The conserved GTPase center and variable region V9 from Saccharomyces cerevisiae 26S rRNA can be replaced by their equivalents from other prokaryotes or eukaryotes without detectable loss of ribosomal function (original) (raw)
Nucleic Acids Research, 2000
The yeast ribosomal GTPase associated center is made of parts of the 26S rRNA domains II and VI, and a number of proteins including P0, P1α, P1β, P2α, P2β and L12. Mapping of the rRNA neighborhood of the proteins was performed by footprinting in ribosomes from yeast strains lacking different GTPase components. The absence of protein P0 dramatically increases the sensitivity of the defective ribosome to degradation hampering the RNA footprinting. In ribosomes lacking the P1/P2 complex, protection of a number of nucleotides is detected around positions 840, 880, 1100, 1220-1280 and 1350 in domain II as well as in several positions in the domain VI α-sarcin region. The protection pattern resembles the one reported for the interaction of elongation factors in bacterial systems. The results exclude a direct interaction of these proteins with the rRNA and are compatible with an increase in the ribosome affinity for EF-2 in the absence of the acidic P proteins. Interestingly, a sordarin derivative inhibitor of EF-2 causes an opposite effect, increasing the reactivity in positions protected by the absence of P1/P2. Similarly, a deficiency in protein L12 exposes nucleotides G1235, G1242, A1262, A1269, A1270 and A1272 to chemical modification, thus situating the protein binding site in the most conserved part of the 26S rRNA, equivalent to the bacterial protein L11 binding site.
Journal of Biological Chemistry, 1998
Protein L12, together with the P0/P1/P2 protein complex, forms the protein moiety of the GTPase domain in the eukaryotic ribosome. In Saccharomyces cerevisiae protein L12 is encoded by a duplicated gene, rpL12A and rpL12B. Inactivation of both copies has been performed and confirmed by Southern and Western analyses. The resulting strains are viable but grow very slowly. Growth rate is recovered upon transformation with an intact copy of the L12 gene. Ribosomes from the disrupted strain lack protein L12 but are able to carry out translation in vitro at about one fourth of the control rate. The L12-deficient ribosomes have also a defective stalk containing standard amounts of the 12-kDa acidic proteins P1 and P2␣, but proteins P1␣ and P2 are drastically reduced. Moreover, the affinity of P0 is reduced in the defective ribosomes. Footprinting of the 26 S rRNA GTPase domain indicates that protein L12 protects in different extent residues G1235, G1242, A1262, A1270, and A1272 from chemical modification. The results in this report indicate that protein L12 is not essential for cell viability but has a relevant role in the structure and stability of the eukaryotic ribosomal stalk.
Biochemical Journal, 2006
We cloned the genes encoding the ribosomal proteins Ph (Pyrococcus horikoshii)-P0, Ph-L12 and Ph-L11, which constitute the GTPase-associated centre of the archaebacterium Pyrococcus horikoshii. These proteins are homologues of the eukaryotic P0, P1/P2 and eL12 proteins, and correspond to Escherichia coli L10, L7/L12 and L11 proteins respectively. The proteins and the truncation mutants of Ph-P0 were overexpressed in E. coli cells and used for in vitro assembly on to the conserved domain around position 1070 of 23S rRNA (E. coli numbering). Ph-L12 tightly associated as a homodimer and bound to the C-terminal half of Ph-P0. The Ph-P0·Ph-L12 complex and Ph-L11 bound to the 1070 rRNA fragments from the three biological kingdoms in the same manner as the equivalent proteins of eukaryotic and eubacterial ribosomes. The Ph-P0·Ph-L12 complex and Ph-L11 could replace L10·L7/L12 and L11 respectively, on the E. coli 50S subunit in vitro. The resultant hybrid ribosome was accessible for eukaryo...
Ribosomal Protein L11 Selectively Stabilizes a Tertiary Structure of the GTPase Center rRNA Domain
Journal of Molecular Biology, 2019
The GTPase Center (GAC) RNA domain in bacterial 23S rRNA is directly bound by ribosomal protein L11, and this complex is essential to ribosome function. Previous cocrystal structures of the 58-nucleotide GAC RNA bound to L11 revealed the intricate tertiary fold of the RNA domain, with one monovalent and several divalent ions located in specific sites within the structure. Here, we report a new crystal structure of the free GAC that is essentially identical to the L11-bound structure, which retains many common sites of divalent ion occupation. This new structure demonstrates that RNA alone folds into its tertiary structure with bound divalent ions. In solution, we find that this tertiary structure is not static, but rather is best described as an ensemble of states. While L11 protein cannot bind to the GAC until the RNA has adopted its tertiary structure, new experimental data show that L11 binds to Mg 2+-dependent folded states, which we suggest lie along the folding pathway of the RNA. We propose that L11 stabilizes a specific GAC RNA tertiary state, corresponding to the crystal structure, and that this structure reflects the functionally critical conformation of the rRNA domain in the fully assembled ribosome.
The EMBO Journal, 2011
The precise functions of most of the B200 assembly factors and 79 ribosomal proteins required to construct yeast ribosomes in vivo remain largely unexplored. To better understand the roles of these proteins and the mechanisms driving ribosome biogenesis, we examined in detail one step in 60S ribosomal subunit assemblyprocessing of 27SA 3 pre-rRNA. Six of seven assembly factors required for this step (A 3 factors) are mutually interdependent for association with preribosomes. These A 3 factors are required to recruit Rrp17, one of three exonucleases required for this processing step. In the absence of A 3 factors, four ribosomal proteins adjacent to each other, rpL17, rpL26, rpL35, and rpL37, fail to assemble, and preribosomes are turned over by Rat1. We conclude that formation of a neighbourhood in preribosomes containing the A 3 factors establishes and maintains stability of functional preribosomes containing 27S pre-rRNAs.
Eukaryotic Cell, 2004
Yeast Rrp5p, one of the fewtrans-acting proteins required for the biogenesis of both ribosomal subunits, has a remarkable two-domain structure. Its C-terminal region consists of seven tetratricopeptide motifs, several of which are crucial for cleavages at sites A0to A2and thus for the formation of 18S rRNA. The N-terminal region, on the other hand, contains 12 S1 RNA-binding motifs, most of which are required for processing at site A3and thus for the production of the short form of 5.8S rRNA. Yeast cells expressing a mutant Rrp5p protein that lacks S1 motifs 10 to 12 (mutantrrp5Δ6) have a normal growth rate and wild-type steady-state levels of the mature rRNA species, suggesting that these motifs are irrelevant for ribosome biogenesis. Here we show that, nevertheless, in therrp5Δ6mutant, pre-rRNA processing follows an alternative pathway that does not include the cleavage of 32S pre-rRNA at site A2. Instead, the 32S precursor is processed directly at site A3, producing exclusively 2...
Structural and functional analysis of 5S rRNA in Saccharomyces cerevisiae
Molecular Genetics and Genomics, 2005
5S rRNA extends from the central protuberance of the large ribosomal subunit, through the A-site finger, and down to the GTPase-associated center. Here, we present a structure-function analysis of seven 5S rRNA alleles which are sufficient for viability in the yeast Saccharomyces cerevisiae when expressed in the absence of wild-type 5S rRNAs, and extend this analysis using a large bank of mutant alleles that show semidominant phenotypes in the presence of wild-type 5S rRNA. This analysis supports the hypothesis that 5S rRNA serves to link together several different functional centers of the ribosome. Data are also presented which suggest that in eukaryotic genomes selection has favored the maintenance of multiple alleles of 5S rRNA, and that these may provide cells with a mechanism to posttranscriptionally regulate gene expression.
Composition and Functional Characterization of Yeast 66S Ribosome Assembly Intermediates
Molecular Cell, 2001
. Yeast 35S pre-rRNA Donald F. Hunt, 3 and John L. Woolford, Jr. 1,5 is present in a 90S preribosomal particle (pre-rRNP), 1 Department of Biological Sciences which is converted to a 43S rRNP containing 20S pre-Carnegie Mellon University rRNA and a series of 66S rRNPs containing 27S or 25.5S Pittsburgh, Pennsylvania 15213 plus 7S pre-rRNAs (Trapman et al., 1975). The 43S rRNP 2 Laboratory of Cellular and Structural Biology is exported from the nucleolus to the cytoplasm, where The Rockefeller University it matures into a 40S ribosomal subunit containing 18S New York, New York 10021 rRNA (Udem and Warner, 1973; Trapman and Planta, 3 Departments of Chemistry and Pathology 1976). The 66S rRNPs are converted to 60S rRNPs con-University of Virginia taining 25S, 5.8S, and 5S rRNA through a series of matu-Charlottesville, Virginia 22901 ration steps in the nucleolus, nucleoplasm, and cyto-4 MDS Proteomics plasm (Eisinger et al., 1997; Kressler et al., 1999a; Ho Charlottesville, Virginia 22903 et al., 2000; Milkereit et al., 2001). Molecular genetic approaches in yeast have identified more than 70 different nonribosomal proteins involved in Summary ribosome biogenesis (reviewed in Kressler et al., 1999a; Venema and Tollervey , 1999). The roles of these proteins The pathway and complete collection of factors that in ribosome biogenesis are best characterized on the orchestrate ribosome assembly are not clear. To adbasis of their mutant phenotype with respect to RNA dress these problems, we affinity purified yeast preribometabolism (Venema and Tollervey, 1999). In many musomal particles containing the nucleolar protein Nop7p tants, processing of certain pre-rRNAs appears to be and developed means to separate their components. blocked, and those RNAs accumulate. In other mutants, Nop7p is associated primarily with 66S preribosomes rRNA-processing intermediates are made but rapidly containing either 27SB or 25.5S plus 7S pre-rRNAs. degraded, presumably due to aberrations in assembly Copurifying proteins identified by mass spectrometry of the rRNPs. While some proteins identified in these include ribosomal proteins, nonribosomal proteins mutant screens have clear functions in rRNA processing previously implicated in 60S ribosome biogenesis, and or modification, there is a large class of so-called "asproteins not known to be involved in ribosome producsembly factors" with no obvious functions. Although it tion. Analysis of strains mutant for eight of these prois presumed that most of these proteins associate with teins not previously implicated in ribosome biogenesis assembling ribosomes in pre-rRNPs, only a few proteins showed that they do participate in this pathway. These have been shown to cosediment on gradients with preresults demonstrate that proteomic approaches in ribosomes Zanchin et al., 1997; de la Cruz et al., 1998; concert with genetic tools provide powerful means to Si and Maitra, 1999; Billy et al., 2000; Bousquet-Antonelli purify and characterize ribosome assembly intermeet al., 2000; Milkereit et al., 2001). Many nonribosomal diates. proteins that might function in ribosome biogenesis also have been found in metazoan pre-rRNPs, although only a few have been studied in detail (reviewed in Piñ ol-Introduction Roma, 1999; Olson et al., 2000) . Most have not been identified, and the specificity of their association with Eukaryotic ribosome assembly is a complex process pre-rRNPs has not been established. occurring primarily in the nucleolus, a subcompartment Thus, we lack an understanding of the functions of of the nucleus where rRNA is transcribed, covalently most proteins involved in eukaryotic ribosome biogenemodified, processed, and assembled with 08ف different sis and the specific ribosome maturation steps in which ribosomal proteins (Woolford and Warner, 1991; Venthey participate. Because many of these proteins may ema and Tollervey, 1999). In Saccharomyces cerevisiae, function in the context of preribosomal particles, it is three of the four rRNAs in mature ribosomal subunits, important to characterize the composition of these ribo-18S, 5.8S, and 25S rRNA, are derived from the 35S rRNA nucleoprotein complexes in detail. To achieve this goal, primary transcript, synthesized by RNA polymerase I. we affinity purified preribosomal particles containing 5S rRNA is transcribed separately by RNA polymerase molecules associated with yeast nucleolar protein III. After transcription, pre-rRNA undergoes multiple Nop7p (C. Adams et al., submitted) and identified their conserved modifications. External and internal tranprotein and RNA constituents. Purified Nop7p is associscribed spacer sequences are removed from 35S preated primarily with 27SB, 25.5S, and 7S pre-rRNAs, indicating that it is present in two consecutive 66S ribosome assembly intermediates in the pathway that produces 5 Correspondence: jw17@andrew.cmu.edu 6 These authors contributed equally to this work. mature 60S ribosomal subunits. Proteins associated Affinity Purification and Identification of Proteins Associated Saccharomyces cerevisiae: DEAD-box proteins and related families. Trends Biochem. Sci. 24, 192-198. with Nop7p The TAP-tag cassette was PCR amplified from pBS1539 using two Deshmukh, M., Tsay, Y.-F., Paulovich, A.G., and Woolford, J.L., Jr. oligonucleotide primers that include sequences upstream and (1993). Yeast ribosomal protein L1 is required for the stability of downstream of the NOP7 stop codon. Amplified DNA was transnewly synthesized 5S rRNA and the assembly of 60S ribosomal formed into yeast; Trp ϩ transformants expressing Nop7p with the subunits. Mol. Cell. Biol. 13, 2835-2845. TAP tag at its carboxyl terminus were identified by immunoblotting Dunbar, D.A., Dragon, F., Lee, S.J., and Baserga, S.J. (2000). A and confirmed by genomic PCR. Cells expressing Nop7p-TAP were nucleolar protein related to ribosomal protein L7 is required for an grown in YEPGlu to 1 ϫ 10 8 cells/ml, and cell-free extracts were early step in large ribosomal subunit biogenesis. Proc. Natl. Acad. prepared as described in Zanchin and Goldfarb (1999), except that Sci. USA 97, 13027-13032. buffer A contained 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Eisinger, D.P., Dick, F.A., Denke, E., and Trumpower, B.L. (1997). EDTA (pH 8), and 0.1% NP-40. Affinity purification was performed Sqt1, which encodes an essential WD domain protein of Saccharoas described in Rigaut et al. (1999), except wash volumes were myces cerevisiae, suppress dominant-negative mutations of the riincreased 10-fold. Proteins were recovered from the final eluate bosomal protein gene QSR1. Mol. Cell. Biol. 17, 5146-5155. by precipitation in 10% TCA, dissolved in SDS sample buffer, and Fabian, G.R., and Hopper, A.K. (1987). RRP1, a Saccharomyces separated by SDS-PAGE on 4%-20% polyacrylamide NOVEX gels cerevisiae gene affecting rRNA processing and production of mature (Invitrogen). ribosomal subunits. J. Bacteriol. 169, 1571-1578. Affinity-purified proteins were separated by formic acid HPLC followed by SDS-PAGE. Each protein sample was dissolved in SDS Fath, S., Milkereit, P., Podtelejnikov, A.V., Bischler, N., Schultz, P., sample buffer and loaded onto a Vydac C4 column preequilibrated Bier, M., Mann, M., and Tschochner, H. (2000). Association of yeast with 20% solvent B (60% [v/v] formic acid in water), 80% solvent A RNA polymerase I with a nucleolar substructure active in rRNA syn-(60% [v/v] formic acid, 33% acetonitrile). A linear gradient of solvent thesis and processing. J. Cell Biol. 149, 575-589. B from 20% to 35% was run over 10 min, then to 100% was run Geerlings, T.H., Vos, J.C., and Raué , H.A. (2000). The final step in the over 270 min, and finally B was kept at 100% for an additional formation of 25S rRNA in Saccharomyces cerevisiae is performed by 20 min. Proteins in fractions were precipitated with TCA/sodium 5Ј → 3Ј exonucleases. RNA 6, 1698-1703. deoxycholate, suspended in SDS sample buffer, and separated by Hadjiolov, A. (1985). The nucleolus and ribosome biogenesis. Cell SDS-PAGE. Gel bands were excised, destained, and enzymatic di-Biol. Monogr. 12, 1-268. gestion was performed in-gel . Resultant peptides were extracted Ho, J.H.-N., Kallstrom, G., and Johnson, A.W. (2000). Nascent 60S and analyzed by mass spectrometry using a PerSeptive Biosystems ribosomal subunits enter the free pool bound by Nmd3p. RNA 6, MALDI-TOF Voyager DE-RP Mass Spectrometer. The search engine 1625-1634. PROWL was used for database search (http://prowl.rockefeller.edu). Huber, M.D., Dworet, J.H., Shire, K., Frappier, L., and McAlear, M.S. (2000). The budding yeast homolog of the human EBNA1-binding Acknowledgments protein 2 (Ebp2p) is an essential nucleolar protein required for pre-rRNA processing. J. Biol. Chem. 275, 28764-28773. We thank N. Zanchin and D. Goldfarb for antibodies versus Nip7p; B. Seraphin for TAP plasmid pBS1539; and L. Visomirski-Robic, P. Jacobs-Anderson, J.S., and Parker, R. (1998). The 3Ј to 5Ј degrada-Antunez de Mayolo, J. Brodsky, J. Lopez, T.G. Kinzy, and J. Warner tion of yeast mRNAs is a general mechanism for mRNA turnover for critical reading of the manuscript. This work was supported by that requires the SK12 DEVH box protein and 3Ј to 5Ј exonucleases grants from the Rita Allen, Sinsheimer,and Hirschl Foundations and of the exosome complex. EMBO J. 17, 1497-1506. the Rockefeller University to M.R.; NIH grant GM28301 to J.L.W.; Kressler, D., Linder, P., and de la Cruz, J. (1999a). Protein trans-NIH grant GM18708 to J.R.; NIH grant GM37537 to D.F.H.; NIH grant acting factors involved in ribosome biogenesis in Saccharomyces GM19937 to E.H.; and by funds from the government of Thailand cerevisiae. Mol. Cell. Biol. 19, 7897-7912. provided to P.H. Kressler, D., Rojo, M., Linder, P., and de la Cruz, J. (1999b). Spb1p is a putative methyltransferase required for 60S ribosomal subunit .H., and the yeast protein similar to the RNA 3Ј-phosphate cyclase, associ-Chartrand, P....
Molecular and cellular biology, 1993
Ribosomal protein L1 from Saccharomyces cerevisiae binds 5S rRNA and can be released from intact 60S ribosomal subunits as an L1-5S ribonucleoprotein (RNP) particle. To understand the nature of the interaction between L1 and 5S rRNA and to assess the role of L1 in ribosome assembly and function, we cloned the RPL1 gene encoding L1. We have shown that RPL1 is an essential single-copy gene. A conditional null mutant in which the only copy of RPL1 is under control of the repressible GAL1 promoter was constructed. Depletion of L1 causes instability of newly synthesized 5S rRNA in vivo. Cells depleted of L1 no longer assemble 60S ribosomal subunits, indicating that L1 is required for assembly of stable 60S ribosomal subunits but not 40S ribosomal subunits. An L1-5S RNP particle not associated with ribosomal particles was detected by coimmunoprecipitation of L1 and 5S rRNA. This pool of L1-5S RNP remained stable even upon cessation of 60S ribosomal subunit assembly by depletion of another...