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

Phenotypic analyses of C2820U and Ψ2922C mutants. (A) Drug disc assays. Overnight yeast cultures were diluted to OD595 = 0.3, and 300 μl of the resulting suspensions were plated onto rich medium. Sterile 0.6 cm diameter filter paper discs were saturated with the indicated amounts of sparsomycin, anisomycin, paromomycin or water and placed onto the plates. Cells were incubated at 30°C for 3 days and the diameters of growth inhibition zones were monitored. (B) UAA termination suppression assays. Suppression of a UAA nonsense codon utilized the pYDL-UAA dual luciferase reporter constructs as previously described (30,31). The [PSI+] prion form of yeast eukaryotic release factor 3 (eRF3) was cured after several passages of cells on YPAD media containing 5 μg/ml of guanidinium hydrochloride (GuHCL). Cured strains were transformed with the pYDL-UAA reporter constructs and analyzed as described above. The insert shows the same data with an expanded Y-axis. Error bars denote standard error.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 4: Phenotypic analyses of C2820U and Ψ2922C mutants. (A) Drug disc assays. Overnight yeast cultures were diluted to OD595 = 0.3, and 300 μl of the resulting suspensions were plated onto rich medium. Sterile 0.6 cm diameter filter paper discs were saturated with the indicated amounts of sparsomycin, anisomycin, paromomycin or water and placed onto the plates. Cells were incubated at 30°C for 3 days and the diameters of growth inhibition zones were monitored. (B) UAA termination suppression assays. Suppression of a UAA nonsense codon utilized the pYDL-UAA dual luciferase reporter constructs as previously described (30,31). The [PSI+] prion form of yeast eukaryotic release factor 3 (eRF3) was cured after several passages of cells on YPAD media containing 5 μg/ml of guanidinium hydrochloride (GuHCL). Cured strains were transformed with the pYDL-UAA reporter constructs and analyzed as described above. The insert shows the same data with an expanded Y-axis. Error bars denote standard error.

Mentions: tRNA-binding defects are often accompanied by hypersensitivity to translational inhibitors. Filter disc assays confirmed that C2820U was resistant to anisomycin as indicated by the lack of a zone of growth inhibition around the anisomycin disc (Figure 4A). Sparsomycin interacts with the P-site and affects interactions between the ribosome and the peptidyl-tRNA (36,46). Consistent with the changes in Kd values for Ac-aa-tRNA, increases in the zones of growth inhibition around the sparsomycin discs showed that both mutants were hypersensitive to this drug relative to wild-type cells (Figure 4A). With regard to the Ψ2922C mutant, this is consistent with a prior report showing that deletion of snr10, the snoRNA that directs pseudouridylation of U2922 also promoted sparsomycin sensitivity (47). Although paromomycin is an aminoglycoside antibiotic that interacts with the decoding center in the small subunit (48), mutations of large subunit rRNAs and proteins have been previously shown to affect sensitivity to this drug (49–51). The very large growth inhibition zones promoted by paromomycin indicated that both mutants were hypersensitive to this antibiotic (Figure 4A), also consistent with paromomycin hypersensitivity of the snr10Δ mutant (52). Paromomycin stimulates termination suppression in yeast (53). Thus, the effects of the two mutants on the ability of ribosomes to recognize the UAA termination codon was assayed using a previously described dual-luciferase reporter system (54). The results demonstrated that the C2820U mutant slightly stimulated translational suppression of the UAA codon, while Ψ2922C was approximately two-fold more accurate (Figure 4B). The actual rates of UAA suppression originally observed in wild-type cells (4.5%) was unusually high however, commensurate with levels normally associated with the presence of [PSI+] (30), the prion form of yeast eRF3 (55). To test this hypothesis and to examine the effects of termination suppression in the absence of the prion, cells were cured of [PSI+] by serial passage in the presence of guanidinium hydrochloride (32). In wild-type cells, this treatment returned rates of UAA suppression down to normal levels (0.27%) (Figure 4B, see inset). Interestingly, the mutants no longer affected termination suppression when cured of [PSI+]. This suggests that the mutants only affect UAA recognition when eRF3 availability is limited.Figure 4.


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

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

Phenotypic analyses of C2820U and Ψ2922C mutants. (A) Drug disc assays. Overnight yeast cultures were diluted to OD595 = 0.3, and 300 μl of the resulting suspensions were plated onto rich medium. Sterile 0.6 cm diameter filter paper discs were saturated with the indicated amounts of sparsomycin, anisomycin, paromomycin or water and placed onto the plates. Cells were incubated at 30°C for 3 days and the diameters of growth inhibition zones were monitored. (B) UAA termination suppression assays. Suppression of a UAA nonsense codon utilized the pYDL-UAA dual luciferase reporter constructs as previously described (30,31). The [PSI+] prion form of yeast eukaryotic release factor 3 (eRF3) was cured after several passages of cells on YPAD media containing 5 μg/ml of guanidinium hydrochloride (GuHCL). Cured strains were transformed with the pYDL-UAA reporter constructs and analyzed as described above. The insert shows the same data with an expanded Y-axis. Error bars denote standard error.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 4: Phenotypic analyses of C2820U and Ψ2922C mutants. (A) Drug disc assays. Overnight yeast cultures were diluted to OD595 = 0.3, and 300 μl of the resulting suspensions were plated onto rich medium. Sterile 0.6 cm diameter filter paper discs were saturated with the indicated amounts of sparsomycin, anisomycin, paromomycin or water and placed onto the plates. Cells were incubated at 30°C for 3 days and the diameters of growth inhibition zones were monitored. (B) UAA termination suppression assays. Suppression of a UAA nonsense codon utilized the pYDL-UAA dual luciferase reporter constructs as previously described (30,31). The [PSI+] prion form of yeast eukaryotic release factor 3 (eRF3) was cured after several passages of cells on YPAD media containing 5 μg/ml of guanidinium hydrochloride (GuHCL). Cured strains were transformed with the pYDL-UAA reporter constructs and analyzed as described above. The insert shows the same data with an expanded Y-axis. Error bars denote standard error.
Mentions: tRNA-binding defects are often accompanied by hypersensitivity to translational inhibitors. Filter disc assays confirmed that C2820U was resistant to anisomycin as indicated by the lack of a zone of growth inhibition around the anisomycin disc (Figure 4A). Sparsomycin interacts with the P-site and affects interactions between the ribosome and the peptidyl-tRNA (36,46). Consistent with the changes in Kd values for Ac-aa-tRNA, increases in the zones of growth inhibition around the sparsomycin discs showed that both mutants were hypersensitive to this drug relative to wild-type cells (Figure 4A). With regard to the Ψ2922C mutant, this is consistent with a prior report showing that deletion of snr10, the snoRNA that directs pseudouridylation of U2922 also promoted sparsomycin sensitivity (47). Although paromomycin is an aminoglycoside antibiotic that interacts with the decoding center in the small subunit (48), mutations of large subunit rRNAs and proteins have been previously shown to affect sensitivity to this drug (49–51). The very large growth inhibition zones promoted by paromomycin indicated that both mutants were hypersensitive to this antibiotic (Figure 4A), also consistent with paromomycin hypersensitivity of the snr10Δ mutant (52). Paromomycin stimulates termination suppression in yeast (53). Thus, the effects of the two mutants on the ability of ribosomes to recognize the UAA termination codon was assayed using a previously described dual-luciferase reporter system (54). The results demonstrated that the C2820U mutant slightly stimulated translational suppression of the UAA codon, while Ψ2922C was approximately two-fold more accurate (Figure 4B). The actual rates of UAA suppression originally observed in wild-type cells (4.5%) was unusually high however, commensurate with levels normally associated with the presence of [PSI+] (30), the prion form of yeast eRF3 (55). To test this hypothesis and to examine the effects of termination suppression in the absence of the prion, cells were cured of [PSI+] by serial passage in the presence of guanidinium hydrochloride (32). In wild-type cells, this treatment returned rates of UAA suppression down to normal levels (0.27%) (Figure 4B, see inset). Interestingly, the mutants no longer affected termination suppression when cured of [PSI+]. This suggests that the mutants only affect UAA recognition when eRF3 availability is limited.Figure 4.

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