scholarly journals Functional and physical interactions of Krr1p, a Saccharomyces cerevisiae nucleolar protein.

2004 ◽  
Vol 51 (1) ◽  
pp. 173-187 ◽  
Author(s):  
Robert Gromadka ◽  
Iwona Karkusiewicz ◽  
Bozenna Rempoła ◽  
Joanna Rytka
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ribosomal Proteins ◽  
Northern Blot Analysis ◽  
Affinity Purification ◽  
Elongation Factor ◽  
Translation Elongation ◽  
Multicopy Suppressors ◽  
Genes Encoding ◽  
Cold Sensitive ◽  
Physical Interactions

The Krr1 protein of Saccharomyces cerevisiae is involved in processing of pre-rRNA and assembly of pre-ribosomal 40S subunits. To further investigate the function of Krr1p we constructed a conditional cold sensitive mutant krr1-21, and isolated seven genes from Schizosaccharomyces pombe whose products suppressed the cold sensitive phenotype of krr1-21 cells. Among the multicopy suppressors we found genes coding for translation elongation factor EF-1alpha, a putative ribose methyltransferase and five genes encoding ribosomal proteins. Using the tandem affinity purification (TAP) method we identified thirteen S. cerevisiae ribosomal proteins interacting with Krr1p. Taken together, these results indicate that Krr1p interacts functionally as well as physically with ribosomal proteins. Northern blot analysis revealed that changes in the level of krr1-21 mRNA were accompanied by similar changes in the level of mRNAs of genes encoding ribosomal proteins. Thus, Krr1p and the genes encoding ribosomal proteins it interacts with seem to be coordinately regulated at the level of transcription.

scholarly journals Increased expression of Saccharomyces cerevisiae translation elongation factor 1 alpha bypasses the lethality of a TEF5 null allele encoding elongation factor 1 beta.

Genetics ◽  
1995 ◽  
Vol 141 (2) ◽  
pp. 481-489 ◽  
Author(s):  
T G Kinzy ◽  
J L Woolford
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Null Allele ◽  
Elongation Factor ◽  
Wild Type ◽  
Translation Elongation Factor ◽  
Gene Encoding ◽  
Translation Elongation ◽  
Dominant Mutant ◽  
Cold Sensitive ◽  
Elongation Factor 1

Abstract Translation elongation factor 1beta (EF-1beta) catalyzes the exchange of bound GDP for GTP on EF-1alpha. The lethality of a null allele of the TEF5 gene encoding EF-1beta in Saccharomyces cerevisiae was suppressed by extra copies of the TEF2 gene encoding EF-1alpha. The strains with tef5::TRP1 suppressed by extra copies of TEF were slow growing, cold sensitive, hypersensitive to inhibitors of translation elongation and showed increased phenotypic suppression of +1 frameshift and UAG nonsense mutations. Nine dominant mutant alleles of TEF2 that cause increased suppression of frameshift mutations also suppressed the lethality of tef5::TRP1. Most of the strains in which tef5::TRP1 is suppressed by dominant mutant alleles of TEF2 grew more slowly and were more antibiotic sensitive than strains with tef5::TRP1 is suppressed by wild-type TEF2. Two alleles, TEF2-4 and TEF2-10, interact with tef5::TRP1 to produce strains that showed doubling times similar to tef5::TRP1 strains containing extra copies of wild-type TEF2. These strains were less cold sensitive, drug sensitive and correspondingly less efficient suppressor of +1 frameshift mutations. These phenotypes indicate that translation and cell growth are highly sensitive to changes in EF-1alpha and EF-1beta activity.

Elongation factor EF-1 alpha gene dosage alters translational fidelity in Saccharomyces cerevisiae

1989 ◽  
Vol 9 (10) ◽  
pp. 4571-4575
Author(s):  
J M Song ◽  
S Picologlou ◽  
C M Grant ◽  
M Firoozan ◽  
M F Tuite ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Gene Dosage ◽  
Elongation Factor ◽  
Nonsense Suppression ◽  
Translational Fidelity ◽  
Genes Encoding ◽  
Alpha Gene ◽  
Nonsense Codons

Changes in the dosage of genes encoding elongation factor EF-1 alpha were shown to cause parallel changes in the misreading of nonsense codons. Higher amounts of EF-1 alpha were correlated with increased nonsense suppression, suggesting that the level of EF-1 alpha is critically involved in translational fidelity.

scholarly journals Translation elongation factor 2 depletion by siRNA in mouse liver leads to mTOR-independent translational upregulation of ribosomal protein genes

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Maxim V. Gerashchenko ◽  
Mikhail V. Nesterchuk ◽  
Elena M. Smekalova ◽  
Joao A. Paulo ◽  
Piotr S. Kowalski ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Mammalian Cells ◽  
Ribosomal Proteins ◽  
Mouse Liver ◽  
Negative Impact ◽  
Elongation Factor ◽  
Ribosome Profiling ◽  
Translation Elongation Factor ◽  
Translation Elongation ◽  
Elongation Factor 2

Abstract Due to breakthroughs in RNAi and genome editing methods in the past decade, it is now easier than ever to study fine details of protein synthesis in animal models. However, most of our understanding of translation comes from unicellular organisms and cultured mammalian cells. In this study, we demonstrate the feasibility of perturbing protein synthesis in a mouse liver by targeting translation elongation factor 2 (eEF2) with RNAi. We were able to achieve over 90% knockdown efficacy and maintain it for 2 weeks effectively slowing down the rate of translation elongation. As the total protein yield declined, both proteomics and ribosome profiling assays showed robust translational upregulation of ribosomal proteins relative to other proteins. Although all these genes bear the TOP regulatory motif, the branch of the mTOR pathway responsible for translation regulation was not activated. Paradoxically, coordinated translational upregulation of ribosomal proteins only occurred in the liver but not in murine cell culture. Thus, the upregulation of ribosomal transcripts likely occurred via passive mTOR-independent mechanisms. Impaired elongation sequesters ribosomes on mRNA and creates a shortage of free ribosomes. This leads to preferential translation of transcripts with high initiation rates such as ribosomal proteins. Furthermore, severe eEF2 shortage reduces the negative impact of positively charged amino acids frequent in ribosomal proteins on ribosome progression.

scholarly journals Elongation factor EF-1 alpha gene dosage alters translational fidelity in Saccharomyces cerevisiae.

1989 ◽  
Vol 9 (10) ◽  
pp. 4571-4575 ◽  
Author(s):  
J M Song ◽  
S Picologlou ◽  
C M Grant ◽  
M Firoozan ◽  
M F Tuite ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Gene Dosage ◽  
Elongation Factor ◽  
Nonsense Suppression ◽  
Translational Fidelity ◽  
Genes Encoding ◽  
Alpha Gene ◽  
Nonsense Codons

Changes in the dosage of genes encoding elongation factor EF-1 alpha were shown to cause parallel changes in the misreading of nonsense codons. Higher amounts of EF-1 alpha were correlated with increased nonsense suppression, suggesting that the level of EF-1 alpha is critically involved in translational fidelity.

scholarly journals Disruption of single-copy genes encoding acidic ribosomal proteins in Saccharomyces cerevisiae.

1990 ◽  
Vol 10 (5) ◽  
pp. 2182-2190 ◽  
Author(s):  
M Remacha ◽  
C Santos ◽  
J P Ballesta
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ribosomal Proteins ◽  
Minimal Medium ◽  
Single Copy ◽  
Diploid Strain ◽  
Tetrad Analysis ◽  
Genes Encoding ◽  
Acidic Proteins ◽  
Dna Fragment ◽  
His3 Gene

Using the cloned genes coding for the ribosomal acidic proteins L44 and L45, constructions were made which deleted part of the coding sequence and inserted a DNA fragment at that site carrying either the URA3 or HIS3 gene. By gene disruption techniques with linearized DNA from these constructions, strains of Saccharomyces cerevisiae were obtained which lacked a functional gene for either protein L44 or protein L45. The disrupted genes in the transformants were characterized by Southern blots. The absence of the proteins was verified by electrofocusing and immunological techniques, but a compensating increase of the other acidic ribosomal proteins was not detected. The mutant lacking L44 grew at a rate identical to the parental strain in complex as well as in minimal medium. The L45-disrupted strain also grew well in both media but at a slower rate than the parental culture. A diploid strain was obtained by crossing both transformants, and by tetrad analysis it was shown that the double transformant lacking both genes is not viable. These results indicated that proteins L44 and L45 are independently dispensable for cell growth and that the ribosome is functional in the absence of either of them.

scholarly journals Purification of Components of the Translation Elongation Factor Complex of Plasmodium falciparum by Tandem Affinity Purification

2007 ◽  
Vol 6 (4) ◽  
pp. 584-591 ◽  
Author(s):  
Sachiko Takebe ◽  
William Harold Witola ◽  
Bernd Schimanski ◽  
Arthur Günzl ◽  
Choukri Ben Mamoun
Keyword(s):  
Plasmodium Falciparum ◽  
Life Cycle ◽  
Protein Interactions ◽  
Affinity Purification ◽  
Elongation Factor ◽  
Tandem Affinity Purification ◽  
Complex Life Cycle ◽  
Spectrometric Analysis ◽  
Translation Elongation Factor ◽  
Translation Elongation

ABSTRACT Plasmodium falciparum is the causative agent of severe human malaria, responsible for over 2 million deaths annually. Of the 5,300 polypeptides predicted to control the parasite life cycle in mosquitoes and humans, 60% are of unknown function. A major challenge of malaria postgenomic biology is to understand how the 5,300 predicted proteins coexist and interact to perform the essential tasks that define the complex life cycle of the parasite. One approach to assign function to these proteins is by identifying their physiological partners. Here we describe the use of tandem affinity purification (TAP) and mass spectrometry for identification of native protein interactions and purification of protein complexes in P. falciparum. Transgenic parasites were generated which express the translation elongation factor PfEF-1β harboring a C-terminal PTP tag which consists of the protein C epitope, a tobacco etch virus protease cleavage site, and two protein A domains. Purification of PfEF-1β-PTP from crude extracts followed by mass spectrometric analysis revealed, in addition to the tagged protein itself, the presence of the native PfEF-1β, the G-protein PfEF-1α, and two new proteins that we named PfEF-1γ and PfEF-1δ based on their homology to other eukaryotic γ and δ translation elongation factor subunits. These data, which constitute the first application of TAP for purification of a protein complex under native conditions in P. falciparum, revealed that the translation elongation complex in this organism contains at least two subunits of PfEF-1β. The success of this approach will set the stage for a systematic analysis of protein interactions in this important human pathogen.

Disruption of single-copy genes encoding acidic ribosomal proteins in Saccharomyces cerevisiae

1990 ◽  
Vol 10 (5) ◽  
pp. 2182-2190
Author(s):  
M Remacha ◽  
C Santos ◽  
J P Ballesta
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ribosomal Proteins ◽  
Minimal Medium ◽  
Single Copy ◽  
Diploid Strain ◽  
Tetrad Analysis ◽  
Genes Encoding ◽  
Acidic Proteins ◽  
Dna Fragment ◽  
His3 Gene

Using the cloned genes coding for the ribosomal acidic proteins L44 and L45, constructions were made which deleted part of the coding sequence and inserted a DNA fragment at that site carrying either the URA3 or HIS3 gene. By gene disruption techniques with linearized DNA from these constructions, strains of Saccharomyces cerevisiae were obtained which lacked a functional gene for either protein L44 or protein L45. The disrupted genes in the transformants were characterized by Southern blots. The absence of the proteins was verified by electrofocusing and immunological techniques, but a compensating increase of the other acidic ribosomal proteins was not detected. The mutant lacking L44 grew at a rate identical to the parental strain in complex as well as in minimal medium. The L45-disrupted strain also grew well in both media but at a slower rate than the parental culture. A diploid strain was obtained by crossing both transformants, and by tetrad analysis it was shown that the double transformant lacking both genes is not viable. These results indicated that proteins L44 and L45 are independently dispensable for cell growth and that the ribosome is functional in the absence of either of them.

scholarly journals Ctk1 Function Is Necessary for Full Translation Initiation Activity in Saccharomyces cerevisiae

2014 ◽  
Vol 14 (1) ◽  
pp. 86-95 ◽  
Author(s):  
Britta Coordes ◽  
Katharina M. Brünger ◽  
Kaspar Burger ◽  
Boumediene Soufi ◽  
Juliane Horenk ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Translation Initiation ◽  
Elongation Factor ◽  
Entry Site ◽  
Translation Elongation ◽  
Internal Ribosome Entry ◽  
Content Type ◽  
Phosphorylation Pattern ◽  
Subunit Joining ◽  
Paralysis Virus

ABSTRACT Translation is a fundamental and highly regulated cellular process. Previously, we reported that the kinase and transcription elongation factor Ctk1 increases fidelity during translation elongation in Saccharomyces cerevisiae . Here, we show that loss of Ctk1 function also affects the initiation step of translation. Translation active extracts from Ctk1-depleted cells show impaired translation activity of capped mRNA, but not mRNA reporters containing the cricket paralysis virus (CrPV) internal ribosome entry site (IRES). Furthermore, the formation of 80S initiation complexes is decreased, which is probably due to reduced subunit joining. In addition, we determined the changes in the phosphorylation pattern of a ribosome enriched fraction after depletion of Ctk1. Thus, we provide a catalogue of phosphoproteomic changes dependent on Ctk1. Taken together, our data suggest a stimulatory function of Ctk1 in 80S formation during translation initiation.

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