Limits...
rRNA mutants in the yeast peptidyltransferase center reveal allosteric information networks and mechanisms of drug resistance.

Rakauskaite R, Dinman JD - Nucleic Acids Res. (2008)

Bottom Line: Here, two viable mutants located in the peptidyltransferase center (PTC) of yeast ribosomes were created using a yeast genetic system that enables stable production of ribosomes containing only mutant rRNAs.We suggest that these structural changes are manifested at the biological level by affecting large ribosomal subunit biogenesis, ribosomal subunit joining during initiation, susceptibility/resistance to peptidyltransferase inhibitors, and the ability of ribosomes to properly decode termination codons.These studies also add to our understanding of how information is transmitted both locally and over long distances through allosteric networks of rRNA-rRNA and rRNA-protein interactions.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Molecular Genetics, University of Maryland, 2135 Microbiology Building, College Park, MD 20742, USA.

ABSTRACT
To ensure accurate and rapid protein synthesis, nearby and distantly located functional regions of the ribosome must dynamically communicate and coordinate with one another through a series of information exchange networks. The ribosome is approximately 2/3 rRNA and information should pass mostly through this medium. Here, two viable mutants located in the peptidyltransferase center (PTC) of yeast ribosomes were created using a yeast genetic system that enables stable production of ribosomes containing only mutant rRNAs. The specific mutants were C2820U (Escherichia coli C2452) and Psi2922C (E. coli U2554). Biochemical and genetic analyses of these mutants suggest that they may trap the PTC in the 'open' or aa-tRNA bound conformation, decreasing peptidyl-tRNA binding. We suggest that these structural changes are manifested at the biological level by affecting large ribosomal subunit biogenesis, ribosomal subunit joining during initiation, susceptibility/resistance to peptidyltransferase inhibitors, and the ability of ribosomes to properly decode termination codons. These studies also add to our understanding of how information is transmitted both locally and over long distances through allosteric networks of rRNA-rRNA and rRNA-protein interactions.

Show MeSH

Related in: MedlinePlus

Generation and preliminary characterization of mutants in the yeast peptidyltransferase center (PTC). (A) 2D map showing locations of bases in the PTC targeted for mutagenesis. All alleles of bases boxed in black with red diagonal slashes were inviable as the sole forms of 25S rRNA. Bases for which viable mutants were obtained are boxed in green (C2820U) and blue (Ψ2922C). This color scheme is used throughout the figures. (B) 3D stereoscopic view of the mutants mapped in panel A based on the Haloarcula marismortui atomic resolution 50S structure (8). The location of Phe-ACC in the A-site of the PTC is shown in red. (C) Dilution spot assays of the effects of the two viable mutants expressed as the sole forms of 25S rRNA from the native RNA polymerase I promoter on cell growth at optimum (30°C), high (37°C), and low (18°C) temperatures. (D) Growth phenotypes of C2820 mutants in the presence of anisomycin. Primary transformants were grown on −trp medium containing 5-FOA to select against wild-type rRNA, hygromycin to select for mutant rRNA, and 20 μg/ml anisomycin. Note that C2820A was only viable in the presence of anisomycin. (E) 60S ribosomal subunit biogenesis and polysome defects caused by expression of C2820U or Ψ2922C mutations transcribed from the native RNA polymerase I promoter. Cytoplasmic extracts from isogenic strains were separated through continuous 7–47% sucrose gradients in an SW41 rotor at 40,000 r.p.m. for 240 min at 4°C, and analyzed by continuous monitoring of A254. Positions of 40S, 60S, 80S, polysomes, and half-mers are indicated.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2275155&req=5

Figure 1: Generation and preliminary characterization of mutants in the yeast peptidyltransferase center (PTC). (A) 2D map showing locations of bases in the PTC targeted for mutagenesis. All alleles of bases boxed in black with red diagonal slashes were inviable as the sole forms of 25S rRNA. Bases for which viable mutants were obtained are boxed in green (C2820U) and blue (Ψ2922C). This color scheme is used throughout the figures. (B) 3D stereoscopic view of the mutants mapped in panel A based on the Haloarcula marismortui atomic resolution 50S structure (8). The location of Phe-ACC in the A-site of the PTC is shown in red. (C) Dilution spot assays of the effects of the two viable mutants expressed as the sole forms of 25S rRNA from the native RNA polymerase I promoter on cell growth at optimum (30°C), high (37°C), and low (18°C) temperatures. (D) Growth phenotypes of C2820 mutants in the presence of anisomycin. Primary transformants were grown on −trp medium containing 5-FOA to select against wild-type rRNA, hygromycin to select for mutant rRNA, and 20 μg/ml anisomycin. Note that C2820A was only viable in the presence of anisomycin. (E) 60S ribosomal subunit biogenesis and polysome defects caused by expression of C2820U or Ψ2922C mutations transcribed from the native RNA polymerase I promoter. Cytoplasmic extracts from isogenic strains were separated through continuous 7–47% sucrose gradients in an SW41 rotor at 40,000 r.p.m. for 240 min at 4°C, and analyzed by continuous monitoring of A254. Positions of 40S, 60S, 80S, polysomes, and half-mers are indicated.

Mentions: Previously, we described a genetic protocol enabling construction of yeast cells stably expressing only mutant rRNA alleles (15). In the first phase of the current study, this method was applied to selected bases of 25S rRNA. Bases were chosen for mutagenesis because of their locations in or near important structural elements of the PTC and because mutations at these positions had been previously shown to promote altered sensitivities to antibiotics in various prokaryotes and archae (36). Yeast 25S rRNA bases targeted for mutagenesis, and their corresponding 23S rRNA bases in E. coli and H. marismortui are shown in Figure 1A and B and Table 1. For consistency, the yeast numbering system is primarily used throughout this report. Mutagenesis of G2621 (the ‘P-loop’), G2814, A2818, A2819, U2947, and A2971 (all in the core of the PTC) to any of the three alternative bases were inviable, supporting the prior observation of a quality-control mechanism capable of detecting and eliminating the defective rRNAs in ribosomes (37). Two alleles did prove to be viable: cytosine 2820 to uracil (C2820U), and pseudouridine 2922 to cytosine (Ψ2922C) (Figure 1A and B). The C2820A mutant was also viable, but only in the presence of high concentrations of anisomycin, i.e. it was anisomycin-dependent for growth (Figure 1D).Figure 1.


rRNA mutants in the yeast peptidyltransferase center reveal allosteric information networks and mechanisms of drug resistance.

Rakauskaite R, Dinman JD - Nucleic Acids Res. (2008)

Generation and preliminary characterization of mutants in the yeast peptidyltransferase center (PTC). (A) 2D map showing locations of bases in the PTC targeted for mutagenesis. All alleles of bases boxed in black with red diagonal slashes were inviable as the sole forms of 25S rRNA. Bases for which viable mutants were obtained are boxed in green (C2820U) and blue (Ψ2922C). This color scheme is used throughout the figures. (B) 3D stereoscopic view of the mutants mapped in panel A based on the Haloarcula marismortui atomic resolution 50S structure (8). The location of Phe-ACC in the A-site of the PTC is shown in red. (C) Dilution spot assays of the effects of the two viable mutants expressed as the sole forms of 25S rRNA from the native RNA polymerase I promoter on cell growth at optimum (30°C), high (37°C), and low (18°C) temperatures. (D) Growth phenotypes of C2820 mutants in the presence of anisomycin. Primary transformants were grown on −trp medium containing 5-FOA to select against wild-type rRNA, hygromycin to select for mutant rRNA, and 20 μg/ml anisomycin. Note that C2820A was only viable in the presence of anisomycin. (E) 60S ribosomal subunit biogenesis and polysome defects caused by expression of C2820U or Ψ2922C mutations transcribed from the native RNA polymerase I promoter. Cytoplasmic extracts from isogenic strains were separated through continuous 7–47% sucrose gradients in an SW41 rotor at 40,000 r.p.m. for 240 min at 4°C, and analyzed by continuous monitoring of A254. Positions of 40S, 60S, 80S, polysomes, and half-mers are indicated.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2275155&req=5

Figure 1: Generation and preliminary characterization of mutants in the yeast peptidyltransferase center (PTC). (A) 2D map showing locations of bases in the PTC targeted for mutagenesis. All alleles of bases boxed in black with red diagonal slashes were inviable as the sole forms of 25S rRNA. Bases for which viable mutants were obtained are boxed in green (C2820U) and blue (Ψ2922C). This color scheme is used throughout the figures. (B) 3D stereoscopic view of the mutants mapped in panel A based on the Haloarcula marismortui atomic resolution 50S structure (8). The location of Phe-ACC in the A-site of the PTC is shown in red. (C) Dilution spot assays of the effects of the two viable mutants expressed as the sole forms of 25S rRNA from the native RNA polymerase I promoter on cell growth at optimum (30°C), high (37°C), and low (18°C) temperatures. (D) Growth phenotypes of C2820 mutants in the presence of anisomycin. Primary transformants were grown on −trp medium containing 5-FOA to select against wild-type rRNA, hygromycin to select for mutant rRNA, and 20 μg/ml anisomycin. Note that C2820A was only viable in the presence of anisomycin. (E) 60S ribosomal subunit biogenesis and polysome defects caused by expression of C2820U or Ψ2922C mutations transcribed from the native RNA polymerase I promoter. Cytoplasmic extracts from isogenic strains were separated through continuous 7–47% sucrose gradients in an SW41 rotor at 40,000 r.p.m. for 240 min at 4°C, and analyzed by continuous monitoring of A254. Positions of 40S, 60S, 80S, polysomes, and half-mers are indicated.
Mentions: Previously, we described a genetic protocol enabling construction of yeast cells stably expressing only mutant rRNA alleles (15). In the first phase of the current study, this method was applied to selected bases of 25S rRNA. Bases were chosen for mutagenesis because of their locations in or near important structural elements of the PTC and because mutations at these positions had been previously shown to promote altered sensitivities to antibiotics in various prokaryotes and archae (36). Yeast 25S rRNA bases targeted for mutagenesis, and their corresponding 23S rRNA bases in E. coli and H. marismortui are shown in Figure 1A and B and Table 1. For consistency, the yeast numbering system is primarily used throughout this report. Mutagenesis of G2621 (the ‘P-loop’), G2814, A2818, A2819, U2947, and A2971 (all in the core of the PTC) to any of the three alternative bases were inviable, supporting the prior observation of a quality-control mechanism capable of detecting and eliminating the defective rRNAs in ribosomes (37). Two alleles did prove to be viable: cytosine 2820 to uracil (C2820U), and pseudouridine 2922 to cytosine (Ψ2922C) (Figure 1A and B). The C2820A mutant was also viable, but only in the presence of high concentrations of anisomycin, i.e. it was anisomycin-dependent for growth (Figure 1D).Figure 1.

Bottom Line: Here, two viable mutants located in the peptidyltransferase center (PTC) of yeast ribosomes were created using a yeast genetic system that enables stable production of ribosomes containing only mutant rRNAs.We suggest that these structural changes are manifested at the biological level by affecting large ribosomal subunit biogenesis, ribosomal subunit joining during initiation, susceptibility/resistance to peptidyltransferase inhibitors, and the ability of ribosomes to properly decode termination codons.These studies also add to our understanding of how information is transmitted both locally and over long distances through allosteric networks of rRNA-rRNA and rRNA-protein interactions.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Molecular Genetics, University of Maryland, 2135 Microbiology Building, College Park, MD 20742, USA.

ABSTRACT
To ensure accurate and rapid protein synthesis, nearby and distantly located functional regions of the ribosome must dynamically communicate and coordinate with one another through a series of information exchange networks. The ribosome is approximately 2/3 rRNA and information should pass mostly through this medium. Here, two viable mutants located in the peptidyltransferase center (PTC) of yeast ribosomes were created using a yeast genetic system that enables stable production of ribosomes containing only mutant rRNAs. The specific mutants were C2820U (Escherichia coli C2452) and Psi2922C (E. coli U2554). Biochemical and genetic analyses of these mutants suggest that they may trap the PTC in the 'open' or aa-tRNA bound conformation, decreasing peptidyl-tRNA binding. We suggest that these structural changes are manifested at the biological level by affecting large ribosomal subunit biogenesis, ribosomal subunit joining during initiation, susceptibility/resistance to peptidyltransferase inhibitors, and the ability of ribosomes to properly decode termination codons. These studies also add to our understanding of how information is transmitted both locally and over long distances through allosteric networks of rRNA-rRNA and rRNA-protein interactions.

Show MeSH
Related in: MedlinePlus