Limits...
Rio1 mediates ATP-dependent final maturation of 40S ribosomal subunits.

Turowski TW, Lebaron S, Zhang E, Peil L, Dudnakova T, Petfalski E, Granneman S, Rappsilber J, Tollervey D - Nucleic Acids Res. (2014)

Bottom Line: Within the Rio1-associated pre-40S particles, in vitro pre-rRNA cleavage was strongly stimulated by ATP and required nucleotide binding by Rio1.Binding sites for Rio1 were within the core of the 18S rRNA, overlapping tRNA interaction sites and distinct from the related kinase Rio2.Site D cleavage occurs within pre-40S-60S complexes and Rio1-associated particles efficiently assemble into these complexes, whereas Pno1 appeared to be depleted relative to Nob1.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Centre for Cell Biology, University of Edinburgh, Michael Swann Building, Kings Buildings, Mayfield Road, Edinburgh EH9 3JR, UK.

Show MeSH

Related in: MedlinePlus

The ATP binding ability of Rio1 is required for growth and site D cleavage. (A) Growth of PGAL::RIO1 strain on permissive galactose medium or repressive glucose medium, without plasmid (empty brackets) or complemented by plasmids expressing functional Rio1-HTP or Rio1 with the D244A or K125R mutations. (B) Comparison of cleavage in pre-ribosomes associated with Rio1, Rio1K125R and Rio1D244A, in the presence of ATP, GTP or non-hydrolysable AMP-PNP each at 1 mM. (C) Quantitation of cleavage efficiencies. Cleavage efficiency was expressed relative to the ATP-treated sample for functional Rio1. Error bars show standard error.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4231747&req=5

Figure 2: The ATP binding ability of Rio1 is required for growth and site D cleavage. (A) Growth of PGAL::RIO1 strain on permissive galactose medium or repressive glucose medium, without plasmid (empty brackets) or complemented by plasmids expressing functional Rio1-HTP or Rio1 with the D244A or K125R mutations. (B) Comparison of cleavage in pre-ribosomes associated with Rio1, Rio1K125R and Rio1D244A, in the presence of ATP, GTP or non-hydrolysable AMP-PNP each at 1 mM. (C) Quantitation of cleavage efficiencies. Cleavage efficiency was expressed relative to the ATP-treated sample for functional Rio1. Error bars show standard error.

Mentions: To test the effects of these mutations, we generated a PGAL::RIO1 strain, in which chromosomal RIO1 gene is expressed on galactose medium but repressed on glucose medium. This strain was transformed with plasmids expressing Rio1 or Rio1K125R or Rio1D244A carrying C-terminal His6-TEV-Protein A (HTP) tags. Growth tests (Figure 2A) showed that expression of Rio1-HTP supported growth of the PGAL::RIO1 strain on glucose medium, whereas neither the K125R nor D244A mutant proteins complement the growth of the Rio1-depleted strain. Pre-ribosomes were purified from the wt and mutant Rio1 strains and tested for in vitro cleavage activity (Figure 2B). Interestingly, the catalytic mutant form of Rio1 (D244A) only partially reduced D cleavage efficiency in vitro, whereas the K125R mutation in the ATP binding pocket almost completely abolished cleavage, showing that nucleotide binding by Rio1 is required for cleavage.


Rio1 mediates ATP-dependent final maturation of 40S ribosomal subunits.

Turowski TW, Lebaron S, Zhang E, Peil L, Dudnakova T, Petfalski E, Granneman S, Rappsilber J, Tollervey D - Nucleic Acids Res. (2014)

The ATP binding ability of Rio1 is required for growth and site D cleavage. (A) Growth of PGAL::RIO1 strain on permissive galactose medium or repressive glucose medium, without plasmid (empty brackets) or complemented by plasmids expressing functional Rio1-HTP or Rio1 with the D244A or K125R mutations. (B) Comparison of cleavage in pre-ribosomes associated with Rio1, Rio1K125R and Rio1D244A, in the presence of ATP, GTP or non-hydrolysable AMP-PNP each at 1 mM. (C) Quantitation of cleavage efficiencies. Cleavage efficiency was expressed relative to the ATP-treated sample for functional Rio1. Error bars show standard error.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4231747&req=5

Figure 2: The ATP binding ability of Rio1 is required for growth and site D cleavage. (A) Growth of PGAL::RIO1 strain on permissive galactose medium or repressive glucose medium, without plasmid (empty brackets) or complemented by plasmids expressing functional Rio1-HTP or Rio1 with the D244A or K125R mutations. (B) Comparison of cleavage in pre-ribosomes associated with Rio1, Rio1K125R and Rio1D244A, in the presence of ATP, GTP or non-hydrolysable AMP-PNP each at 1 mM. (C) Quantitation of cleavage efficiencies. Cleavage efficiency was expressed relative to the ATP-treated sample for functional Rio1. Error bars show standard error.
Mentions: To test the effects of these mutations, we generated a PGAL::RIO1 strain, in which chromosomal RIO1 gene is expressed on galactose medium but repressed on glucose medium. This strain was transformed with plasmids expressing Rio1 or Rio1K125R or Rio1D244A carrying C-terminal His6-TEV-Protein A (HTP) tags. Growth tests (Figure 2A) showed that expression of Rio1-HTP supported growth of the PGAL::RIO1 strain on glucose medium, whereas neither the K125R nor D244A mutant proteins complement the growth of the Rio1-depleted strain. Pre-ribosomes were purified from the wt and mutant Rio1 strains and tested for in vitro cleavage activity (Figure 2B). Interestingly, the catalytic mutant form of Rio1 (D244A) only partially reduced D cleavage efficiency in vitro, whereas the K125R mutation in the ATP binding pocket almost completely abolished cleavage, showing that nucleotide binding by Rio1 is required for cleavage.

Bottom Line: Within the Rio1-associated pre-40S particles, in vitro pre-rRNA cleavage was strongly stimulated by ATP and required nucleotide binding by Rio1.Binding sites for Rio1 were within the core of the 18S rRNA, overlapping tRNA interaction sites and distinct from the related kinase Rio2.Site D cleavage occurs within pre-40S-60S complexes and Rio1-associated particles efficiently assemble into these complexes, whereas Pno1 appeared to be depleted relative to Nob1.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Centre for Cell Biology, University of Edinburgh, Michael Swann Building, Kings Buildings, Mayfield Road, Edinburgh EH9 3JR, UK.

Show MeSH
Related in: MedlinePlus