Localization of Five Antibiotic Resistances at the Subunit Level in Chloroplast Ribosomes of Chlamydomonas

The chloroplast ribosomes from five antibiotic resistant strains of Chlamydomonas, each carrying one mutant gene mapping in chloroplast DNA, have been shown to be resistant to the corresponding antibiotic in a poly(U)-directed amino-acid incorporating assay system. The alteration conferring resistance was localized to the 30S subunit in ribosomes from streptomycin, neamine, and spectinomycin resistant strains, and to the 50S subunit in ribosomes from cleocin and carbomycin resistant strains. Spectinomycin resistant ribosomes showed no cross-resistance to any other drugs, but limited cross-resistance was noted with the other mutant ribosomes. The similarity between these findings and results reported by others with bacterial ribosomes supports our hypothesis that at least some chloroplast ribosomal proteins are coded by genes in chloroplast DNA.     

Erythromycin resistance due to a mutation in a ribosomal RNA operon of Escherichia coli.

There are seven ribosomal RNA operons (rrn operons) in Escherichia coli. A single rrn operon was amplified by use of a multicopy recombinant plasmid containing a complete rrnH operon. rrnH thereby has the potential to contribute a greater fraction of the rRNA found in ribosomes. Erythromycin-resistant mutants were isolated from cells containing the plasmid, and at least one mutation to resistance was shown to reside in rrnH on the plasmid. Erythromycin resistance was retained when a major deletion was introduced into the 16S rRNA gene and was abolished by deletions that affect the 16S and 23S rRNA genes but do not alter the 5S rRNA gene or non-rrnH DNA. Cell-free S30 protein-synthesizing extracts from cells containing the mutant plasmid have an increased resistance to erythromycin. The selection procedure used to isolate erythromycin-resistance mutations in rrnH may allow, with minor modifications, the isolation of mutations in rrn operons that change resistance of the ribosome to other antibiotics or that alter other properties of ribosomes.     

Cytoplasmic and chloroplast synthesis of phycobilisome polypeptides

In vivo labeling of eukaryotic phycobilisomes in the presence of inhibitors of translation on 70S and 80S ribosomes demonstrates that some of the polypeptides of this light-harvesting complex are synthesized in the cytoplasm while others are synthesized in the chloroplast. The major pigmented polypeptides, the α and β subunits of the biliproteins (molecular weights between 15,000 and 20,000) and the anchor protein (molecular weight about 90,000) are translated on 70S ribosomes. This suggests that these polypeptides are made within the algal chloroplast. Because the α and β subunits comprise a group of closely related polypeptides, the genes encoding these polypeptides may reside in the plastid genome as a multigene family. Other prominent phycobilisome polypeptides, including a nonpigmented polypeptide that may be involved in maintaining the structural integrity of the complex, are synthesized on cytoplasmic ribosomes. Because the synthesis of phycobilisomes appears to require the expression of genes in two subcellular compartments, this system may be an excellent model for: (i) examining interaction between nuclear and plastid genomes: (ii) elucidating the molecular processes involved in the evolution of plastid genes: (iii) clarifying the events in the synthesis and assembly of macromolecular complexes in the chloroplast.     

Structural and Functional Properties of Ribosomes Crosslinked with Dimethylsuberimidate

To test whether a 30S ribosomal subunitformylmethionyl-tRNA-mRNA complex is an obligatory intermediate in protein synthesis, 70S ribosomes from Escherichia coli were crosslinked with the bifunctional imidoester, dimethylsuberimidate. Crosslinked ribosomes contained covelently joined 30S and 50S subunits, as judged by their inability to dissociate at low Mg(2+) concentrations. Treatment of 70S ribosomes with high salt (1 M NH(4)Cl), either before or after reaction with the crosslinking reagent, produced two different crosslinked ribosomal particles, one of "60 S" and the other "70 S." Preliminary evidence indicates that both particles can bind N-acetylphenylalanyl-tRNA at low Mg(2+) concentrations and are active for polyphenylalanine syntheses.Crosslinked ribosomes were functional when tested with poly(U) as an mRNA in systems requiring initiation factors and N-acetylphenylalanyl-tRNA for activity. Under optimal crosslinking conditions, they retained 80% of the activity of unmodified ribosomes for polyphenylalanine synthesis. Despite the maintenance of these functional capacities, such ribosomes had a sharply reduced ability to bind fMet-tRNA and were completely inactive in protein synthesis with bacteriophage f2 RNA as a messenger. We conclude that 70S ribosomes must dissociate into subunits to initiate protein synthesis with natural mRNAs.     

Cryo-EM study of the spinach chloroplast ribosome reveals the structural and functional roles of plastid-specific ribosomal proteins

Protein synthesis in the chloroplast is carried out by chloroplast ribosomes (chloro-ribosome) and regulated in a light-dependent manner. Chloroplast or plastid ribosomal proteins (PRPs) generally are larger than their bacterial counterparts, and chloro-ribosomes contain additional plastid-specific ribosomal proteins (PSRPs); however, it is unclear to what extent these proteins play structural or regulatory roles during translation. We have obtained a three-dimensional cryo-EM map of the spinach 70S chloro-ribosome, revealing the overall structural organization to be similar to bacterial ribosomes. Fitting of the conserved portions of the x-ray crystallographic structure of the bacterial 70S ribosome into our cryo-EM map of the chloro-ribosome reveals the positions of PRP extensions and the locations of the PSRPs. Surprisingly, PSRP1 binds in the decoding region of the small (30S) ribosomal subunit, in a manner that would preclude the binding of messenger and transfer RNAs to the ribosome, suggesting that PSRP1 is a translation factor rather than a ribosomal protein. PSRP2 and PSRP3 appear to structurally compensate for missing segments of the 16S rRNA within the 30S subunit, whereas PSRP4 occupies a position buried within the head of the 30S subunit. One of the two PSRPs in the large (50S) ribosomal subunit lies near the tRNA exit site. Furthermore, we find a mass of density corresponding to chloro-ribosome recycling factor; domain II of this factor appears to interact with the flexible C-terminal domain of PSRP1. Our study provides evolutionary insights into the structural and functional roles that the PSRPs play during protein synthesis in chloroplasts.     

Interaction of rabbit reticulocyte ribosomes with bacteriophage f1 mRNA and of Escherichia coli ribosomes with rabbit globin mRNA

We have compared the behavior of a prokaryotic mRNA in a eukaryotic ribosome binding system and of a eukaryotic mRNA in a prokaryotic ribosome binding system. Using (32)P- and (125)I-labeled bacteriophage f1 mRNA, we have shown that rabbit reticulocyte 80S ribosomes can protect specific sequences from pancreatic RNase digestion, including those sequences protected by Escherichia coli ribosomes. We have also found that E. coli ribosomes fail to protect any region of (125)I-labeled globin mRNA. Iodination of the mRNA appeared to have little or no effect on the specificity of binding or protection by the ribosomes of either system.The eukaryotic and prokaryotic systems differ markedly in the ability of the small ribosomal subunits to protect mRNA from nuclease digestion. The regions of phage f1 mRNA protected by E. coli 30S subunits are virtually identical to those protected by the 70S ribosomes. By contrast, rabbit reticulocyte 40S subunits protect substantially larger fragments of mRNA from nuclease digestion than do the 80S ribosomes. These 40S-protected fragments are specific in the case of globin mRNA and overlap the shorter region protected by the 80S ribosomes. However, the 40S-protected fragments of phage f1 mRNA were found to be extremely heterogeneous, reflecting perhaps an important difference between the initial interactions made by these two mRNAs with the ribosomes.     

Biogenesis of Mitochondria, XVIII. A New Class of Cytoplasmically Determined Antibiotic Resistant Mutants in Saccharomyces cerevisiae

New mutant yeasts resistant to the antibiotics chloramphenicol and mikamycin were isolated. They are mitochondrial mutants, characterized by several criteria as cytoplasmically determined. Biochemical studies show that amino acid incorporation into protein in vitro by mitochondria isolated from cells resistant or sensitive to mikamycin or chloramphenicol is inhibited by these antibiotics. Although aerobically-grown resistant strains of Saccharomyces cerevisiae are not affected by mikamycin or chloramphenicol, it is found that the mitochondrial protein-synthesizing system of anaerobically grown cells is inhibited in vivo. Cross resistance among the antibiotics chloramphenicol, mikamycin, erythromycin, lincomycin, carbomycin, and spiramycin is reported. All erythromycin resistant mutants, unlike the others, are resistant to erythromycin in vivo and in vitro. The results indicate that some of the cytoplasmic mutations (mikamycin and chloramphenicol resistance) are expressed at the mitochondrial membrane, whereas others (erythromycin resistance) possibly reflect changes in mitochondrial ribosomal proteins. We further suggest that conformational changes, either in the membranes or ribosomes, are likely to account for the observed antibiotic cross resistances.     

MUTANTS IN Escherichia coli RIBOSOMES: A NEW SELECTION

Only a few ribosome genes have been identified. Increasing the available kinds of ribosome mutants would thus facilitate the analysis of specification and function of the organelle. In an attempt to reveal a number of ribosome genes, we sought mutants that could not form functional ribosomes at a nonpermissive temperature (43 degrees ). Cells were depleted of ribosomes by nutrient starvation at 43 degrees , and 98 strains that could not recover from the starvation were then isolated. Of these 98, many satisfied auxiliary criteria for ribosome mutants; 23 showed increased sensitivity to antibiotics that affect ribosome function; and 8 of 20 representative strains had crude extracts more temperature sensitive than that of the parental strain when assayed for polyphenylalanine formation in the presence of excess soluble enzymes (S100) of the parental strain. In further tests with two of these strains, the temperature sensitivity of the washed ribosomes, compared to that of the parental strain, was confirmed.The variety of phenotypes among the mutants suggests that this selection is capable of revealing mutants in many ribosome genes.     

FUNCTIONAL INTERDEPENDENCE OF RIBOSOMAL COMPONENTS OF Escherichia coli

Ribosomes are made up of parts that interact so strongly that a mutation in one of them can mask the effect of mutation in another. For example, when a mutation to neomycin resistance, which is a ribosome mutation, is introduced into cells carrying a ribosome mutation to spectinomycin resistance, some of the doubly mutant strains were phenotypically sensitive to spectinomycin, even though the mutation to spectinomycin resistance is still intact and recoverable in appropriate crosses. The neomycin mutant alleles that cause masking were shown by genetic tests to be in an identified locus that affects ribosomes. Protein synthesis in cell-free extracts of a double mutant strain was as sensitive to the action of spectinomycin as was the extract of the doubly sensitive parental strain. Thus, the masking effect of neomycin mutations on the spectinomycin mutation is exerted at the level of the ribosomes.We conclude that the genetic analysis of an organelle like the ribosome is likely to be severely complicated by pleiotropic effects and by interactions among its component parts and that the ribosomal binding sites for spectinomycin and neomycin are partially interdependent. These results suggest that the function of the ribosome requires a very precise conformation of all its elements, some of which are interdependent. Modification in one element can thus alter the function of another or even render the entire structure nonfunctional.     

A protein residing at the subunit interface of the bacterial ribosome.

Surface labeling of Escherichia coli ribosomes with the use of the tritium bombardment technique has revealed a minor unidentified ribosome-bound protein (spot Y) that is hidden in the 70S ribosome and becomes highly labeled on dissociation of the 70S ribosome into subunits. In the present work, the N-terminal sequence of the protein Y was determined and its gene was identified as yfia, an ORF located upstream the phe operon of E. coli. This 12.7-kDa protein was isolated and characterized. An affinity of the purified protein Y for the 30S subunit, but not for the 50S ribosomal subunit, was shown. The protein proved to be exposed on the surface of the 30S subunit. The attachment of the 50S subunit resulted in hiding the protein Y, thus suggesting the protein location at the subunit interface in the 70S ribosome. The protein was shown to stabilize ribosomes against dissociation. The possible role of the protein Y as ribosome association factor in translation is discussed.     

Biogenic membranes of the chloroplast in Chlamydomonas reinhardtii

The polypeptide subunits of the photosynthetic electron transport complexes in plants and algae are encoded by two genomes. Nuclear genome-encoded subunits are synthesized in the cytoplasm by 80S ribosomes, imported across the chloroplast envelope, and assembled with the subunits that are encoded by the plastid genome. Plastid genome-encoded subunits are synthesized by 70S chloroplast ribosomes directly into membranes that are widely believed to belong to the photosynthetic thylakoid vesicles. However, in situ evidence suggested that subunits of photosystem II are synthesized in specific regions within the chloroplast and cytoplasm of Chlamydomonas. Our results provide biochemical and in situ evidence of biogenic membranes that are localized to these translation zones. A “chloroplast translation membrane” is bound by the translation machinery and appears to be privileged for the synthesis of polypeptides encoded by the plastid genome. Membrane domains of the chloroplast envelope are located adjacent to the cytoplasmic translation zone and enriched in the translocons of the outer and inner chloroplast envelope membranes protein import complexes, suggesting a coordination of protein synthesis and import. Our findings contribute to a current realization that biogenic processes are compartmentalized within organelles and bacteria.     

Purification, primary structure, and homology relationships of a chloroplast ribosomal protein

A chloroplast ribosomal protein that showed immunological homology to Escherichia coli ribosomal protein L12 was purified from spinach (Spinacia oleracea) leaves and its primary structure was determined by manual micro Edman degradation. The protein is composed of 130 amino acid residues and has M_r 13,576. It shows structural features characteristic of the L12 proteins of eubacterial 70S ribosomes (e.g., identical amino acid residues in about 50% of the sequence) but no apparent homology to the L12-type proteins of eukaryotic cytoplasmic 80S ribosomes. The homology to eubacterial proteins is highest in the COOH-terminal region (70%) and low in the NH₂-terminal region (<20%).     

Differential Effects of Chronic Ethanol Consumption on Hepatic Mitochondrial and Cytoplasmic Ribosomes

The effects of chronic ethanol consumption on the properties of mitochondrial and cytoplasmic ribosomes were investigated in rat liver. Sedimentation properties of purified mitochondrial (55s) and cytoplasmic (80s) ribosomes were determined by analyses on sucrose density gradients. Mitochondrial ribosomes from control animals moved further in the gradients than did those isolated from ethanol-fed rats, which suggests that ethanol ribosomes have a lower molecular weight. In addition, mitochondria from ethanol-fed animals contained a lower percentage of ribosomes present as the intact monosome, suggesting that ethanol may have an effect on the stability of the functional mitochondrial ribosomes. This was confirmed by the presence of the larger 39s subunit in preparations from ethanol-fed animals. No such ethanol-related alterations were seen with cytoplasmic ribosomes. The protein composition of mitochondrial and cytoplasmic ribosomes was investigated using two-dimensional gel electrophoresis, followed by two-dimensional densitometry. As indicated by differences in protein staining intensity, ethanol consumption seemed to alter the concentration of seven mitochondrial ribosomal proteins. In contrast, no such changes were observed in the protein pattern from cytoplasmic ribosomes. Observations in this study provide for the possibility that alterations in the amounts of selected proteins in the mitochondrial ribosome lead to impaired assembly of the ribosome. These ethanol-related structural changes may be responsible for the decreased activity of mitochondrial ribosomes that results in impaired hepatic mitochondrial protein synthesis (W. B. Coleman and C. C. Cunningham, Biochim. Bio-phys. Acta 1058:178–186, 1991). Furthermore, this study re-emphasizes the increased susceptibility of the hepatic mitochondrial translation system, compared with the cytoplasmic system to chronic ethanol consumption.     

Structure and probable genetic location of a "ribosome modulation factor" associated with 100S ribosomes in stationary-phase Escherichia coli cells.

The decrease in overall translation activity occurring concomitantly with the transition from the exponential growth phase to the stationary phase of Escherichia coli cells was found to be accompanied by the appearance of 100S ribosomes (dimers of 70S ribosome monomers). Analysis of ribosomal proteins by the radical-free and highly reducing method of two-dimensional gel electrophoresis indicated that a protein, designated protein E, was exclusively associated with 100S ribosomes. From the results, we propose that protein E is a "ribosome modulation factor" (RMF), which associates with 70S ribosomes and converts them to a dimeric form. A homology search of the partial amino acid sequence of RMF using the DNA sequence data bases revealed that the rmf gene, which encodes RMF, is located next to the fabA gene at 21.8 min on the E. coli chromosome.     

Can a non-Mendelian mutation affect both chloroplast and mithchondrial ribosomes?

Chloroplast ribosomes isolated from a spectinomycin-resistant mutant (spr-1-27-3) of Chlamydomonas reinhardtii that displays non-Mendelian inheritance fail to bind labeled antibiotic, in contrast to ribosomes from wild-type cells. In vitro resistance of this mutant appears to result from the absence of a specific protein in the small subunit of the chloroplast ribosome. However, chloroplast protein synthesis in the mutant and wild type shows identical sensitivity to spectinomycin in short-term in vivo experiments where ribulosediphosphate carboxylase serves as the marker. Long-term experiments demonstrate that the mutant can grow in the presence of spectinomycin only when acetate is supplied as a carbon source. Mitochondrial structure and function of the mutant are not affected by the antibiotic, whereas chloroplast structure and function are. Apparently, the mitochondrion, rather than the chloroplast, of this mutant is resistant to spectinomycin in vivo. We hypothesize that the gene product of the spr locus is a protein common to both chloroplast and mitochondrial ribosomes. The mutant gene product, in vivo, confers resistance on mitochondrial, but not chloroplast, ribosomes. We suppose that the mutant spr protein loosely attaches to chloroplast ribosomes in vivo so that the antibiotic is bound and blocks protein synthesis, but it dissociates during isolation, resulting in loss of the binding site.     

Identification of a soluble protein that stimulates peptide bond synthesis.

A soluble protein factor was isolated, free of elongation factor (EF)-T and EF-G, based on its ability to stimulate the synthesis of peptide bonds using ribosomal bound 70S-AUG-N-formyl-[35S]methionyl-tRNA complex and added puromycin as substrates. Over 90% of this activity was found in the ribosome-free cytoplasm of Escherichia coli extracts. Otherfeatures such as molecular weight, purification properties, and catalytic activities distinguish this factor from ribosomal proteins and known activators of translation. The factor requires all components needed for peptide bond synthesis and is inhibited by antibiotics known to specifically block the peptidyl transferase activity of ribosomes. The factor increases the binding affinity of the ribosome for the aminoacyl-tRNA analog puromycin about 10-fold. We suggest that this extraribosomal factor modulates the intrinsic activity of ribosomes to catalyze peptide-bond synthesis, and regard it as a new factor required for peptide chain elongation, which we call EF-P.     

Elongation factor Tu resistant to kirromycin in an Esherichia coli mutant altered in both tuf genes

A mutant of Escherichia coli is described that displays kirromycin resistance in a cell-free system by virtue of an altered elongation factor Tu (EF-Tu). In poly(U)-directed poly(Phe) synthesis the kirromycin resistance of the crystallized enzyme ranged between a factor of 80 and 700, depending on temperature. Similarly, kirromycin-induced EF-Tu GTPase activity uncoupled from ribosomes and aminoacyl-tRNA required correspondingly higher concentrations of the antibiotic. Resistance of EF-Tu to kirromycin is a consequence of a modified enzyme structure as indicated by its altered fingerprint pattern.P1 transduction experiments showed that the kirromycin-resistant EF-Tu is coded by an altered tufB gene (tufB1). The known existence of two genes coding for EF-Tu would interfere with the recognition of a mutant altered in only one of those genes, if the mutation were recessive. Because kirromycin blocks EF-Tu release from the ribosome, kirromycin sensitivity is dominant, as shown by the failure of a mixed EF-Tu population to express resistance in vitro. Therefore, phenotypic expression of kirromycin resistance in vivo appears to be only possible if the EF-Tu mutant lacks an active tufA gene, a property likely to be inherited from the parental D22 strain. The observations that introduction of a tufA(+) region makes the resistant strain sensitive to the antibiotic and that transduction of tufB1 into a recipient other than E. coli D22 yields kirromycin-sensitive progeny support these conclusions.     

Localization of the Stringent Protein of Escherichia coli on the 50S Ribosomal Subunit

The "stringent" protein of the ribosome, required for its synthesis of (p)ppGpp, is readily lost during zonal centrifugation. However, enough can be retained to permit its qualitative localization. It is found in native 50S subunits, runoff 70S ribosomes, and polysomes, but not in native 30S subunits. This protein, therefore, appears to be attached to the 50S moiety of the ribosome, and it may be a constant (though easily removed) component of that structure rather than a factor that joins and leaves during the ribosome cycle.     

23S rRNA base pair 2057-2611 determines ketolide susceptibility and fitness cost of the macrolide resistance mutation 2058A-->G

The 23S rRNA A2058G alteration mediates macrolide, lincosamide, and streptogramin B resistance in the bacterial domain and determines the selectivity of macrolide antibiotics for eubacterial ribosomes, as opposed to eukaryotic ribosomes. However, this mutation is associated with a disparate resistance phenotype: It confers high-level resistance to ketolides in mycobacteria but only marginally affects ketolide susceptibility in streptococci. We used site-directed mutagenesis of nucleotides within domain V of 23S rRNA to study the molecular basis for this disparity. We show that mutational alteration of the polymorphic 2057-2611 base pair from A-U to G-C in isogenic mutants of Mycobacterium smegmatis significantly affects susceptibility to ketolides but does not influence susceptibility to other macrolide antibiotics. In addition, we provide evidence that the 2057-2611 polymorphism determines the fitness cost of the 23S rRNA A2058G resistance mutation. Supported by structural analysis, our results indicate that polymorphic nucleotides mediate the disparate phenotype of genotypically identical resistance mutations and provide an explanation for the large species differences in the epidemiology of defined drug resistance mutations.     

Mutations Altering Chloroplast Ribosome Phenotype in Chlamydomonas, II. A New Mendelian Mutation

A new mutation of Chlamydomonas reinhardi, cr-1, is characterized. The mutation exhibits Mendelian inheritance and affects the sedimentation velocity and formation of intact chloroplast ribosomes. The mutant grows reasonably well when supplied with sodium acetate as a carbon source, but poorly when forced to grow photosynthetically using carbon dioxide. Since the mutant cr-1 accumulates large subunits of the chloroplast ribosome, we postulate that it is blocked in the formation of the small subunit. A tentative model explaining the behavior of the several mutants in Chlamydomonas now known to have altered chloroplast ribosomal phenotypes is presented.