Mature Small Subunit Research Articles

Co-Expression of a Precursor and the Mature Protein of Wheat Ribulose-1, 5-bisphosphate Carboxylase Small Subunit from a Single Gene inEscherichia coli

The cDNA encoding a precursor of wheat ribulose-1,5-bisphosphate carboxylase/oxygenase was inserted in-phase with prokaryotic expression elements in four different vectors. Five expression vectors encoding the small subunit precursors were cloned in Escherichia coli. None of these constructs expressed detectable amounts of the precursor protein, but all directed synthesis of the mature small subunit. The expression of the small subunit was a consequence of an independent, intragenic Shine-Dalgarno sequence optimally located upstream from an ATG specifying the first codon of the mature small subunit portion in the precursor transcript. Similar internal translation signals have been identified in the nuclear-encoded cDNAs of the small-subunit precursors of numerous higher plant genes. The 5' end of the wheat small-subunit precursor was linked with a consensus E. coli DNA sequence such that the modified gene encoded a partial hybrid precursor carrying four additional residues at its amino terminus. The resultant construct, pEI-W3, directed abundant synthesis of both the partially hybrid small-subunit precursor and the mature small subunit, constituting as much as 10% of the total bacterial protein. The bacterially synthesized small subunit precursor was purified to homogeneity. The authenticity of the recombinant protein was verified by its size, immunological properties, amino-terminal sequence, and amino acid composition.

Molecular evolution of the small subunit of ribulose bisphosphate carboxylase: nucleotide substitution and gene conversion.

The nucleotide sequences encoding the mature portion of 31 ribulose 1.5-bisphosphate carboxylase small subunit (SSU) genes from 17 genera of plants, green algae and cyanobacteria were examined. Among the 465 pairwise sequence comparisons, SSU multigene family members within the same species were more similar to each other in nonsynonymous or replacement nucleotide substitutions (RNS) than they were to SSU sequences in any other organism. The concerted evolution of independent SSU gene lineages within closely related plant species suggests that homogenization of RNS positions has occurred at least once in the life of each genus. The rate of expected RNS among mature SSU sequences was calculated to be 1.25 X 10(-9)/site/yr for the first 70 million years (MY) of divergence with a significant slowing to 0.13 X 10(-9)/site/yr for the next 1,400 MY. The data suggest that mature SSU sequences do not accumulate more than 20% differences in the RNS positions without compensatory changes in other components of this enzyme system. During the first 70 MY of divergence between species, the rate of expected synonymous or silent nucleotide substitutions (SNS) is approximately 6.6 X 10(-9)/site/yr. This is five times the RNS rate and is similar to the silent rate observed in animals. In striking contrast, SNS and RNS do not show this correlation among SSU gene family members within a species. A mechanism involving gene conversion within the exons followed by selection for biased gene conversion products with conservation of RNS positions and divergence of SNS positions is discussed. A SSU gene tree based on corrected RNS for 31 SSU sequences is presented and agrees well with a species tree based on morphological and cytogenetic traits for the 17 genera examined. SSU gene comparisons may be useful in predicting phylogenetic relationships and in some cases divergence times of various plant, algal and cyanobacterial species.

Identification of an assembly domain in the small subunit of ribulose-1,5-bisphosphate carboxylase.

The mature small subunit (SSU) of ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) in higher plants contains a highly conserved sequence of 16 amino acids that is absent in the SSUs of cyanobacteria. To determine whether this region of the SSU of higher plants has a specific function, portions of the SSU genes (rbcS) of pea (Pisum sativum) and the cyanobacterium Anacystis nidulans were fused to create chimeric genes that either lacked or contained the coding sequence for the 16 conserved amino acids. Precursor proteins synthesized in vitro from the chimeric genes were incubated with isolated pea chloroplasts to assay import and assembly into the holoenzyme. Fusion proteins lacking the 16-amino acid sequence were imported and processed but failed to assemble with endogenous large subunit. Addition of the region from a pea rbcS containing the 16 amino acids to the rbcS of Anacystis enabled the imported SSU fusion protein to assemble with pea large subunit. This 16-amino acid sequence is encoded by a separate exon in certain rbcS genes of higher plants. We propose that the conserved 16-amino acid sequence constitutes a domain acquired to facilitate assembly of the eukaryotic holoenzyme.

Chloroplast transport of a ribulose bisphosphate carboxylase small subunit-5-enolpyruvyl 3-phosphoshikimate synthase chimeric protein requires part of the mature small subunit in addition to the transit peptide.

Ribulose bisphosphate carboxylase small subunit protein is synthesized in the cytoplasm as a precursor and transported into the chloroplast where the amino-terminal portion, the transit peptide, is removed proteolytically. To obtain chloroplast delivery of the 43-kDa 5-enolpyruvyl 3-phosphoshikimate (EPSP) synthase of Salmonella typhimurium, we constructed fusion proteins between the bacterial EPSP synthase and the ribulose bisphosphate carboxylase small subunit. A fusion protein consisting of the transit peptide fused to the EPSP synthase was not transported in vitro or in vivo into chloroplasts. A second fusion protein consisting of the transit peptide and 24 amino acids of the mature small subunit fused to the EPSP synthase was transported both in vitro and in vivo into chloroplasts. It was processed into two polypeptides of 46 and 47 kDa, respectively. This heterogeneity in processing was not caused by the presence of the aroA start codon, since its removal resulted in the same pattern. Substituting 24 different amino acids for the 24 amino acids of the mature small subunit resulted in a fusion protein that was not transported into the chloroplast. It was concluded that a portion of the mature small subunit was needed for efficient chloroplast delivery.

Mitochondrial gene expression in a nuclear mutant of Neurospora deficient in large subunits of mitochondrial ribosomes

A new cytochrome a and b deficient nuclear mutant of Neurospora crassa, cyt-U-28, is defective in the assembly of large subunits of mitochondrial ribosomes. Nonetheless, this mutant overproduces apparently normal small subunits of mitochondrial ribosomes, even though it should be deficient for the S5 ribosomal protein required for assembly of the particles beyond the CAP30S stage. The mitochondria of cyt-U-28 indeed synthesize only small amounts of most mitochondrial polypeptides, including cytochrome oxidase subunits I, II, and III, contain very low amounts of the normal seven-polypeptide cytochrome oxidase complex, and, unlike the organelles from other cytochrome a deficient mutants, do not accumulate the nuclear-encoded cytochrome oxidase subunits 5 and 6. Nonetheless, the mutant markedly oversynthesizes a mitochondrial protein that comigrates with subunit 9 of the mitochondrial ATPase on S DS–polyacrylamide electrophoresis gels. The overproduction of this protein and the accumulation of mature small subunits of mitochondrial ribosomes indicate that the cyt-U-28 mutant preferentially expresses two mitochondrial genes, one coding for ATPase subunit 9, the other for the S5 ribosomal protein.Key words: Neurospora, mitochondria, ribosomes, protein synthesis.

Complete processing of a small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase from pea requires the amino acid sequence Ile-Thr-Ser.

Chloroplast import and processing of two precursor proteins with mutations in the carboxyl-terminal region of the transit peptide were examined in vitro. Deletion mutations were introduced into the 57-amino acid transit peptide of a chloroplast protein, the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase, from pea. A mutant, PSd48/57, in which nine carboxyl-terminal amino acids of the transit peptide had been deleted, was imported and processed to a series of 13- to 18-kDa polypeptides including the 14-kDa mature small subunit. In contrast, processing of a mutant, PSd45/57, in which an additional three amino acids had been removed, resulted in a series of polypeptides which did not include the mature small subunit. Whereas PSd48/57 was imported as efficiently as the wild-type precursor, import of PSd45/57 was only 25% as efficient as that of the authentic precursor. The mutant precursor proteins PSd48/57 and PSd45/57 are distinguished by a three-amino acid sequence, Ile-Thr-Ser, located in the carboxyl-terminal region of the transit peptide. We show that all or part of this sequence is required for correct processing.

Co-expression of both the maize large and wheat small subunit genes of ribulose-bisphosphate carboxylase in Escherichia coli.

A cDNA clone for the precursor form of the small subunit of wheat ribulose-bisphosphate carboxylase has been modified to allow the expression in Escherichia coli of a mature form of small subunit that lacks the transit peptide. Synthesis of the protein is controlled by a lac promoter, and translation is initiated from a lacZ ribosome binding site, giving rise to a small subunit with several beta-galactosidase amino acids fused to its N-terminus. A plasmid has been constructed that enables both wheat small subunits and maize large subunits to be synthesized in the bacterial cell, but using different promoters to allow independent expression of the rbcS and rbcL genes. When the small subunit is synthesized in the absence of the large subunit, it is found in the soluble fraction but the polypeptide is unstable and has a half-life of less than 15 min. Its size on sucrose gradients indicates a monomeric or dimeric form. When large subunit synthesis is induced in cells containing the small subunit, both subunits are found predominantly in the insoluble fraction and are fully stable for more than 120 min, suggesting that aggregation of the subunits may occur. The two subunits do not assemble together to form an active holoenzyme in vivo, even when nascent large subunits ware synthesized in a pool of mature small subunits. This indicates that other factors may be required to mediate the assembly of the higher plant enzyme.

Synthesis of the small subunit of ribulose-bisphosphate carboxylase from genes cloned into plasmids containing the SP6 promoter.

DNA sequences encoding ribulose 1,5-bisphosphate carboxylase small subunit precursor from Pisum sativum L. have been transcribed from plasmids containing the SP6 promoter, and translated in a wheat germ cell-free system. The small subunit precursor polypeptide, its N-terminal leader sequence (transit peptide) and the mature small subunit have each been synthesized independently from three different plasmid constructs. The precursor polypeptide is imported into isolated pea chloroplasts and processed to the mature small subunit by a stromal proteinase. The mature polypeptide is neither imported, nor subject to proteolysis by stromal extracts. The transit peptide alone is very rapidly degraded by a stromal proteinase activity which can be inhibited by EDTA or 1,10-phenanthroline. The use of these gene constructs helps to establish the crucial role of the transit peptide in protein import into the chloroplast.

Targeting of protein to chloroplasts in transgenic tobacco by fusion to mutated transit peptide

Transport of foreign proteins into chloroplasts was studied in a transgenic plant expressing two different fusion proteins, the transit peptide (TP) of ribulose-bisphosphate carboxylase small subunit (SS) fused to neomycin phosphotransferase (TP-NPT II) and, the same transit peptide plus the amino-terminal 23 amino acids of mature SS linked to NPT II. The second fusion protein (TP-SS-NPT II) was found in isolated chloroplasts but accumulated to a lesser degree than the first (TP-NPT II). This finding does not support the hypothesis that the highly conserved amino acid sequence surrounding the cleavage site between the transit peptide (TP) and mature SS is required for efficient transport. This cleavage region shows a markedly higher conservation than either the mature protein or the TP sequences in SS genes from different plant species. Evidence is presented indicating that the transport of the TP-SS-NPT II precursor is diminished as a result of competition between the rate of its uptake and the rate of its degradation by cytosolic proteases. In an attempt to identify further regions in the TP involved in transport and processing, we designed derivatives of both the TP-SS-NPT II and TP-NPT II precursors. A derivative of TP-SS-NPT II lacking the amino acids at the processing site was expressed in plants and was shown to be transported and processed. A derivative of TP-NPT II comprising the first 41 amino acids (out of 57) of the transit peptide linked to NPT II was also expressed in plants. This protein was not imported into the organelles; however a significant amount of partially processed fusion protein was found to be attached to the outer membrane of the chloroplast.

The importance of the transit peptide and the transported protein for protein import into chloroplasts

We compared the transport in vitro of fusion proteins of neomycin phosphotransferase II (NPTII) with either the transit peptide of the small subunit (SSU) of ribulose-1,5-bisphosphate carboxylase/oxygenase or the transit peptide and the 23 aminoterminal amino acids of the mature small subunit. The results showed that the transit peptide is sufficient for import of NPTII. However, transport of the fusion protein consisting of the transit peptide linked directly to NPTII was very inefficient. In contrast, the fusion protein containing a part of the mature SSU was imported with an efficiency comparable to that of the authentic SSU precursor. We conclude from these results that other features of the precursor protein in addition to the transit peptide are important for transport into chloroplasts. In order to identify functional regions in the transit peptide, we analyzed the transport of mutant fusion proteins. We found that the transport of fusion proteins with large deletions in the aminoterminal, or central part was drastically reduced. In contrast, duplication of a part of the transit peptide led to a marked increase in transport.

Use of chimaeric genes harbouring small subunit transit peptide sequences to study transport in chloroplasts

A sequence coding for the transit peptide part of the precursor of the ribulose bisphosphate carboxylase small subunit can be used to construct chimaeric genes coding for easily detectable marker proteins. Chloroplasts of transgenic plants containing such chimaeric genes were found to contain the marker proteins. In addition, measurements of uptake by isolated chloroplasts of various precursor proteins, synthesized in vitro , demonstrated that most if not all of the transit peptide sequence was necessary and sufficient to obtain transport into chloroplasts. However, quantitative measurements of uptake indicated that high efficiency of transport required domains from both the transit peptide and the mature small subunit protein.

Length variation in eukaryotic rRNAs: small subunit rRNAs from the protists Acanthamoeba castellanii and Euglena gracilis

We have sequenced the region of the Acanthamoeba castellanii ribosomal RNA transcription unit which encodes the mature small subunit ribosomal RNA (SSU rRNA). It, like the SSU rRNA coding regions of Euglena gracilis and kinetoplastids, is approx. 30% larger than those reported from other eukaryotes. The extra nucleotides are present in highly variable regions of the rRNA genes. Direct sequence analysis of the corresponding variable regions in the rRNA of A. castellanii and E. gracilis demonstrates that the extra nucleotides are present in the mature rRNA; no post-transcriptional modification of the rRNAs occurs to reduce them to a size more typical of eukaryotes. The extra elements present in the rRNAs of these two organisms are not homologous; they have independent evolutionary origins.

Defective assembly of the mitochondrial ribosomes in yeast cells grown in the presence of mitochondrial protein synthesis inhibitors

The involvement of mitochondrial protein synthesis in the assembly of the mitochondrial ribosomes was investigated by studying the extent to which the assembly process can proceed in the presence of mitochondrial protein synthesis inhibitors erythromycin and chloramphenicol. Yeast cells grown in the presence of erythromycin (2 mg/ml) do not appear to contain any detectable amounts of the mitochondrial small (37 S) ribosomal subunit. Instead, a ribonucleoparticle with a sedimentation coefficient of 30 S was observed; this particle could be shown to be related to the mitochondrial small ribosomal subunit by two-dimensional gel electrophoretic analysis of its protein components. Since the var1 protein is the only mitochondrial translation product known to be associated with the mitochondrial ribosome, our results suggest that this protein is essential for the assembly of the mature small subunit, and that the var1 protein enters the pathway for the assembly of the small subunit at a late step. In at least one strain of yeast the accumulation of the 30-S particle appears to be very sensitive to catabolite repression. When yeast cells are grown in the presence of chloramphenicol instead of erythromycin, assembly of the small subunit appears to be only partially inhibited, and the presence of the 30-S particle could not be clearly demonstrated. This observation is consistent with the fact that in yeast, chloramphenicol inhibits mitochondrial protein synthesis by about 95% only and that the synthesis of the var1 protein appears to be the least sensitive to this inhibition.

The formation of a defective small subunit of the mitochondrial ribosomes in petite mutants of Saccharomyces cerevisiae

The involvement of mitochondrial protein synthesis in the assembly of the mitochondrial ribosomes was investigated by studying the extent to which the assembly process can proceed in petite mutants of Saccharomyces cerevisiae which lack mitochondrial protein synthetic activity due to the deletion of some tRNA genes and/or one of the rRNA genes on the mtDNA. Petite strains which retain the 15-S rRNA gene can synthesize this rRNA species, but do not contain any detectable amounts of the small mitochondrial ribosomal subunit. Instead, a ribonucleoparticle with a sedimentation coefficient of 30 S (instead of 37 S) was observed. This ribonucleoparticle contained all the small ribosomal subunit proteins with the exception of the var1 and three to five other proteins, which indicates that the 30-S ribonucleoparticle is related to the small mitochondrial ribosomal subunit (37 S). Reconstitution experiments using the 30-S particle and the large mitochondrial ribosomal subunit from a wild-type yeast strain indicate that the 30-S particle is not active in translating the artificial message poly(U). The large mitochondrial ribosomal subunit was present in petite strains retaining the 21-S rRNA gene. The petite 54-S subunit is biologically active in the translation of poly(U) when reconstituted with the small subunit (37 S) from a wild-type strain. The above results indicate that mitochondrial protein synthetic activity is essential for the assembly of the mature small ribosomal subunit, but not for the large subunit. Since the var1 protein is the only mitochondrial translation product known to date to be associated with the mitochondrial ribosomes, the results suggest that this protein is essential for the assembly of the mature small subunit.

Structural Analysis of Nuclear Genes Coding for the Precursor to the Small Subunit of Wheat Ribulose–1,5–Bisphosphate Carboxylase

We have isolated and characterized a full–length cDNA clone encoding the precursor to the small subunit of wheat ribulose–1,5–bisphosphate carboxylase. From the nucleotide sequence, we deduce that the precursor contains the mature small subunit of 128 amino acid residues and an amino–terminal transit sequence of 47 amino acid residues. Southern blot analysis reveals that the small subunit precursor is encoded by a multigene family. One member of the family has been isolated. Nucleotide sequence and R–loop analyses show that it contains only one intron which separates the coding sequence of the transit peptide from that of the mature protein. This gene is expressed in vivo, as shown by mRNA hybridization to a specific probe from its 3′ non–coding region.

Nucleotide sequences of two pea cDNA clones encoding the small subunit of ribulose 1,5-bisphosphate carboxylase and the major chlorophyll a/b-binding thylakoid polypeptide.

Two major chloroplast proteins are encoded by nuclear genes and synthesized on free cytoplasmic ribosomes: the small subunit of ribulose 1,5-bisphosphate carboxylase and the apoprotein components of the chlorophyll a/b light harvesting complex. We have recently reported the isolation of two cDNA clones from pea which encode both the small subunit of ribulose 1,5-bisphosphate carboxylase (pSS15) and the polypeptide 15 (pAB96), the major chlorophyll a/b binding protein (Broglie, R., Bellemare, G., Bartlett, S., Chua, N.-H., and Cashmore, A. R. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 7304-7308). To further characterize these clones, we determined their nucleotide sequence. Clone pSS15 contains a 691-base pair cDNA insert which encodes the entire 123 amino acids of the mature small subunit protein. In addition, this clone also encodes 33 amino acids of the NH2-terminal transit peptide extension and 148 nucleotides of the 3' noncoding region preceding the poly(A)tail. A second cDNA clone (pAB96) contains an 833-nucleotide insert which encodes most of polypeptide 15. The DNA sequence of this cloned cDNA was used to deduce the previously undetermined amino acid sequence of this integral thylakoid membrane protein. The nucleotide sequence of the cDNA clone, pSS15, should provide information concerning the role of the transit sequence in the transport of cytoplasmically synthesized chloroplast proteins. Similarly, the deduced amino acid sequence of polypeptide 15 will provide information for predicting its orientation in thylakoid membranes as well as its role in binding chlorophyll.

Mitochondrial ribosome assembly in Neurospora. Structural analysis of mature and partially assembled ribosomal subunits by equilibrium centrifugation in CsCl gradients.

In Neurospora, one protein associated with the mitochondrial small ribosomal subunit (S-5, Mr 52,000) is synthesized intramitochondrially and is assumed to be encoded by mtDNA. When mitochondrial protein synthesis is inhibited, either by chloramphenicol or by mutation, cells accumulate incomplete mitochondrial small subunits (CAP-30S and INC-30S particles) that are deficient in S-5 and several other proteins. To gain additional insight into the role of S-5 in mitochondrial ribosome assembly, the structures of Neurospora mitochondrial ribosomal subunits, CAP-30S particles, and INC-30S particles were analyzed by equilibrium centrifugation in CsCl gradients containing different concentrations of Mg+2. The results show (a) that S-5 is tightly associated with small ribosomal subunits, as judged by the fact that it is among the last proteins to be dissociated in CsCl gradients as the Mg+2 concentration is decreased, and (b) that CAP-30S and INC-30S particles, which are deficient in S-5, contain at most 12 proteins that are bound as tightly as in mature small subunits. The CAP-30S particles isolated from sucrose gradients contain a number of proteins that appear to be loosely bound, as judged by dissociation of these proteins in CsCl gradients under conditions in which they remain associated with mature small subunits. The results suggest that S-5 is required for the stable binding of a subset of small subunit ribosomal proteins.

Regulation of the synthesis of ribulose-1,5-bisphosphate carboxylase and its subunits in the flagellate Chlorogonium elongatum. Different levels of translatable messenger RNAs for the large and the small subunits in autotrophic and heterotrophic cells as determined by immunological techniques.

Poly(A)-rich and poly(A)-free RNAs were isolated from autotrophic and heterotrophic cells of the phytoflagellate Chlorogonium elongatum and translated in an mRNA-depleted reticulocyte lysate system. Immunoprecipitation methods were improved to detect large and small subunits of the chloroplast enzyme ribulose-1,5-bisphosphate carboxylase synthesized in vitro. Large-subunit polypeptides were shown to be the translation products of poly(A)-free RNA having the same molecular weight as large subunits made in vivo. Small-subunit polypeptides were synthesized when poly(A)-rich RNA was used as a template. They were made in vitro as a precursor, with an Mr about 6000 larger than mature small subunits. Cells growing heterotrophically in the dark with acetate are provided with lower levels of mRNA activities for the large and the small subunits is at least partially controlled by the amounts of translatable mRNAs.

Mitochondrial ribosome assembly in Neurospora. Two-dimensional gel electrophoretic analysis of mitochondrial ribosomal proteins.

Recent results with Neurospora crassa show that one protein (S-5, mol wt 52,000) associated with the mitochondrial (mit) small ribosomal subunit is translated within the mitochondria (Lambowitz et al. 1976. J. Mol. Biol. 107:223-253). In the present work, Neurospora mit ribosomal proteins were analyzed by two-dimensional gel electrophoresis using a modification of the gel system of Mets and Bogorad. The results show that S-5 is present in near stoichiometric concentrations in high salt (0.5 MKCl)-washed mit small subunits from wild-type strains. S-5 is among the most basic mit ribosomal proteins (pI greater than 10) and has a high affinity for RNA under the conditions of the urea-containing gel buffers. The role of S-5 in mit ribosome assembly was investigated by an indirect method, making use of chloramphenicol to specifically inhibit mit protein synthesis. Chloramphenicol was found to rapidly inhibit the assembly of mit small subunits leading to the formation of CAP-30S particles which sediment slightly behind mature small subunits (LaPolla and Lambowitz. 1977. J. Mol. 116: 189-205). Two-dimensional gel analysis shows that the more slowly sedimentaing CAP-30S particles are deficient in S-5 and in several other proteins, whereas these proteins are present in normal concentrations in mature small subunits from the same cells. Because S-5 is the only mit ribosomal protein whose synthesis is directly inhibited by chloramphenicol, the results tentatively suggest that S-5 plays a role in the assembly of mit small subunits. In addition, the results are consistent with the idea that S-5 stabilizes the binding of several other mit small subunit proteins. Two-dimensional gel electrophoresis was used to examine mit ribosomal proteins from [poky] and six additional extra-nuclear mutants with defects in the assembly of mit small subunits. The electrophoretic mobility of S-5 is not detectably altered in any of the mutants. However, [poky] mit small subunits are deficient in S-5 and also contain several other proteins in abnormally low or high concentrations. These and other results are consistent with a defect in a mit ribosomal constituent in [poky].

Mitochondrial ribosome assembly in Neurospora crassa: Chloramphenicol inhibits the maturation of small ribosomal subunits

Recent results suggest that, in Neurospora crassa, one small subunit mitochondrial ribosomal protein (S-4a, M r 52,000) is synthesized intramitochondrially (Lambowitz et al., 1976). We now find that, when wild-type cells are treated with chloramphenicol to block mitochondrial protein synthesis, the maturation of 30 S mitochondrial ribosomal subunits is rapidly inhibited and there is an accumulation of CAP-30 S particles which sediment slightly behind mature small subunits. Electrophoretic analysis suggests that the CAP-30 S particles are deficient in several proteins including S-4a and that they are enriched in a precursor RNA species that is slightly longer than 19 S RNA. Chloramphenicol also appears to inhibit the maturation of 50 S ribosomal subunits, but this effect is much less pronounced. Continued incubation in chloramphenicol leads to a decrease in the proportion of total mitochondrial ribonucleoprotein present as monomers, possibly reflecting the depletion of competent subunits. After long-term (17 h) growth in chloramphenicol, mitochondrial ribosome profiles from wild-type cells show decreased ratios of small to large subunits, a feature which is also characteristic of the poky (mi-1) mutant. Pulse-labeling experiments combined with electrophoretic analysis show that the synthesis of mitochondrial ribosomal RNAs is relatively unaffected by chloramphenicol and that, despite the deficiency of small subunits, 19 S and 25 S RNA are present in normal ratios in whole mitochondria. By contrast, 19 S RNA in poky mitochondria is rapidly degraded leading to a decreased ratio of 19 S to 25 S RNA. The significance of these results with respect to the etiology of the poky mutation is discussed and a model of mitochondrial ribosome assembly that incorporates all available data is presented.