scholarly journals Ribosomal Protein L3 Mutants Alter Translational Fidelity and Promote Rapid Loss of the Yeast Killer Virus

1999 ◽  
Vol 19 (1) ◽  
pp. 384-391 ◽  
Author(s):  
Stuart W. Peltz ◽  
Amy B. Hammell ◽  
Ying Cui ◽  
Jason Yasenchak ◽  
Lara Puljanowski ◽  
...  
Keyword(s):  
Ribosomal Protein ◽  
Missense Mutations ◽  
Ribosomal Frameshift ◽  
Virus Propagation ◽  
Particle Assembly ◽  
Ribosomal Frameshifting ◽  
Peptidyl Transfer ◽  
Yeast Killer ◽  
Peptidyltransferase Center ◽  
Killer Virus

ABSTRACT Programmed −1 ribosomal frameshifting is utilized by a number of RNA viruses as a means of ensuring the correct ratio of viral structural to enzymatic proteins available for viral particle assembly. Altering frameshifting efficiencies upsets this ratio, interfering with virus propagation. We have previously demonstrated that compounds that alter the kinetics of the peptidyl-transfer reaction affect programmed −1 ribosomal frameshift efficiencies and interfere with viral propagation in yeast. Here, the use of a genetic approach lends further support to the hypothesis that alterations affecting the ribosome’s peptidyltransferase activity lead to changes in frameshifting efficiency and virus loss. Mutations in theRPL3 gene, which encodes a ribosomal protein located at the peptidyltransferase center, promote approximately three- to fourfold increases in programmed −1 ribosomal frameshift efficiencies and loss of the M1 killer virus of yeast. Themak8-1 allele of RPL3 contains two adjacent missense mutations which are predicted to structurally alter the Mak8-1p. Furthermore, a second allele that encodes the N-terminal 100 amino acids of L3 (called L3Δ) exerts atrans-dominant effect on programmed −1 ribosomal frameshifting and killer virus maintenance. Taken together, these results support the hypothesis that alterations in the peptidyltransferase center affect programmed −1 ribosomal frameshifting.

scholarly journals The Mof2/Sui1 Protein Is a General Monitor of Translational Accuracy

1998 ◽  
Vol 18 (3) ◽  
pp. 1506-1516 ◽  
Author(s):  
Ying Cui ◽  
Jonathan D. Dinman ◽  
Terri Goss Kinzy ◽  
Stuart W. Peltz
Keyword(s):  
Site Selection ◽  
Start Site ◽  
Wild Type ◽  
Virus Propagation ◽  
Ribosomal Frameshifting ◽  
Reading Frame ◽  
Translational Accuracy ◽  
Its Gene ◽  
Killer Virus

ABSTRACT Although it is essential for protein synthesis to be highly accurate, a number of cases of directed ribosomal frameshifting have been reported in RNA viruses, as well as in procaryotic and eucaryotic genes. Changes in the efficiency of ribosomal frameshifting can have major effects on the ability of cells to propagate viruses which use this mechanism. Furthermore, studies of this process can illuminate the mechanisms involved in the maintenance of the normal translation reading frame. The yeast Saccharomyces cerevisiae killer virus system uses programmed −1 ribosomal frameshifting to synthesize its gene products. Strains harboring the mof2-1 allele demonstrated a fivefold increase in frameshifting and prevented killer virus propagation. In this report, we present the results of the cloning and characterization of the wild-type MOF2 gene.mof2-1 is a novel allele of SUI1, a gene previously shown to play a role in translation initiation start site selection. Strains harboring the mof2-1 allele demonstrated a mutant start site selection phenotype and increased efficiency of programmed −1 ribosomal frameshifting and conferred paromomycin sensitivity. The increased frameshifting observed in vivo was reproduced in extracts prepared from mof2-1 cells. Addition of purified wild-type Mof2p/Sui1p reduced frameshifting efficiencies to wild-type levels. Expression of the human SUI1 homolog in yeast corrects all of the mof2-1 phenotypes, demonstrating that the function of this protein is conserved throughout evolution. Taken together, these results suggest that Mof2p/Sui1p functions as a general modulator of accuracy at both the initiation and elongation phases of translation.

scholarly journals Delayed rRNA Processing Results in Significant Ribosome Biogenesis and Functional Defects

2003 ◽  
Vol 23 (5) ◽  
pp. 1602-1613 ◽  
Author(s):  
Arturas Meskauskas ◽  
Jennifer L. Baxter ◽  
Edward A. Carr ◽  
Jason Yasenchak ◽  
Jennifer E. G. Gallagher ◽  
...  
Keyword(s):  
Histone Deacetylase ◽  
Ribosome Biogenesis ◽  
Recessive Mutation ◽  
Rrna Processing ◽  
Wild Type ◽  
Ribosomal Frameshifting ◽  
Peptidyl Transfer ◽  
Subsequent Loss ◽  
Killer Virus ◽  
Functional Defects

ABSTRACT mof6-1 was originally isolated as a recessive mutation in Saccharomyces cerevisiae which promoted increased efficiencies of programmed −1 ribosomal frameshifting and rendered cells unable to maintain the killer virus. Here, we demonstrate that mof6-1 is a unique allele of the histone deacetylase RPD3, that the deacetylase function of Rpd3p is required for controlling wild-type levels of frameshifting and virus maintenance, and that the closest human homolog can fully complement these defects. Loss of the Rpd3p-associated histone deacetylase function, either by mutants of rpd3 or loss of the associated gene product Sin3p or Sap30p, results in a delay in rRNA processing rather than in an rRNA transcriptional defect. This results in production of ribosomes having lower affinities for aminoacyl-tRNA and diminished peptidyltransferase activities. We hypothesize that decreased rates of peptidyl transfer allow ribosomes with both A and P sites occupied by tRNAs to pause for longer periods of time at −1 frameshift signals, promoting increased programmed −1 ribosomal frameshifting efficiencies and subsequent loss of the killer virus. The frameshifting defect is accentuated when the demand for ribosomes is highest, suggesting that rRNA posttranscriptional modification is the bottleneck in ribosome biogenesis.

Evolution of defective-interfering double-stranded RNAs of the yeast killer virus.

1979 ◽  
Vol 32 (2) ◽  
pp. 692-696 ◽  
Author(s):  
W P Kane ◽  
D F Pietras ◽  
J A Bruenn
Keyword(s):  
Yeast Killer ◽  
Killer Virus

Genetic analysis of maintenance and expression of L and M double-stranded RNAs from yeast killer virus K28

Yeast ◽  
1992 ◽  
Vol 8 (5) ◽  
pp. 373-384 ◽  
Author(s):  
Manfred J. Schmitt ◽  
Donald J. Tipper
Keyword(s):  
Genetic Analysis ◽  
Yeast Killer ◽  
Killer Virus

Ribosomal Protein S19 and Diamond Blackfan Anemia.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2839-2839
Author(s):  
Steven R. Ellis ◽  
Carlos Arce-Lara ◽  
Jacqueline M. Caffrey ◽  
Diana A. Alvarez-Arias
Keyword(s):  
Ribosomal Protein ◽  
Single Gene ◽  
Yeast Cells ◽  
Functional Testing ◽  
Missense Mutations ◽  
Candidate Locus ◽  
Yeast Saccharomyces Cerevisiae ◽  
Growth Defects ◽  
Diamond Blackfan Anemia ◽  
Ribosomal Protein S19

Abstract Diamond Blackfan Anemia (DBA) is one of several bone marrow failures that have been linked to defects in ribosome synthesis. 25% of DBA cases are linked to mutations in ribosomal protein S19 (Rps19). The etiology of the remaining cases is unknown. To gain a better understanding of the function of the Rps19 family of proteins we have characterized members of this protein family in the yeast, Saccharomyces cerevisiae. In yeast, Rps19 is encoded by duplicated genes, RPS19A and RPS19B. Yeast cells lacking both RPS19 genes are not viable, whereas those lacking a single gene are viable but have growth defects. These latter strains are defective in a specific step in rRNA processing that preferentially affects the maturation of 40S ribosomal subunits. We scanned other yeast strains with mutations in genes for 40S subunit proteins for processing phenotypes similar to RPS19 mutants. Several have phenotypes that overlap with RPS19 mutants, but only RPS18 stands out as being virtually identical to RPS19 mutants. The human RPS18 gene is therefore a candidate locus for pathogenic mutations in DBA patients with normal RPS19. We are currently developing strategies to sequence RPS18 genes from DBA patients with normal RPS19 to determine if mutations in RPS18 are associated with DBA. We have also developed a yeast system for the functional testing of mutant alleles of RPS19 found in DBA patients. In general, a mutation is considered pathogenic if it is not found in unaffected family members and in the general population. We have found, however, that several missense mutations classified as pathogenic in DBA patients do not affect Rps19 function in the yeast system. The failure of these mutations to affect Rps19 function in yeast points to a need for functional testing of RPS19 mutant alleles in human cells.

Role of Ribosomal Protein L27 in Peptidyl Transfer†

Biochemistry ◽  
2008 ◽  
Vol 47 (17) ◽  
pp. 4898-4906 ◽  
Author(s):  
Stefan Trobro ◽  
Johan Åqvist
Keyword(s):  
Ribosomal Protein ◽  
Peptidyl Transfer

scholarly journals Achieving a Golden Mean: Mechanisms by Which Coronaviruses Ensure Synthesis of the Correct Stoichiometric Ratios of Viral Proteins

2010 ◽  
Vol 84 (9) ◽  
pp. 4330-4340 ◽  
Author(s):  
Ewan P. Plant ◽  
Rasa Rakauskaitė ◽  
Deborah R. Taylor ◽  
Jonathan D. Dinman
Keyword(s):  
Hepatitis Virus ◽  
Biological Significance ◽  
Open Reading Frames ◽  
Nonstructural Proteins ◽  
Virus Propagation ◽  
Ribosomal Frameshifting ◽  
Golden Mean ◽  
Encoded Proteins ◽  
Upstream Open Reading Frames ◽  
Reading Frames

ABSTRACT In retroviruses and the double-stranded RNA totiviruses, the efficiency of programmed −1 ribosomal frameshifting is critical for ensuring the proper ratios of upstream-encoded capsid proteins to downstream-encoded replicase enzymes. The genomic organizations of many other frameshifting viruses, including the coronaviruses, are very different, in that their upstream open reading frames encode nonstructural proteins, the frameshift-dependent downstream open reading frames encode enzymes involved in transcription and replication, and their structural proteins are encoded by subgenomic mRNAs. The biological significance of frameshifting efficiency and how the relative ratios of proteins encoded by the upstream and downstream open reading frames affect virus propagation has not been explored before. Here, three different strategies were employed to test the hypothesis that the −1 PRF signals of coronaviruses have evolved to produce the correct ratios of upstream- to downstream-encoded proteins. Specifically, infectious clones of the severe acute respiratory syndrome (SARS)-associated coronavirus harboring mutations that lower frameshift efficiency decreased infectivity by >4 orders of magnitude. Second, a series of frameshift-promoting mRNA pseudoknot mutants was employed to demonstrate that the frameshift signals of the SARS-associated coronavirus and mouse hepatitis virus have evolved to promote optimal frameshift efficiencies. Finally, we show that a previously described frameshift attenuator element does not actually affect frameshifting per se but rather serves to limit the fraction of ribosomes available for frameshifting. The findings of these analyses all support a “golden mean” model in which viruses use both programmed ribosomal frameshifting and translational attenuation to control the relative ratios of their encoded proteins.

Yeast killer virus transcription initiation in vitro

Virology ◽  
1992 ◽  
Vol 187 (1) ◽  
pp. 333-337 ◽  
Author(s):  
Francis P. Barbone ◽  
Teresa L. Williams ◽  
Michael J. Leibowitz
Keyword(s):  
Transcription Initiation ◽  
Virus Transcription ◽  
Yeast Killer ◽  
Killer Virus

scholarly journals The Pokeweed Antiviral Protein Specifically Inhibits Ty1-Directed +1 Ribosomal Frameshifting and Retrotransposition in Saccharomyces cerevisiae

1998 ◽  
Vol 72 (2) ◽  
pp. 1036-1042 ◽  
Author(s):  
Nilgun E. Tumer ◽  
Bijal A. Parikh ◽  
Ping Li ◽  
Jonathan D. Dinman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Viral Particle ◽  
Protein Translation ◽  
Pokeweed Antiviral Protein ◽  
Ribosomal Frameshift ◽  
Single Chain ◽  
Antiviral Protein ◽  
Vitro Assay ◽  
Ribosomal Frameshifting

ABSTRACT Programmed ribosomal frameshifting is a molecular mechanism that is used by many RNA viruses to produce Gag-Pol fusion proteins. The efficiency of these frameshift events determines the ratio of viral Gag to Gag-Pol proteins available for viral particle morphogenesis, and changes in ribosomal frameshift efficiencies can severely inhibit virus propagation. Since ribosomal frameshifting occurs during the elongation phase of protein translation, it is reasonable to hypothesize that agents that affect the different steps in this process may also have an impact on programmed ribosomal frameshifting. We examined the molecular mechanisms governing programmed ribosomal frameshifting by using two viruses of the yeast Saccharomyces cerevisiae. Here, we present evidence that pokeweed antiviral protein (PAP), a single-chain ribosomal inhibitory protein that depurinates an adenine residue in the α-sarcin loop of 25S rRNA and inhibits translocation, specifically inhibits Ty1-directed +1 ribosomal frameshifting in intact yeast cells and in an in vitro assay system. Using an in vivo assay for Ty1 retrotransposition, we show that PAP specifically inhibits Ty1 retrotransposition, suggesting that Ty1 viral particle morphogenesis is inhibited in infected cells. PAP does not affect programmed −1 ribosomal frameshift efficiencies, nor does it have a noticeable impact on the ability of cells to maintain the M1-dependent killer virus phenotype, suggesting that −1 ribosomal frameshifting does not occur after the peptidyltransferase reaction. These results provide the first evidence that PAP has viral RNA-specific effects in vivo which may be responsible for the mechanism of its antiviral activity.

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