter enables a marker cassette to be removed after integration into the loci of interest and, thereafter, the tagged protein is expressed under its endogenous promoter. Specifically for fission yeast, high copy pREP plasmids containing the E2a epitope tag as an N-terminal or C-terminal tag were constructed. The properties of E2a and E2b epitopes and the sensitivity of two anti-E2 monoclonal antibodies (5E11 and 3F12) were tested using several S. cerevisiae and Sz. pombe E2-tagged strains.

Plasmids with E2 epitope tags: tagging modules for N- and C-terminal PCR-based gene targeting in both budding and fission yeast, and inducible expression vectors for fission yeast.

The -1 programmed ribosomal frameshifting (-1 PRF) mechanism utilized by many viruses is dependent on a heptanucleotide slippery sequence and a downstream secondary structure element. In the current study, the RNA structure downstream from the slippery site of cocksfoot mottle sobemovirus (CfMV) was proven to be a 12bp stem-loop with a single bulge and a tetranucleotide loop. Several deletion and insertion mutants with altered stem-loop structures were tested in wheat germ extract (WGE) for frameshifting efficiency. The impact of the same mutations on virus infectivity was tested in oat plants. Mutations shortening or destabilizing the stem region reduced significantly but did not abolish -1 PRF in WGE. The same mutations proved to be deleterious for virus infection. However, extending the loop region to seven nucleotides had no significant effect on frameshifting efficiency in WGE and did not hamper virus replication in infected leaves. This is the first report about the experimentally proven RNA secondary structure directing -1 PRF of sobemoviruses.

Stem-loop structure of Cocksfoot mottle virus RNA is indispensable for programmed -1 ribosomal frameshifting.

Schizosaccharomyces pombe, the fission yeast, has been a popular and useful model system for investigating the mechanisms of biological processes for a long time. To facilitate purification, localization, and functional analysis of gene products, a wide range of expression vectors have been developed. Several of these vectors utilize the inducible/repressible promoter systems and enable the episomal expression of proteins as fusion proteins with epitope tags attached to their N terminus or C terminus.This chapter provides a detailed protocol for expression of the epitope-tagged proteins from thiamine-regulatable nmt promoter in fission yeast. The yeast culture conditions and procedures for yeast transformation, expression induction, preparation of whole-cell extracts, and analysis of epitope-tagged protein expression by Western blotting are described.

A thiamine-regulatable epitope-tagged protein expression system in fission yeast.

Saccharomyces cerevisiae mitochondrial DNA polymerase (Mip1) contains a C-terminal extension (CTE) of 279 amino acid residues. The CTE is required for mitochondrial DNA maintenance in yeast but is absent in higher eukaryotes. Here we use recombinant Mip1 C-terminal deletion mutants to investigate functional importance of the CTE. We show that partial removal of the CTE in Mip1Δ216 results in strong preference for exonucleolytic degradation rather than DNA polymerization. This disbalance in exonuclease and polymerase activities is prominent at suboptimal dNTP concentrations and in the absence of correctly pairing nucleotide. Mip1Δ216 also displays reduced ability to synthesize DNA through double-stranded regions. Full removal of the CTE in Mip1Δ279 results in complete loss of Mip1 polymerase activity, however the mutant retains its exonuclease activity. These results allow us to propose that CTE functions as a part of Mip1 polymerase domain that stabilizes the substrate primer end at the polymerase active site, and is therefore required for efficient mitochondrial DNA replication in vivo.

C-terminal extension of the yeast mitochondrial DNA polymerase determines the balance between synthesis and degradation.

Mitochondrial metabolism is targeted by conserved signaling pathways that mediate external information to the cell. However, less is known about whether mitochondrial dysfunction interferes with signaling and thereby modulates the cellular response to environmental changes. In this study, we analyzed defective filamentous and invasive growth of the yeast Saccharomyces cerevisiae strains that have a dysfunctional mitochondrial genome (rho mutants). We found that the morphogenetic defect of rho mutants was caused by specific downregulation of FLO11, the adhesin essential for invasive and filamentous growth, and did not result from general metabolic changes brought about by interorganellar retrograde signaling. Transcription of FLO11 is known to be regulated by several signaling pathways, including the filamentous-growth-specific MAPK and cAMP-activated protein kinase A (cAMP-PKA) pathways. Our analysis showed that the filamentous-growth-specific MAPK pathway retained functionality in respiratory-deficient yeast cells. In contrast, the cAMP-PKA pathway was downregulated, explaining also various phenotypic traits observed in rho mutants. Thus, our results indicate that dysfunctional mitochondria modulate the output of the conserved cAMP-PKA signaling pathway.

Dysfunctional mitochondria modulate cAMP-PKA signaling and filamentous and invasive growth of Saccharomyces cerevisiae.

Structural studies have revealed that the core of the ribosome structure is conserved among ribosomes of all kingdoms. Kingdom-specific ribosomal proteins (r-proteins) are located in peripheral parts of the ribosome. In this work, the interactions between rRNA and r-proteins of eukaryote Saccharomyces cerevisiae ribosome were investigated applying LiCl induced splitting and quantitative mass spectrometry. R-proteins were divided into four groups according to their binding properties to the rRNA. Most yeast r-proteins are removed from rRNA by 0.5-1 M LiCl. Eukaryote-specific r-proteins are among the first to dissociate. The majority of the strong binders are known to be required for the early ribosome assembly events. As compared to the bacterial ribosome, yeast r-proteins are dissociated from rRNA at lower ionic strength. Our results demonstrate that the nature of protein-RNA interactions in the ribosome is not conserved between different kingdoms.

Stepwise splitting of ribosomal proteins from yeast ribosomes by LiCl.

During translation, the two eukaryotic ribosomal subunits remain associated through 17 intersubunit bridges, five of which are eukaryote specific. These are mainly localized to the peripheral regions and are believed to stabilize the structure of the ribosome. The functional importance of these bridges remains largely unknown. Here, the essentiality of the eukaryote-specific bridge eB12 has been investigated. The main component of this bridge is ribosomal protein eL19 that is composed of an N-terminal globular domain, a middle region, and a long C-terminal α-helix. The analysis of deletion mutants demonstrated that the globular domain and middle region of eL19 are essential for cell viability, most likely functioning in ribosome assembly. The eB12 bridge, formed by contacts between the C-terminal α-helix of eL19 and 18S rRNA in concert with additional stabilizing interactions involving either eS7 or uS17, is dispensable for viability. Nevertheless, eL19 mutants impaired in eB12 bridge formation displayed slow growth phenotypes, altered sensitivity/resistance to translational inhibitors, and enhanced hyperosmotic stress tolerance. Biochemical analyses determined that the eB12 bridge contributes to the stability of ribosome subunit interactions in vitro. 60S subunits containing eL19 variants defective in eB12 bridge formation failed to form 80S ribosomes regardless of Mg(2+) concentration. The reassociation of 40S and mutant 60S subunits was markedly improved in the presence of deacetylated tRNA, emphasizing the importance of tRNAs during the subunit association. We propose that the eB12 bridge plays an important role in subunit joining and in optimizing ribosome functionality.

The Functional Role of eL19 and eB12 Intersubunit Bridge in the Eukaryotic Ribosome.

Interactions between subunits in the Saccharomyces cerevisiae ribosome are mediated by universal and eukaryote-specific intersubunit bridges. Universal bridges are positioned close to the ribosomal functional centers, while eukaryote-specific bridges are mainly located on the periphery of the ribosome. Two bridges, eB13 and B6, are formed by the ribosomal protein eL24. The eukaryotic eL24 is composed of an N-terminal domain, a linker region and a C-terminal α-helix. Here, the functions of different domains of eL24 in the S. cerevisiae ribosome were evaluated. The C-terminal domain and the linker region of the eL24 form eukaryote-specific eB13 bridge. Phenotypic characterization of the eL24 deletion mutants indicated that the functional integrity of the eB13 bridge mainly depends on the protein-protein contacts between eL24 and eS6. Further investigation showed importance of the eB13 bridge in the subunit joining in vivo and in vitro. In vitro translation assay demonstrated the role of the eB13 bridge in both initiation and elongation steps of translation. Intriguingly, results of in vitro translation experiment suggest involvement of the N-terminal domain of eL24 in the translation initiation. Therefore, eL24 performs number of tasks required for the optimal ribosome functionality.

Ribosomal protein eL24, involved in two intersubunit bridges, stimulates translation initiation and elongation.

Ribosomes of Archaea and Eukarya share higher homology with each other than with bacterial ribosomes. For example, there is a set of 35 r-proteins that are specific only for archaeal and eukaryotic ribosomes. Three of these proteins-eL19, eL24, and eL41-participate in interactions between ribosomal subunits. The eukaryote-specific extensions of r-proteins eL19 and eL24 form two intersubunit bridges eB12 and eB13, which are present only in eukaryotic ribosomes. The third r-protein, eL41, forms bridge eB14. Notably, eL41 is found in all eukaryotes but only in some Archaea. It has been shown that bridges eB12 and eB13 are needed for efficient translation, while r-protein eL41 plays a minor role in ribosome function. Here, the functional interactions between intersubunit bridges were studied using budding yeast strains lacking different combinations of the abovementioned bridges/proteins. The growth phenotypes, levels of  translation, ribosome-polysome profiles, and  association of ribosomal subunits were analyzed. The results show a genetic interaction between r-protein eL41 and the eB12 bridge-forming region of eL19, and between r-proteins eL41 and eL24. It was possible to construct viable yeast strains with Archaea-like ribosomes lacking two or three eukaryote-specific bridges. These strains display slow growth and a poor translation phenotype. In addition, bridges eB12 and eB13 appear to cooperate during ribosome subunit association. These results indicate that nonessential structural elements of r-proteins become highly important in the context of disturbed subunit interactions. Therefore, eukaryote-specific bridges may contribute to the evolutionary success of eukaryotic translation machinery.

Functional Interactions of Ribosomal Intersubunit Bridges in .

The elongation step of translation is a key contributor to the abundance, folding and quality of proteins and to the stability of mRNA. However, control over translation elongation has not been thoroughly investigated. In this study, a Renilla-firefly luciferase fusion reporter system was further developed to investigate the in vitro elongation rate and processivity of ribosomes independent of the initiation and termination steps. The reporter mRNA was constructed to contain a single ORF encoding in-frame Renilla luciferase, a specific domain moiety and firefly luciferase. Such a reporter structure enables the quantitative and individual evaluation of the synthesis of a specific domain. As a proof of principle, the synthesis of three protein domains of different lengths and structures was analyzed. Using a cell-free translation assay, both the elongation rate and processivity of ribosomes were shown to vary depending on the domain synthesized. Additionally, a stalling sequence consisting of ten rare arginine codons notably reduced the elongation rate and the processivity of the ribosomes. All these results are consistent with the previously known dynamics of elongation in vivo. Overall, the methodology presented in this report provides a framework for studying aspects that contribute to the elongation step of translation.

Luciferase-based reporter system for in vitro evaluation of elongation rate and processivity of ribosomes.

In three domains of life, proteins are synthesized by large ribonucleoprotein particles called ribosomes. All ribosomes are composed of ribosomal RNAs (rRNA) and numerous ribosomal proteins (r-protein). The three-dimensional shape of ribosomes is mainly defined by a tertiary structure of rRNAs. In addition, rRNAs have a major role in decoding the information carried by messenger RNAs and catalyzing the peptide bond formation. R-proteins are essential for shaping the network of interactions that contribute to a various aspects of the protein synthesis machinery, including assembly of ribosomes and interaction of ribosomal subunits. Structural studies have revealed that many key components of ribosomes are conserved in all life domains. Besides the core structure, ribosomes contain domain-specific structural features that include additional r-proteins and extensions of rRNA and r-proteins. This review focuses specifically on those r-proteins that are found only in archaeal and eukaryotic ribosomes. The role of these archaea/eukaryote specific r-proteins in stabilizing the ribosome structure is discussed. Several examples illustrate their functions in the formation of the internal network of ribosomal subunits and interactions between the ribosomal subunits. In addition, the significance of these r-proteins in ribosome biogenesis and protein synthesis is highlighted.

Archaea/eukaryote-specific ribosomal proteins - guardians of a complex structure.

Yeasts are single-celled fungi that are widespread around the globe. They are part of a community of microorganisms that use a wide variety of habitats, including fruit surfaces. This study aimed to characterise the culturable epiphytic yeasts associated with apple fruits. The isolated yeast strains were identified by sequencing the 5.8S-ITS region and D1/D2 region of the large subunit ribosomal RNA gene and maintained for long-term storage. A total of 230 yeast isolates belonging to 33 species were recovered. Most of the collected isolates belonged to the phylum Basidiomycota. Members of genera  and  were most frequently isolated. Over half of the species were isolated on only one to three occasions. In seven of the species obtained, the isolates were considerably divergent from their closest relatives and may therefore represent new distinct species. The results of this study demonstrate a high diversity of yeast species associated with apple fruits.

Richness of yeast community associated with apple fruits in Estonia.

Ribosomal RNAs (rRNAs) are extensively modified during the transcription and subsequent maturation. Three types of modifications, 2'-O-methylation of ribose moiety, pseudouridylation, and base modifications, are introduced either by a snoRNA-driven mechanism or by stand-alone enzymes. Modified nucleotides are clustered at the functionally important sites, including peptidyl transferase center (PTC). Therefore, it has been hypothesised that the modified nucleotides play an important role in ensuring the functionality of the ribosome. In this study, we demonstrate that seven 25S rRNA modifications, including four evolutionarily conserved modifications, in the proximity of PTC can be simultaneously depleted without loss of cell viability. Yeast mutants lacking three snoRNA genes (,  and ) and/or expressing enzymatically inactive variants of  and  were constructed. The results show that rRNA modifications in PTC contribute collectively to efficient translation in eukaryotic cells. The deficiency of seven modified nucleotides in 25S rRNA resulted in reduced cell growth, cold sensitivity, decreased translation levels, and hyperaccurate translation, as indicated by the reduced missense and nonsense suppression. The modification mC2870 is crucial in the absence of the other six modified nucleotides. Thus, the pattern of rRNA-modified nucleotides around the PTC is essential for optimal ribosomal translational activity and translational fidelity.

Loss of Conserved rRNA Modifications in the Peptidyl Transferase Center Leads to Diminished Protein Synthesis and Cell Growth in Budding Yeast.

Isolation of amplifiable genomic DNA is a prerequisite for the implementation of PCR-based techniques. Here we present a protocol for isolating the genomic DNA from a variety of wild yeast species. This can be completed in approximately 1 h and does not require sophisticated laboratory equipment. We describe steps for growing yeast cells, genomic data extraction, and downstream assay for amplification of specific sequences from the genomic DNA. We then detail procedures for gel electrophoresis and analysis of the results. For complete details on the use and execution of this protocol, please refer to Kristjuhan et al..

Article	Year
Plasmids with E2 epitope tags: tagging modules for N- and C-terminal PCR-based gene targeting in both budding and fission yeast, and inducible expression vectors for fission yeast. Yeast (Chichester, England)\| PubMed ID: 19180640	2009
Stem-loop structure of Cocksfoot mottle virus RNA is indispensable for programmed -1 ribosomal frameshifting. Virus research\| PubMed ID: 19748532	2009
A thiamine-regulatable epitope-tagged protein expression system in fission yeast. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 22160912	2012
C-terminal extension of the yeast mitochondrial DNA polymerase determines the balance between synthesis and degradation. PloS one\| PubMed ID: 22432028	2012
Dysfunctional mitochondria modulate cAMP-PKA signaling and filamentous and invasive growth of Saccharomyces cerevisiae. Genetics\| PubMed ID: 23172851	2013
Stepwise splitting of ribosomal proteins from yeast ribosomes by LiCl. PloS one\| PubMed ID: 24991888	2014
The Functional Role of eL19 and eB12 Intersubunit Bridge in the Eukaryotic Ribosome. Journal of molecular biology\| PubMed ID: 27038511	2016
Ribosomal protein eL24, involved in two intersubunit bridges, stimulates translation initiation and elongation. Nucleic acids research\| PubMed ID: 30407570	2019
Functional Interactions of Ribosomal Intersubunit Bridges in . Genetics\| PubMed ID: 31649153	2019
Luciferase-based reporter system for in vitro evaluation of elongation rate and processivity of ribosomes. Nucleic acids research\| PubMed ID: 33684199	2021
Archaea/eukaryote-specific ribosomal proteins - guardians of a complex structure. Computational and structural biotechnology journal\| PubMed ID: 36817958	2023
Richness of yeast community associated with apple fruits in Estonia. Heliyon\| PubMed ID: 38545165	2024
Loss of Conserved rRNA Modifications in the Peptidyl Transferase Center Leads to Diminished Protein Synthesis and Cell Growth in Budding Yeast. International journal of molecular sciences\| PubMed ID: 38791231	2024
Protocol for rapid and cost-effective extraction of genomic DNA from a wide range of wild yeast species for use in PCR-based applications. STAR protocols\| PubMed ID: 39232988	2024
Translon: a single term for translated regions. Nature methods\| PubMed ID: 40890551	2025

Tiina Tamm

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