Ribosomal protein L15 as a probe of 50 S ribosomal subunit structure

L15, a 15 kDa protein of the large ribosomal subunit, interacts with over ten other proteins during 50 S assembly in vitro. We have probed the interaction L15 with 23 S rRNA in 50 S ribosomal subunits by chemical footprinting, and have used localized hydroxyl radical probing, generated from Fe(II) tethered to unique sites of L15, to characterize the three-dimensional 23 S rRNA environment of L15. Footprinting of L15 was done by reconstituting purified, recombinant L15 with core particles derived from Escherichia coli 50 S subunits by treatment with 2 M LiCl. The cores migrate as compact 50 S-like particles in sucrose gradients, contain 23 S and 5 S rRNA, and lack a subset of the 50 S proteins, including L15. Using both Fe(II).EDTA and dimethyl sulfate, we have identified a strong footprint for L15 in the region spanning nucleotides 572-654 in domain II of 23 S rRNA. This footprint cannot be detected when L15 is incubated with "naked" 23 S rRNA, indicating that formation of the L15 binding site requires a partially assembled particle.Protein-tethered hydroxyl radical probing was done using mutants of L15 containing single cysteine residues at amino acid positions 68, 71 and 115. The mutant proteins were derivatized with 1-[p-(bromo-acetamido)benzyl]-EDTA. Fe(II), bound to core particles, and hydroxyl radical cleavage was initiated. Distinct but overlapping sets of cleavages were obtained in the footprinted region of domain II, and in specific regions of domains I, IV and V of 23 S rRNA. These data locate L15 in proximity to several 23 S rRNA elements that are dispersed in the secondary structure, consistent with its central role in the latter stages of 50 S subunit assembly. Furthermore, these results indicate the proximity of these rRNA regions to one another, providing constraints on the tertiary folding of 23 S rRNA.     

Yeast proteins related to the p40/laminin receptor precursor are required for 20S ribosomal RNA processing and the maturation of 40S ribosomal subunits

Numerous studies have linked the overexpression of the Mr 37,000 laminin receptor precursor (37-LRP) to tumor cell growth and proliferation. The role of this protein in carcinogenesis is generally considered in the context of its putative role as a precursor for the Mr 67,000 high-affinity laminin receptor. Recent studies have shown that 37-LRP, also termed p40, is a component of the small ribosomal subunit indicating that it may be a multifunctional protein. The p40/37-LRP protein is highly conserved phylogenetically, and closely related proteins have been identified in species as evolutionarily distant as humans and the yeast, Saccharomyces cerevisiae. Yeast homologues of p40/37-LRP are encoded by a duplicated pair of genes, RPS0A and RPS0B. The Rps0 proteins are essential components of the 40S ribosomal subunit. Previous results have shown that cells disrupted in either of the RPS0 genes have a reduction in growth rate and reduced amounts of 40S ribosomal subunits relative to wild-type cells. Here, we show that the Rps0 proteins are required for the processing of the 20S rRNA-precursor to mature 18S rRNA, a late step in the maturation of 40S ribosomal subunits. Immature subunits that are depleted of Rps0 protein that contain the 20S rRNA precursor are preferentially excluded from polysomes, which indicates that their activity in protein synthesis is dramatically reduced relative to mature 40S ribosomal subunits. These data demonstrate that the assembly of Rps0 proteins into immature 40S subunits and the subsequent processing of 20S rRNA represent critical steps in defining the translational capacity of yeast cells. If the function of these yeast proteins is representative of other members of the p40/37-LRP family of proteins, then the role of these proteins as key components of the protein synthetic machinery should also be considered as a basis for the linkage between the their overexpression and tumor cell growth and proliferation.     

Association of a group I intron with its splice junction in 50S ribosomes: implications for intron toxicity

The effect of genetic context on splicing of group I introns is not well understood at present. The influence of ribosomal RNA conformation on splicing of rDNA introns in vivo was investigated using a heterologous system in which the Tetrahymena group I intron is inserted into the homologous position of the Escherichia coli 23S rRNA. Mutations that block splicing in E. coli result in accumulation of unspliced 23S rRNA that is assembled into 50S complexes, but not 70S ribosomes. The data indicate that accommodation of the intron structure on the surface of the 50S subunit inhibits interactions with the small ribosomal subunit. Spliced intron RNA also remains noncovalently bound to 50S subunits on sucrose gradients. This interaction appears to be mediated by base pairing between the intron guide sequence and the 23S rRNA, because the fraction of bound intron RNA is reduced by point mutations in the IGS or deletion of the P1 helix. Association of the intron with 50S subunits correlates with slow cell growth. The results suggest that group I introns have the potential to inhibit protein synthesis in prokaryotes by direct interactions with ribosomes.     

Directed hydroxyl radical probing of 16S ribosomal RNA in ribosomes containing Fe(II) tethered to ribosomal protein S20

The 16S ribosomal RNA neighborhood of ribosomal protein S20 has been mapped, in both 30S subunits and 70S ribosomes, using directed hydroxyl radical probing. Cysteine residues were introduced at amino acid positions 14, 23, 49, and 57 of S20, and used for tethering 1-(p-bromoacetamidobenzyl)-Fe(II)-EDTA. In vitro reconstitution using Fe(II)-derivatized S20, together with the remaining small subunit ribosomal proteins and 16S ribosomal RNA (rRNA), yielded functional 30S subunits. Both 30S subunits and 70S ribosomes containing Fe(II)-S20 were purified and hydroxyl radicals were generated from the tethered Fe(II). Hydroxyl radical cleavage of the 16S rRNA backbone was monitored by primer extension. Different cleavage patterns in 16S rRNA were observed from Fe(II) tethered to each of the four positions, and these patterns were not significantly different in 30S and 70S ribosomes. Cleavage sites were mapped to positions 160-200, 320, and 340-350 in the 5' domain, and to positions 1427-1430 and 1439-1458 in the distal end of the penultimate stem of 16S rRNA, placing these regions near each other in three dimensions. These results are consistent with previous footprinting data that localized S20 near these 16S rRNA elements, providing evidence that S20, like S17, is located near the bottom of the 30S subunit.     

Structure of 5S rRNA within the Escherichia coli ribosome: iodine-induced cleavage patterns of phosphorothioate derivatives

The protection patterns of 5S rRNA in solution, within the ribosomal 50S subunit, 70S ribosomes, and functional complexes, were assessed with the phosphorothioate method. About 20% of the analyzed positions (G9-G107) showed strong assembly defects: A phosphorothioate at one of these positions significantly impaired the incorporation of 5S rRNA into 50S particles. The reverse has also been observed: A phosphorothioate is preferred over a phosphate residue in the assembly process at a few positions. The results further demonstrate that 5S rRNA undergoes conformational changes during the assembly in the central protuberance of the 50S subunit and upon association with the small ribosomal subunit forming a 70S ribosome. In striking contrast, when the 70S ribosomes are once formed, the contact pattern of the 5S rRNA is the same in various functional states such as initiation-like complexes and pre- and posttranslocational states.     

Fal1p is an essential DEAD-box protein involved in 40S-ribosomal-subunit biogenesis in Saccharomyces cerevisiae

A previously uncharacterized Saccharomyces cerevisiae gene, FAL1, was found by sequence comparison as a homolog of the eukaryotic translation initiation factor 4A (eIF4A). Fal1p has 55% identity and 73% similarity on the amino acid level to yeast eIF4A, the prototype of ATP-dependent RNA helicases of the DEAD-box protein family. Although clearly grouped in the eIF4A subfamily, the essential Fal1p displays a different subcellular function and localization. An HA epitope-tagged Fal1p is localized predominantly in the nucleolus. Polysome analyses in a temperature-sensitive fal1-1 mutant and a Fal1p-depleted strain reveal a decrease in the number of 40S ribosomal subunits. Furthermore, these strains are hypersensitive to the aminoglycoside antibiotics paromomycin and neomycin. Pulse-chase labeling of pre-rRNA and steady-state-level analysis of pre-rRNAs and mature rRNAs by Northern hybridization and primer extension in the Fal1p-depleted strain show that Fal1p is required for pre-rRNA processing at sites A0, A1, and A2. Consequently, depletion of Fal1p leads to decreased 18S rRNA levels and to an overall deficit in 40S ribosomal subunits. Together, these results implicate Fal1p in the 18S rRNA maturation pathway rather than in translation initiation.     

Experience of intravascular malignant lymphomatosis with urinary incontinence and gait disturbance

Nucleotide residue U89 in the D loop of Escherichia coli 5S rRNA is adjacent to two domains of 23S rRNA in the large ribosomal subunit [Dokudovskaya et al., RNA 2 (1996) 146-152]. 50S ribosomal subunits were reconstituted containing U89(C, G or A) mutants of 5S rRNAs and the activities of the corresponding 70S ribosomes were studied. The U89C mutant behaves similarly to the wild-type 5S rRNA. Replacement of the pyrimidine base at position U89 by more bulky purine bases impairs the incorporation of 5S rRNA into 50S subunits, whereas the particles formed showed full activities in poly(U)-dependent poly(Phe) synthesis in the presence of either U89G or U89A 5S rRNA mutants. The activity of the reconstituted particles depends on the incorporation of 5S rRNA in agreement with early observations.     

Spb4p, an essential putative RNA helicase, is required for a late step in the assembly of 60S ribosomal subunits in Saccharomyces cerevisiae

Spb4p is a putative ATP-dependent RNA helicase that is required for synthesis of 60S ribosomal subunits. Polysome analyses of strains genetically depleted of Spb4p or carrying the cold-sensitive spb4-1 mutation revealed an underaccumulation of 60S ribosomal subunits. Analysis of pre-rRNA processing by pulse-chase labeling, northern hybridization, and primer extension indicated that these strains exhibited a reduced synthesis of the 25S/5.8S rRNAs, due to inhibition of processing of the 27SB pre-rRNAs. At later times of depletion of Spb4p or following transfer of the spb4-1 strain to more restrictive temperatures, the early pre-rRNA processing steps at sites A0, Al, and A2 were also inhibited. Sucrose gradient fractionation showed that the accumulated 27SB pre-rRNAs are associated with a high-molecular-weight complex, most likely the 66S pre-ribosomal particle. An HA epitope-tagged Spb4p is localized to the nucleolus and the adjacent nucleoplasmic area. On sucrose gradients, HA-Spb4p was found almost exclusively in rapidly sedimenting complexes and showed a peak in the fractions containing the 66S pre-ribosomes. We propose that Spb4p is involved directly in a late and essential step during assembly of 60S ribosomal subunits, presumably by acting as an rRNA helicase.     

Interaction of yeast elongation factor 3 with polynucleotides, ribosomal RNA and ribosomal subunits

In addition to the two usual eukaryotic elongation factors (EF-1 alpha and EF-2) fungal ribosomes need a third protein, elongation factor 3, for translation. EF-3 is essential for in vivo and in vitro protein synthesis. Functionally, EF-3 stimulates EF-1 alpha dependent binding of aminoacyl-tRNA to the ribosomal A site when E site is occupied by deacylated tRNA. EF-3 has intrinsic ATPase activity which is regulated by the functional state of the ribosome. EF-3 ATPase is activated by both 40S and 60S ribosomal subunits. However intact 80S ribosomes are needed for efficient activation of EF-3 ATPase. EF-3 appears to be an RNA binding protein with high affinity for polynucleotides containing guanosine rich sequences. To determine whether guanosine rich sequence of ribosomal RNA is involved in EF-3 binding, an antisense oligonucleotide dC6 was used to block EF-3 interaction with the ribosome. The oligonucleotide suppresses activation of EF-3 ATPase by 40S ribosomal subunit and not by the 60S or the 80S particles. Poly(U)-directed polyphenylalanine synthesis by yeast ribosomes is inhibited by dC6. To define the binding site of the oligonucleotide and presumably of EF-3 on 18S ribosomal RNA, hydrolysis of rRNA by RNase H was followed in the presence of dC6. These experiments reveal an RNase H cleavage site at 1094GGGGGG1099 sequence of 18S ribosomal RNA. This guanosine rich sequence of rRNA is suggested to be involved in EF-3 binding to yeast ribosome. Data presented in this communication suggest that the activity of EF-3 involved a direct interaction with the guanosine rich sequence of rRNA.     

Directed hydroxyl radical probing of 16S ribosomal RNA in 70S ribosomes from internal positions of the RNA

Directed hydroxyl radical probing of 16S ribosomal RNA from Fe(II) tethered to specific sites within the RNA was used to determine RNA-RNA proximities in 70S ribosomes. We have transcribed 16S ribosomal RNA in vitro as two separate fragments, covalently attached an Fe(II) probe to a 5'-guanosine-alpha-phosphorothioate at the junction between the two fragments, and reconstituted 30S subunits with the two separate pieces of RNA and the small subunit proteins. Reconstituted 30S subunits capable of association with 50S subunits were selected by isolation of 70S ribosomes. Hydroxyl radicals, generated in situ from the tethered Fe(II), cleaved sites in the 16S rRNA backbone that were close in three-dimensional space to the Fe(II), and a primer extension was used to identify these sites of cleavage. Two sets of 16S ribosomal RNA fragments, 1-360/361-1542 and 1-448/449-1542, were reconstituted into active 30S subunits. Fe(II) tethered to position 361 results in cleavage of 16S rRNA around nucleotides 34, 160, 497, 512, 520, 537, 552, and 615, as well as around positions 1410, 1422, 1480, and 1490. Fe(II) tethered to position 449 induces cleavage around nucleotide 488 and around positions 42 and 617. Fe(II) tethered to the 5' end of 16S rRNA induces cleavage of the rRNA around nucleotides 5, 601, 615, and 642. These results provide constraints for the positioning of these regions of 16S rRNA, for which there has previously been only limited structural information, within the 30S subunit.     

Protein-rRNA binding features and their structural and functional implications in ribosomes as determined by cross-linking studies

We have investigated protein-rRNA cross-links formed in 30S and 50S ribosomal subunits of Escherichia coli and Bacillus stearothermophilus at the molecular level using UV and 2-iminothiolane as cross-linking agents. We identified amino acids cross-linked to rRNA for 13 ribosomal proteins from these organisms, namely derived from S3, S4, S7, S14, S17, L2, L4, L6, L14, L27, L28, L29 and L36. Several other peptide stretches cross-linked to rRNA have been sequenced in which no direct cross-linked amino acid could be detected. The cross-linked amino acids are positioned within loop domains carrying RNA binding features such as conserved basic and aromatic residues. One of the cross-linked peptides in ribosomal protein S3 shows a common primary sequence motif--the KH motif--directly involved in interaction with rRNA, and the cross-linked amino acid in ribosomal protein L36 lies within the zinc finger-like motif of this protein. The cross-linked amino acids in ribosomal proteins S17 and L6 prove the proposed RNA interacting site derived from three-dimensional models. A comparison of our structural data with mutations in ribosomal proteins that lead to antibiotic resistance, and with those from protein-antibiotic cross-linking experiments, reveals functional implications for ribosomal proteins that interact with rRNA.     

The active sites of the eukaryotic 20 S proteasome and their involvement in subunit precursor processing

The 26 S proteasome is the central protease involved in ubiquitin-mediated protein degradation and fulfills vital regulatory functions in eukaryotes. The proteolytic core of the complex is the 20 S proteasome, a cylindrical particle with two outer rings each made of 7 different alpha-type subunits and two inner rings made of 7 different beta-type subunits. In the archaebacterial 20 S proteasome ancestor proteolytically active sites reside in the 14 uniform beta-subunits. Their N-terminal threonine residues, released by precursor processing, perform the nucleophilic attack for peptide bond hydrolysis. By directed mutational analysis of 20 S proteasomal beta-type proteins of Saccharomyces cerevisiae, we identified three active site-carrying subunits responsible for different peptidolytic activities as follows: Pre3 for post-glutamyl hydrolyzing, Pup1 for trypsin-like, and Pre2 for chymotrypsin-like activity. Double mutants harboring only trypsin-like or chymotrypsin-like activity were viable. Mutation of two potentially active site threonine residues in the Pre4 subunit excluded its catalytic involvement in any of the three peptidase activities. The generation of different, incompletely processed forms of the Pre4 precursor in active site mutants suggested that maturation of non-active proteasomal beta-type subunits is exerted by active subunits and occurs in the fully assembled particle. This trans-acting proteolytic activity might also account for processing intermediates of the active site mutated Pre2 subunit, which was unable to undergo autocatalytic maturation.     

Effect of ribosomal proteins on synthesis and assembly of preribosomal particles in isolated rat liver nuclei

Ribosomal proteins are rapidly taken up by isolated rat liver nuclei. The proteins are localized mainly in the nucleolus and are found associated with a nucleolar 80 S particle containing newly synthesized 45 S RNA. Ribosomal proteins in the nucleoplasm were associated with particles containing 18 and 28 S RNAs. In the absence of ribosomal proteins in the incubations, there was a decrease in the amount of newly synthesized 45, 18, and 28 S RNAs and an increase in low molecular weight RNA in both the nucleolus and nucleoplasm. This in vitro system appears to be useful for studies on the formation of ribosomal particles in the nucleus.     

Characterization of cDNAs encoding the p44 and p35 subunits of human translation initiation factor eIF3

Eukaryotic translation initiation factor 3 (eIF3) is a large multisubunit complex that plays a central role in the initiation of translation. It binds to 40 S ribosomal subunits resulting in dissociation of 80 S ribosomes, stabilizes initiator methionyl-tRNA binding to 40 S subunits, and is required for mRNA binding. eIF3 has an aggregate molecular mass of approximately 600 kDa and comprises at least 10 subunits. The cDNAs encoding eight of the subunits have been cloned previously (p170, p116, p110, p66, p48, p47, p40, and p36). Here we report the cloning and characterization of human cDNAs encoding two more subunits of human eIF3, namely eIF3-p44 and eIF3-p35. These proteins are immunoprecipitated by affinity-purified anti-eIF3-p170 antibodies, indicating they are components of the eIF3 complex. Far Western analysis shows that eIF3-p44 interacts strongly and specifically with the eIF3-p170 subunit, and weakly with p116/p110, p66, p40, and itself. eIF3-p44 contains an RNA recognition motif near its C terminus. Northwestern blotting shows that eIF3-p44 binds 18 S rRNA and beta-globin mRNA. Possession of cloned cDNAs encoding all 10 subunits of eIF3 provides the tools necessary to elucidate the functions of the individual subunits and the structure of the eIF3 complex.     

Variable regions V13 and V3 of Saccharomyces cerevisiae contain structural features essential for normal biogenesis and stability of 5.8S and 25S rRNA

The homologous ribosomal RNA species of all organisms can be folded into a common "core" secondary structure. In addition, eukaryotic rRNAs contain a large number of segments, located at fixed positions, that are highly variable in size and sequence from one organism to another. We have investigated the role of the two largest of these variable regions in Saccharomyces cerevisiae 25S rRNA, V13, and V3, by mutational analysis in a yeast strain that can be rendered completely dependent on the synthesis of mutant (pre-)rRNA. We found that approximately half of variable region V13 can be deleted without any phenotypic effect. The remaining portion, however, contains multiple structural features whose disturbance causes serious growth defects or lethality. Accumulation of 25S rRNA is strongly reduced by these mutations, at least in part because they inhibit processing of ITS2. Removal of even a relatively small portion of V3 also strongly reduces the cellular growth rate and larger deletions are lethal. Interestingly, some of the deletions in V3 cause accumulation of 27S(A) pre-rRNA and, moreover, appear to interfere with the close coupling between the processing cleavages at sites A3 and B1(S). These results demonstrate that both variable regions play an important role in 60S subunit formation.     

23S rRNA positions essential for tRNA binding in ribosomal functional sites

rRNA plays an important role in function of peptidyl transferase, the catalytic center of the ribosome responsible for the peptide bond formation. Proper placement of the peptidyl transferase substrates, peptidyl-tRNA and aminoacyl-tRNA, is essential for catalysis of the transpeptidation reaction and protein synthesis. In this report, we define a small set of rRNA nucleotides that are most likely directly involved in binding of tRNA in the functional sites of the large ribosomal subunit. By binding biotinylated tRNA substrates to randomly modified large ribosomal subunits from Escherichia coli and capturing resulting complexes on the avidin resin, we identified four nucleotides in the large ribosomal subunit rRNA (positions G2252, A2451, U2506, and U2585) whose modifications prevent binding of a peptidyl-tRNA analog in the P site and one residue (U2555) whose modification interferes with transfer of peptidyl moiety to puromycin. These nucleotides represent a subset of positions protected by tRNA analogs from chemical modification and significantly narrow the number of 23S rRNA nucleotides that may be directly involved in tRNA binding in the ribosomal functional sites.     

Saccharomyces cerevisiae Nip7p is required for efficient 60S ribosome subunit biogenesis

The Saccharomyces cerevisiae temperature-sensitive (ts) allele nip7-1 exhibits phenotypes associated with defects in the translation apparatus, including hypersensitivity to paromomycin and accumulation of halfmer polysomes. The cloned NIP7+ gene complemented the nip7-1 ts growth defect, the paromomycin hypersensitivity, and the halfmer defect. NIP7 encodes a 181-amino-acid protein (21 kDa) with homology to predicted products of open reading frames from humans, Caenorhabditis elegans, and Arabidopsis thaliana, indicating that Nip7p function is evolutionarily conserved. Gene disruption analysis demonstrated that NIP7 is essential for growth. A fraction of Nip7p cosedimented through sucrose gradients with free 60S ribosomal subunits but not with 80S monosomes or polysomal ribosomes, indicating that it is not a ribosomal protein. Nip7p was found evenly distributed throughout the cytoplasm and nucleus by indirect immunofluorescence; however, in vivo localization of a Nip7p-green fluorescent protein fusion protein revealed that a significant amount of Nip7p is present inside the nucleus, most probably in the nucleolus. Depletion of Nip7-1p resulted in a decrease in protein synthesis rates, accumulation of halfmers, reduced levels of 60S subunits, and, ultimately, cessation of growth. Nip7-1p-depleted cells showed defective pre-rRNA processing, including accumulation of the 35S rRNA precursor, presence of a 23S aberrant precursor, decreased 20S pre-rRNA levels, and accumulation of 27S pre-rRNA. Delayed processing of 27S pre-rRNA appeared to be the cause of reduced synthesis of 25S rRNA relative to 18S rRNA, which may be responsible for the deficit of 60S subunits in these cells.