A temperature-sensitive mutation affecting the mammalian 60 S ribosome.

A temperature-sensitive Chinese hamster cell mutant, ts14, is unable to synthesize protein in tissue culture at 39 degrees. That mutant's protein biosynthetic machinery has been characterized in cell-free, biologically active extracts. Similar to the mutant's phenotype in tissue culture, ts14 extracts cease protein synthesis in vitro within 15 min at 40 degrees. In contrast, at 25 degrees both ts14 and wild type extracts synthesize protein for more than 2 hours. Fractionation of mutant extracts and complementation with comparable wild type preparations indicate that ts14 possesses a thermolabile component associated with its polyribosomes. In preparation of ts14 ribosomes that are free of mRNA and bound protein factors, the defective factor is complemented functionally only by 60 S ribosomal subunits prepared from the wild type parent. Sedimentation analyses in sucrose gradients demonstrate that ts14's mutation specifically affects stability of the mutant's 60 S ribosome. Treatment with high ionic strength buffers preferentially disrupts the mutant's 60 S ribosomal subunit and results in preparations of mutant ribosomes that contain biologically active 40 S subunits only. These studies demonstrate the applicability of a genetic approach to analyzing structure-function relationships in the eukaryotic ribosome.     

The role of 5-S RNA in temperature-sensitive ribosomal RNA maturation.

The synthesis of 5-S RNA was found to be unchanged at both the permissive (33.5 degrees C) and non-permissive (38.5 degrees C) temperatures in a temperature-sensitive Baby Hamster Kidney cell line (BHK 21 ts 422 E) as measured relative to synthesis of 18-S rRNA. The 5-S RNA is shown to be associated with nucleolar ribonucleoprotein particles even though rRNA processing does not yield a functional 28-S rRNA at the non-permissive temperature. The amount of 5-S RNA found associated with the 80-S ribonucleoprotein particles was the same at the permissive and non-permissive temperatures, indicating that an aberrant 5-S RNA contribution to rRNA processing is not a primary cause for the temperature-sensitive lesion of rRNA maturation in this mutant cell line. The amount of 5-S RNA in nucleolar 80-S RNA particles indicated that the association of 5-S RNA with the rRNA precursor particle occurs before the cleavage step at which 32-S precursor RNA is produced.     

Defect in polyamine metabolism in a BHK cell mutant temperature-sensitive for rRNA maturation

A mutant of BHK cells (ts422E) temperature-sensitive for processing 32S rRNA to 28S rRNA (Toniolo et al., '73) also loses the ability to synthesize polyamines and 5.8S rRNA when shifted to the non-permissive temperature (39 degrees). The activity of several enzymes not involved with polyamine synthesis, methylation of 32S rRNA, and small nuclear RNA production are apparently unaffected after at least 24 hours at 39 degrees. When cultures are returned to the permissive temperature (33 degrees), polyamine synthesizing capacity returns to normal as mature rRNA production resumes.     

Secondary structure features of ribosomal RNA species within intact ribosomal subunits and efficiency of RNA-protein interactions in thermoacidophilic (Caldariella acidophila, Bacillus acidocaldarius) and mesophilic (Escherichia coli) bacteria

Ribosomal subunits of Caldariella acidophila (max.growth temp., 90 degrees C) have been compared to subunits of Bacillus acidocaldarius (max. growth temp., 70 degrees C) and Escherichia coli (max. growth temp., 47 degrees C) with respect to (a) bihelical content of rRNA; (b) G . C content of bihelical domains and (c) tightness of rRNA-protein interactions. The principal results are as follows. Subunits of C. acidophilia ribosomes (Tm = 90-93 degrees C) exhibit considerable thermal tolerance over their B. acidocaldarius (Tm = 77 degrees C) and E. coli counterparts (Tm = 72 degrees C). Based on the "melting' hyperchromicities of the intact ribosomal subunits a 51-55% fraction of the nucleotides appears to participate in hydrogen-bonded base pairing regardless of ribosome source, whereas a larger fraction, 67-70%, appears to be involved in hydrogen bonding in the naked rRNA species. The G . C content of bihelical domains of both free and ribosome-bound rRNA increases with increasing thermophily; based on hyperchromicity dispersion spectra of intact subunits and free rRNA, the bihelical parts of C. acidophila rRNA are estimated to contain 63-64% G . C, compared to 58.5% G . C for B. acidocaldarius and 55% G . C for E. coli. The increment of ribosome Tm values with increasing thermophily is greater than the increase in Tm for the free rRNA, indicating that within ribosomes bihelical domains of the thermophile rRNA species are stabilized more efficiently than their mesophile counterparts by proteins or/ and other component(s). The efficiency of the rRNA-protein interactions in the mesophile and thermophile ribosomes has been probed by comparing the releases, with LiCl-urea, of the rRNA species from the corresponding ribosomal subunits stuck to a Celite column through their protein moiety; it has been established that the release of C. acidophila rRNA from the Celite-bound ribosomes occurs at salt-urea concentrations about 4-fold higher than those required to release rRNA from Celite-bound E. coli ribosomes. Compared to E. coli the C. acidophila 50 and 30 S ribosomal subunits are considerably less susceptible to treatment designed to promote ribosome unfolding through depletion of magnesium ions.     

Serum-mediated regulation of amino acid transport in cultured chick embryo fibroblasts

Three different temperature sensitive mutants derived from the Syrian hamster cell line BHK 21 were found to have greatly reduced DNA synthesis at the non-permissive temperature. These mutants are distinct by complementation analysis and behave at the non-permissive temperature as cell cycle traverse defective mutants. Microfluorometric analysis of mutant populations arrested at the non-permissive temperature shows an accumulation of cells with G1 DNA content. Mutants ts 13 and ts HJ4 synchronized in G1 by serum or isoleucine deprivation and shifted to the non-permissive temperature at the time of release do not enter the S phase, while in the case of mutant ts 11 preincubation at the non-permissive temperature before release is required to completely prevent its entry into S. Ts 13 and ts 11 are able to traverse the S phase at the non-permissive temperature when synchronized at the boundary G1/S; in this case, preincubation of ts 11 at the non-permissive temperature before release does not affect the ability of these cells to perform DNA synthesis. On the other hand, ts HJ4 appears to traverse S only partially when tested under similar conditions. Temperature shift experiments of mutant populations at different times after isoleucine synchronization suggest that ts 13 and ts 11 are blocked at the non-permissive temperature in early G1, whereas ts HJ4 is probably affected near the initiation of DNA synthesis, or in some early S function.     

Effects of ribosomal proteins S1, S2 and the DeaD/CsdA DEAD-box helicase on translation of leaderless and canonical mRNAs in Escherichia coli

Leaderless mRNAs beginning with the AUG initiating codon occur in all kingdoms of life. It has been previously reported that translation of the leaderless cI mRNA is stimulated in an Escherichia coli rpsB mutant deficient in ribosomal protein S2. Here, we have studied this phenomenon at the molecular level by making use of an E. coli rpsB(ts) mutant. The analysis of the ribosomes isolated under the non-permissive conditions revealed that in addition to ribosomal protein S2, ribosomal protein S1 was absent, demonstrating that S2 is essential for binding of S1 to the 30S ribosomal subunit. In vitro translation assays and the selective translation of a leaderless mRNA in vivo at the non-permissive temperature corroborate and extend previous in vitro ribosome binding studies in that S1 is indeed dispensable for translation of leaderless mRNAs. The deaD/csdA gene, encoding the "DeaD/CsdA" DEAD-box helicase, has been isolated as a multicopy suppressor of rpsB(ts) mutations. Here, we show that expression of a plasmid-borne DeaD/CsdA gene restores both S1 and S2 on the ribosome at the non-permissive temperature in the rpsB(ts) strain, which in turn leads to suppression of the translational defect affecting canonical mRNSa. These data are discussed in terms of a model, wherein DeaD/CsdA is involved in ribosome biogenesis rather than acting directly on mRNA.     

Isolation and characterization of active ribosomal subunits from human placenta.

We have developed a method for the large-scale isolation of active ribosomal subunits from human placenta. The technique involves incubating crude ribosomes for 15 min at 37 degrees C with 0.2 mM puromycin in 50 mM Tris-HCl buffer, pH 7.6, 500 mM KCl and 3 mM MgCl2 followed by centrifugation at 5 degrees C in a BXV zonal rotor using an equivolumetric sucrose gradient in the same buffer, upon which 80--90% of all ribosomes are dissociated into subunits. The purified subunits differ in their chemical composition, the 60-S particle containing no more than 36% protein whereas the 40-S subunit consists of 43% protein. In poly(U)-directed protein synthesis, tested in a completely homologous cell-free system, one recombined couple polymerizes at 37 degrees C 12 to 17 phenylalanine residues at an initial rate of 0.7 residues per minute. However, free 80-S ribosomes obtained by puromycin treatment of the crude ribosomes and reassociation of the subunits without prior isolation, have an even higher incorporating activity (20--25 mol phenylalanine/mol of ribosome). At least 55% of the subunits were estimated to actively participate in the polyphenylalanine synthesis.     

The Caulobacter crescentus GTPase CgtAC is required for progression through the cell cycle and for maintaining 50S ribosomal subunit levels

The Obg subfamily of bacterial GTP-binding proteins are biochemically distinct from Ras-like proteins raising the possibility that they are not controlled by conventional guanine nucleotide exchange factors (GEFs) and/or guanine nucleotide activating proteins (GAPs). To test this hypothesis, we generated mutations in the Caulobacter crescentus obg gene (cgtAC) which, in Ras-like proteins, would result in either activating or dominant negative phenotypes. In C. crescentus, a P168V mutant is not activating in vivo, although in vitro, the P168V protein showed a modest reduction in the affinity for GDP. Neither the S173N nor N280Y mutations resulted in a dominant negative phenotype. Furthermore, the S173N was significantly impaired for GTP binding, consistent with a critical role of this residue in GTP binding. In general, conserved amino acids in the GTP-binding pocket were, however, important for function. To examine the in vivo consequences of depleting CgtAC, we generated a temperature-sensitive mutant, G80E. At the permissive temperature, G80E cells grow slowly and have reduced levels of 50S ribosomal subunits, indicating that CgtAC is important for 50S assembly and/or stability. Surprisingly, at the non-permissive temperature, G80E cells rapidly lose viability and yet do not display an additional ribosome defect. Thus, the essential nature of the cgtAC gene does not appear to result from its ribosome function. G80E cells arrest as predivisional cells and stalkless cells. Flow cytometry on synchronized cells reveals a G1-S arrest. Therefore, CgtAC is necessary for DNA replication and progression through the cell cycle.     

Assembly of the mitochondrial ribosomes in a temperature-conditional mutant of Saccharomyces cerevisiae defective in the synthesis of the var1 protein

An investigation of the role of the var1 protein in the assembly of the yeast mitochondrial ribosomes was carried out in a temperature conditional mutant, strain h56, which contains a mutation (tsv1) just upstream of the structural gene for the var1 protein. The mutation results in a marked decrease in the synthesis of the var1 protein at the permissive temperature of 28 degrees C and an apparently complete absence of var1 synthesis at the restrictive temperature of 36 degrees C. Long-term growth of strain h56 at the non-permissive temperature was found to result in the loss of the small (37 S) ribosomal subunit and the appearance of a novel 30 S ribonucleoparticle. Both the small (37 S) and the large (54 S) mitochondrial ribosomal subunits were found to be assembled in strain h56 for at least 3 h after transfer to the non-permissive temperature.     

Suppression of an Escherichia coli secAts mutant by a gene cloned from Staphylococcus carnosus

Escherichia coli mutant MM52 (secA(ts)) was transformed with a cosmid library from Staphylococcus carnosus, and a recombinant cosmid (pBO23) allowing growth at the non-permissive temperature (42 degrees C) was isolated. pBO23 also restored the growth defects of E. coli mutants IQ85 (secY(ts)) and IT41 (lep(ts)). Nucleotide sequencing revealed that the DNA fragment responsible for the suppression effect codes for a S. carnosus protein highly homologous to the ribosomal protein L13 of E. coli. The staphylococcal L13 protein was efficiently incorporated into E. coli ribosomes. Possible explanations for the effect of this polypeptide on the growth of temperature-sensitive E. coli secretion mutants are discussed.     

TRANSPORT OF VIRAL RNA IN KB CELLS INFECTED WITH ADENOVIRUS TYPE 2

Messenger RNA transport was studied in KB cells infected with the nuclear DNA virus adenovirus type 2. Addition of 0.04 µg/ml of actinomycin completes the inhibition of ribosome synthesis normally observed late after infection and apparently does not alter the pattern of viral RNA synthesis: Hybridization-inhibition experiments indicate that similar viral RNA sequences are transcribed in cells treated or untreated with actinomycin. The polysomal RNA synthesized during a 2 hr labeling period in the presence of actinomycin is at least 60% viral specific. Viral messenger RNA transport can occur in the absence of ribosome synthesis. When uridine-³H is added to a late-infected culture pretreated with actinomycin, viral RNA appears in the cytoplasm at 10 min, but the polysomes do not receive viral RNA-³H until 30 min have elapsed. Only 25% of the cytoplasmic viral RNA is in polyribosomes even when infected cells have been labeled for 150 min. The nonpolysomal viral RNA in cytoplasmic extracts sediments as a broad distribution from 10S to 80S and does not include a peak cosedimenting with 45S ribosome subunits. The newly formed messenger RNA that is ribosome associated is not equally distributed among the ribosomes; by comparison to polyribosomes, 74S ribosomes are deficient at least fivefold in receipt of new messenger RNA molecules.     

The binding of Met-tRNAf to isolated 40-S ribosomal subunits and the formation of Met-tRNAf - 80-S-ribosome initiation complexes.

Different forms of 40-S ribosomal subunit, distinguishable by their buoyant densities on CsCl equilibrium density gradients, are formed when derived 40-S ribosomal subunits are incubated with partially purified reticulocyte ribosomal wash proteins. One of these subunits, the 1.37-g-cm-3 form is not present in the cell but the other two forms, the 1.40-g-cm-3 and 1.40-g-cm-3 subunits, are present in cell extracts. 35S label is bound to 1.37-g-cm-3 and 1.40-g-cm-s subunits when [35S]Met-tRANf, GTP and poly(A,U,G) are included in the incubations. The 35S-labelled 40-S subunits recovered, and the amount of 35S label bound to them, are changed if the [35S]Met-tRNAf-40-S-subunit-poly(A,U,G) complexes are first purified on sucrose gradients before analysing them on CsCl. The 1.37-g-cm-3 particle is no longer seen and the total quantity of 35S label on the 40-S subunits is 90% lower after sucrose gradient purification. Between 30% and 40% of the 40-S subunits bind [35S]Met-tRNAf when 1 mM GTP, an excess of ribosomal wash proteins and [35S]Met-tRNAf over derived 40-S subunits, and poly(A,U,G) or AUG is included in the incubations. The omission of poly(A,U,G) or AUG from the incubations substantially lowers the amount of subunit-bound 35S label ultimately recovered. With these incubations less than 10% of the 40-S subunits have bound [35S]Met-tRNAf. [35S]Met-tRNAf binding is affected by the nature of the RNA added. The addition of poly(U), rRNA and native 9-S golbin mRNA is without effect, whereas denatured globin mRNA is stimulatory. Maximum binding is obtained however with AUG. Poly(A,U,G) is less stimulatory than AUG but more stimulatory than denatured mRNA, suggesting that the number as well the accessibility of the AUG initiations condons determines the amount of 35S label bound. Similar results are obtained for the ribosomal-wash-dependent binding of [35S]Met-tRNAf to 80-S ribosomes. Contrary to the binding results, the ability of mRNA to stimulate protein synthesis is dependent on the integrity of the mRNA. Thus, native 9-S globin mRNA but not poly(A,U,G) stimulatex protein synthesis in the wheat germ system. HCHO-treated globin mRNA, although stimulatory, is 45% less effective than native mRNA. The addition of AUG, derived 60-S subunits and extra ribosomal wash is required for the formation of [35S]Met-tRNAf-80-S-ribosome complexes from sucrose-gradient-purified [35S]Met-tRNAf-40-S-subunit complexes. The 80-S ribosome complexes are able to form peptide bonds. Thus, if puromycin is added to the full incubations at zero time, no 35S label is present on the 80-S ribosome. 35S label is released as methionyl-puromycin. If the [35S]Met-tRNAf-40-S-subunit complexes are assembled with poly(A,U,G) or AUG in the incubations and then purified, only derived 60-S subunits are required to form [35S]Met-tRNAf-80-S-ribosome complexes. 35S label is not released from them when puromycin is added to the incubations unless extra ribosomal wash is also added.     

DnaK-facilitated ribosome assembly in Escherichia coli revisited

Assembly helpers exist for the formation of ribosomal subunits. Such a function has been suggested for the DnaK system of chaperones (DnaK, DnaJ, GrpE). Here we show that 50S and 30S ribosomal subunits from an Escherichia coli dnaK-null mutant (containing a disrupted dnaK gene) grown at 30 degrees C are physically and functionally identical to wild-type ribosomes. Furthermore, ribosomal components derived from mutant 30S and 50S subunits are fully competent for in vitro reconstitution of active ribosomal subunits. On the other hand, the DnaK chaperone system cannot circumvent the necessary heat-dependent activation step for the in vitro reconstitution of fully active 30S ribosomal subunits. It is therefore questionable whether the requirement for DnaK observed during in vivo ribosome assembly above 37 degrees C implicates a direct or indirect role for DnaK in this process.     

Differential features of ribosomes and of poly(U)-programmed cell-free systems derived from sulphur-dependent archaebacterial species

The properties of poly(U)-directed cell-free systems developed from the sulphur-dependent, thermophilic archaebacteria Desulfurococcus mobilis, Thermoproteus tenax, Sulfolobus solfataricus, Thermococcus celer and Thermoplasma acidophilum have been compared. All systems are truly thermophilic in requiring incubation at temperatures close to the physiological optimum for cell growth. Under optimized conditions the error frequency in tRNA selection is less than 0.4% at 80 degrees C, and synthetic efficiencies (Phe residues polymerized per ribosome in 40 min) span from 4 for Tp. tenax, to 10 for Tc. celer, to 20-25 for D. mobilis and T. acidophilum and to 40 for S. solfataricus. According to requirements for polypeptide synthesis and to degree of stability of the ribosomal subunits' association, sulphur-dependent thermophiles cluster into two groups. Group I organisms (D. mobilis, Tp. tenax, S. solfataricus) harbour 70-S monomers composed of weakly associated subunits, whose poly(Phe)-synthesizing capacity is totally dependent on added spermine while being drastically inhibited by monovalent cations. Group II organisms (Tc. celer and T. acidophilum) contain 70-S particles composed of tightly bonded subunits, whose synthetic capacity is independent of spermine while being totally dependent on monovalent cations. Spermine promotes poly(Phe) synthesis on ribosomes of group I organisms by converting the peptidyltransferase center into an active conformation, while monovalent cations are inhibitory by preventing the interaction between the free ribosomal subunits. The closeness between Tc. celer and T. acidophilum ribosomes provides new insight on the phylogenetic placement of Thermococcaceae.     

Functional analysis of the invariant residue G791 of Escherichia coli 16S rRNA

The nucleotide at position 791(G791) of E. coli 16S rRNA was previously identified as an invariant residue for ribosomal function. In order to characterize the functional role of G791, base substitutions were introduced at this position, and mutant ribosomes were analyzed with regard to their protein synthesis ability, via the use of a specialized ribosome system. These ribosomal RNA mutations attenuated the ability of ribosomes to conduct protein synthesis by more than 65%. A transition mutation (G to A) exerted a moderate effect on ribosomal function, whereas a transversion mutation (G to C or U) resulted in a loss of protein synthesis ability of more than 90%. The sucrose gradient profiles of ribosomes and primer extension analysis showed that the loss of protein-synthesis ability of mutant ribosomes harboring a base substitution from G to U at position 791 stems partially from its inability to form 70S ribosomes. These findings show the involvement of the nucleotide at position 791 in the association of ribosomal subunits and protein synthesis steps after 70S formation, as well as the possibility of using 16S rRNA mutated at position 791 for the selection of second-site revertants in order to identify ligands that interact with G791 in protein synthesis.     

Quantitative analysis of the protein composition of yeast ribosomes

The molecular weights of the individual yeast ribosomal proteins were determined. The ribosomal proteins from the 40-S subunit have molecular weights ranging from 11 800 to 31 000 (average molecular weight = 21 300). The molecular weights of the 60-S subunit proteins range from 10 000 to 48 400 (average molecular weight = 21 800). Stoichiometric measurements, performed by densitometric scanning on ribosomal proteins extracted from high-salt dissociated subunits revealed that isolated ribosomal subunits contain, besides some protein species occurring in submolar amounts, a number of protein species which are present in multiple copies: S13, S27, L22, L31, L33, L34 and L39. The mass fractions of the ribosomal proteins which were found to be present on isolated ribosomes in non-unimolar amounts, were re-examined by using an isotope dilution technique. Applying this method to proteins extracted from mildely isolated 80-S ribosomes, we found that some protein species such as S32, S34 and L43 still are present in submolar amounts. On the other hand, however, we conclude that some other ribosomal proteins, in particular the strongly acidic proteins L44 and L45 get partially lost during ribosome dissociation. Proteins L44/L45 appears to be present on 80-S ribosomes in three copies.     

Processing of ribosomal RNA in a temperature sensitive mutant of BHK cells.

The processing of ribosomal RNA has been studied in a temperature sensitive mutant of the Syrian hamster cell line BHK 21. At 39 degrees C, these cells are unable to synthesize 28S RNA, and 60S ribosomal subunits, while 18S RNA, and 40S subunits are produced at both temperatures. At 39 degrees C the 45S RNA precursor is transcribed and processed as in wild type cells. The processing of the RNA precursors becomes defective after the cleavage of the 41S RNA, and the separation of the 18S and 28S RNAs sequences in two different RNA molecules. The 36S RNA precursor, which is always present in very small quantity in the nucleoli of wild type cells and of the mutant at 33 degrees C, is found in very large amounts in the mutant at 39 degrees C. The 36S RNA can be, however, slowly processed to 32S RNA. The 32S RNA cannot be processed at 39 degrees C, and it is degraded soon after its formation. Only a small proportion accumulates in the nucleoli. The 32S RNA synthesized at 39 degrees C cannot be processed to 28S RNA upon shift to the permissive temperature, even when the processing of the newly synthesized rRNA has returned to normal. The data suggest that the 36S and 32S RNAs are contained in aberrant ribonucleoprotein particles, leading to a defective processing of the particles as a whole.     

Protein synthesis defects in temperature-sensitive mutants of Escherichia coli with altered ribosomal proteins

The ribosomes from four temperature-sensitive mutants of Escherichia coli have been examined for defects in cell-free protein synthesis. The mutants examined had alterations in ribosomal proteins S10, S15, or L22 (two strains). Ribosomes from each mutant showed a reduced activity in the translation of phage MS2 RNA at 44 degrees C and were more rapidly inactivated by heating at this temperature compared to control ribosomes. Ribosomal subunits from three of the mutants demonstrated a partial or complete inability to reassociate at 44 degrees C. 70-S ribosomes from two strains showed a reducton in messenger RNA binding. tRNA binding to the 30 S subunit was reduced in the strains with altered 30-S proteins and binding to the 50 S subunit was affected in the mutants with a change in 50 S protein L22. The relation between ribosomal protein structure and function in protein synthesis in these mutants is discussed.     

Cold-sensitive ribosome assembly in an Escherichia coli mutant lacking a single methyl group in ribosomal protein L3

Ribosomal protein methylation has been well documented but its function remains unclear. We have examined this phenomenon using an Escherichia coli mutant (prmB2), which fails to methylate glutamine residue number 150 of ribosomal protein L3. This mutant exhibits a cold-sensitive phenotype: its growth rate at 22 degrees C is abnormally low in complete medium. In addition, strains with this mutation accumulate abnormal and unstable ribosomal particles; 50-S and 30-S subunits are formed, but at a lower rate. Once assembled, ribosomes with unmethylated L3 are fully active by several criteria. (a) Protein synthesis in vitro with purified 70-S prmB2 ribosomes is as active as wild-type using either a natural (R17) or an artificial [poly(U)] messenger. (b) The induction of beta-galactosidase in vivo exhibits normal kinetics and the enzyme has a normal rate of thermal denaturation. (c) These ribosomes are standard when exposed in vitro to a low magnesium concentration or increasing molarities of LiCl. Efficient methylation of L3 in vitro requires either unfolded ribosomes or a mixture of ribosomal protein and RNA. We suggest that the L3-specific methyltransferase may qualify as one of the postulated 'assembly factors' of the E. coli ribosome.     

30S ribosomal subunits can be assembled in vivo without primary binding ribosomal protein S15

Assembly of 30S ribosomal subunits from Escherichia coli has been dissected in detail using an in vitro system. Such studies have allowed characterization of the role for ribosomal protein S15 in the hierarchical assembly of 30S subunits; S15 is a primary binding protein that orchestrates the assembly of ribosomal proteins S6, S11, S18, and S21 with the central domain of 16S ribosomal RNA to form the platform of the 30S subunit. In vitro S15 is the sole primary binding protein in this cascade, performing a critical role during assembly of these four proteins. To investigate the role of S15 in vivo, the essential nature of rpsO, the gene encoding S15, was examined. Surprisingly, E. coli with an in-frame deletion of rpsO are viable, although at 37 degrees C this DeltarpsO strain has an exaggerated doubling time compared to its parental strain. In the absence of S15, the remaining four platform proteins are assembled into ribosomes in vivo, and the overall architecture of the 30S subunits formed in the DeltarpsO strain at 37 degrees C is not altered. Nonetheless, 30S subunits lacking S15 appear to be somewhat defective in subunit association in vivo and in vitro. In addition, this strain is cold sensitive, displaying a marked ribosome biogenesis defect at low temperature, suggesting that under nonideal conditions S15 is critical for assembly. The viability of this strain indicates that in vivo functional populations of 70S ribosomes must form in the absence of S15 and that 30S subunit assembly has a plasicity that has not previously been revealed or characterized.