Depletion of free 30S ribosomal subunits in Escherichia coli by expression of RNA containing Shine-Dalgarno-like sequences.

We have constructed synthetic coding sequences for the expression of poly(alpha,L-glutamic acid) (PLGA) as fusion proteins with dihydrofolate reductase (DHFR) in Escherichia coli. These PLGA coding sequences use both GAA and GAG codons for glutamic acid and contain sequence elements (5'-GAGGAGG-3') that resemble the consensus Shine-Dalgarno (SD) sequence found at translation initiation sites in bacterial mRNAs. An unusual feature of DHFR-PLGA expression is that accumulation of the protein is inversely related to the level of induction of its mRNA. Cellular protein synthesis was inhibited >95% by induction of constructs for either translatable or untranslatable PLGA RNAs. Induction of PLGA RNA resulted in the depletion of free 30S ribosomal subunits and the appearance of new complexes in the polyribosome region of the gradient. Unlike normal polyribosomes, these complexes were resistant to breakdown in the presence of puromycin. The novel complexes contained 16S rRNA, 23S rRNA, and PLGA RNA. We conclude that multiple noninitiator SD-like sequences in the PLGA RNA inhibit cellular protein synthesis by sequestering 30S small ribosomal subunits and 70S ribosomes in nonfunctional complexes on the PLGA mRNA.     

Puromycin photoaffinity labels small- and large-subunit proteins at the A site of the Drosophila ribosome

[3H]Puromycin covalently incorporates into the protein and to a much lesser extent into the RNA components of Drosophila ribosomes in the presence of 254-nm light. The photoincorporation reaction takes place with a small number of large- (L2 and L17) and small- (S8 and S22) subunit proteins as determined by two-dimensional gel analysis. More quantitative one-dimensional gel results show that puromycin reacts with each of these proteins in a functional site specific manner. The small percentage of the total labeling that occurs with rRNA also appears to be site specific. The rRNA labeling arises from a puromycin-mediated cross-linking of ribosomal protein and rRNA. Ionic conditions shift the pattern of puromycin-labeled ribosomal proteins. These results suggest that puromycin can occupy two distinct sites on Drosophila 80S ribosomes. The pattern of ribosomal proteins labeled by puromycin is affected by the presence of other antibiotics such as emetine, anisomycin, and trichodermin.     

Do ribosomal RNAs act merely as scaffold for ribosomal proteins?

Investigations that are being carried out in various laboratories including ours clearly provide the answer which is in the negative. Only the direct evidences obtained in this laboratory will be presented and discussed. It has been unequivocally shown that the interaction between 16S and 23S RNAs plays the primary role in the association of ribosomal subunits. Further, 23S RNA is responsible for the Binding of 5S RNA to 16S.23S RNA complex with the help of three ribosomal proteins, L5, L18, L15/L25. The 16S.23S RNA complex is also capable of carrying out the following ribosomal functions, although to small but significant extents, with the help of a very limited number of ribosomal proteins and the factors involved in protein synthesis: (a) poly U-Binding, (B) poly U-dependent Binding of phenylalanyl tRNA, (c) EF-G-dependent GTPase activity, (d) initiation complex formation, (e) peptidyl transferase activity (puromycin reaction) and (f) polyphenylalanine synthesis. These results clearly indicate the direct involvement of rRNAs in the various steps of protein synthesis. Very recently it has Been demonstrated that the conformational change of 23S RNA is responsible for the translocation of peptidyl tRNA from the aminoacyl (A) site to the peptidyl (P) site. A model has Been proposed for translocation on the Basis of direct experimental evidences. The new concept that ribosomal RNAs are the functional components in ribosomes and proteins act as control switches may eventually turn out to Be noncontroversial.     

Ribosomal activity of the 16 S.23 S RNA complex

It has been demonstrated in this laboratory that 16 S and 23 S RNAs form a binary complex like 30 S and 50 S ribosomes under certain specific conditions, and 5 S RNA can be incorporated into the complex in stoichiometric amounts in presence of three ribosomal proteins, L5, L18, and L15/25. These studies raised the basic question of whether such complex will have biological activity. Therefore, the following steps in protein synthesis were examined with the complex in place of the ribosomes: (i) poly-U-dependent binding of phenylalanyl tRNA; (ii) EF-G-dependent GTPase activity; (iii) initiation complex formation; (iv) peptidyl transferase activity; and (v) poly-U-dependent polyphenylalanine synthesis. All the steps could be unequivocally demonstrated by the addition of a limited number of proteins although the complex had comparatively much less activity than 70 S ribosomes. It appears that rRNAs are directly involved in various steps of protein synthesis. Furthermore, the 16 S.23 S RNA complex might have acted as a primitive ribosome, as suggested by Crick and Orgel.     

RIBOSOME-MEMBRANE INTERACTION : Nondestructive Disassembly of Rat Liver Rough Microsomes into Ribosomal and Membranous Components

In a medium of high ionic strength, rat liver rough microsomes can be nondestructively disassembled into ribosomes and stripped membranes if nascent polypeptides are discharged from the bound ribosomes by reaction with puromycin. At 750 mM KCl, 5 mM MgCl₂, 50 mM Tris·HCl, pH 7 5, up to 85% of all bound ribosomes are released from the membranes after incubation at room temperature with 1 mM puromycin. The ribosomes are released as subunits which are active in peptide synthesis if programmed with polyuridylic acid. The ribosome-denuded, or stripped, rough microsomes (RM) can be recovered as intact, essentially unaltered membranous vesicles Judging from the incorporation of [³H]puromycin into hot acid-insoluble material and from the release of [³H]leucine-labeled nascent polypeptide chains from bound ribosomes, puromycin coupling occurs almost as well at low (25–100 mM) as at high (500–1000 mM) KCl concentrations. Since puromycin-dependent ribosome release only occurs at high ionic strength, it appears that ribosomes are bound to membranes via two types of interactions: a direct one between the membrane and the large ribosomal subunit (labile at high KCl concentration) and an indirect one in which the nascent chain anchors the ribosome to the membrane (puromycin labile). The nascent chains of ribosomes specifically released by puromycin remain tightly associated with the stripped membranes. Some membrane-bound ribosomes (up to 40%) can be nondestructively released in high ionic strength media without puromycin; these appear to consist of a mixture of inactive ribosomes and ribosomes containing relatively short nascent chains. A fraction (~15%) of the bound ribosomes can only be released from membranes by exposure of RM to ionic conditions which cause extensive unfolding of ribosomal subunits, the nature and significance of these ribosomes is not clear.     

30S ribosomal subunits with fragmented 16S RNA: a new approach for structure and function study of ribosomes.

A new approach for function and structure study of ribosomes based on oligodeoxyribonucleotide-directed cleavage of rRNA with RNase H and subsequent reconstitution of ribosomal subunits from fragmented RNA has been developed. The E coli 16S rRNA was cleaved at 9 regions belonging to different RNA domains. The deletion of 2 large regions was also produced by cleaving 16S rRNA in the presence of 2 or 3 oligonucleotides complementary to different RNA sites. Fragmented and deleted RNA were shown to be efficiently assembled with total ribosomal protein into 30S-like particles. The capacity to form 70S ribosomes and translate both synthetic and natural mRNA of 30S subunits reconstituted from intact and fragmented 16S mRNA was compared. All 30S subunits assembled with fragmented 16S rRNA revealed very different activity: the fragmentation of RNA at the 781-800 and 1392-1408 regions led to the complete inactivation of ribosomes, whereas the RNA fragmentation at the regions 296-305, 913-925, 990-998, 1043-1049, 1207-1215, 1499-1506, 1530-1539 did not significantly influence the ribosome protein synthesis activity, although it was also reduced. These findings are mainly in accordance with the data on the functional activity of some 16S rRNA sites obtained by other methods. The relations between different 16S RNA functional sites are discussed.     

Unbalanced rRNA gene dosage and its effects on rRNA and ribosomal-protein synthesis.

The synthesis of rRNA was unbalanced by the introduction of plasmids containing rRNA operons with large internal deletions. Significant unbalanced synthesis was achieved only when the deletions affected both 16S and 23S RNA genes or when the deletions affected the 23S RNA gene alone. Although large imbalances in rRNA synthesis resulted from deletions affecting 16S and 23S RNA genes or only 23S RNA genes, excess 16S RNA and defective rRNA species were rapidly degraded. Large imbalances in the synthesis of regions of rRNA did not result in significantly unbalanced synthesis of ribosomal proteins. It therefore is probable that excess intact 16S RNA is degraded because ribosomal proteins are not available for packaging the RNA into ribosomes. Defective RNA species also may be degraded for this reason or because proper ribosome assembly is prevented by the defects in RNA structure. We propose two possible explanations for the finding that unbalanced overproduction of binding sites for feedback ribosomal protein does not result in significant unbalanced translational feedback depression of ribosomal protein mRNAs.     

The Synthesis of Ribosomes in E. coli: IV. The Synthesis of Ribosomal Protein and the Assembly of Ribosomes

The incorporation of C¹⁴ leucine into the protein moiety of ribosomes has been studied as a sequel to the studies of ribosomal RNA synthesis. In contrast to the latter studies, labeled leucine is incorporated directly into 50S and 30S ribosomes without measurable delay by precursor stages. There is, however, evidence of some transfer of radioactivity from the 43S group of particles to the 50S. The inhibition of protein synthesis by chloramphenicol results in the accumulation of material similar to the eosome—the primary precursor in ribosome synthesis. There is also evidence for the synthesis of some neosome. The results of the studies of ribosomal RNA and protein synthesis are combined into a model of ribosome synthesis. Finally, consideration is made of the significance of these studies of ribosome synthesis for general problems of protein synthesis and information transfer.     

NUCLEOLAR NECKLACES IN CHICK EMBRYO MYOBLASTS FORMED BY LACK OF ARGININE

The round nucleoli of chick embryo myoblasts, when grown in a culture medium devoid of arginine, unravel in several days into 5–20 µ long, beaded strands termed nucleolar necklaces (NN). Addition of arginine reverses this change. The NN contain protein, RNA, and traces of DNA as determined cytochemically by enzyme digestion and by acridine-orange fluorescent staining. When a cell containing the beaded strand is treated with agents, such as actinomycin D, that prevent rRNA polymerase action, the strand collapses and condenses into a small dense nucleolus with segregated regions of ribonucleoprotein (RNP) and deoxyribonucleoprotein (DNP). The properties of the NN appear to resemble those of the nucleolar necklaces of amphibian oocytes. Cycloheximide or puromycin inhibition of general protein synthesis does not lead to NN formation. We suggest that NN formation during arginine starvation may be a result of a singular depletion of some rapidly turning over, arginine-rich proteins that normally attach to ribosomal RNA precursor molecules during their synthesis in the processing towards maturation of the ribosomes.     

Functional Inactivation and Appearance of Breaks in RNA Chains Caused by Gamma Irradiation of Escherichia coli Ribosomes

70S ribosomes and 30S ribosomal subunits from Escherichia coli MRE 600 were exposed to gamma irradiation at -80szC. Exponential decline of activity with dose was observed when the ability of ribosomes to support the synthesis of polyphenylalanine was assayed. Irradiated ribosomes showed also an increased thermal lability. D₃₇ values of 2.2 MR and 4.8 MR, corresponding to radiation-sensitive molecular weights of 3.1 × 10⁵ and 1.4 × 10⁵, were determined for inactivation of 70S ribosomes and 30S subunits, respectively. Zone sedimentation analysis of RNA isolated from irradiated bacteria or 30S ribosomal subunits showed that at average, one chain scission occurs per four hits into ribosomal RNA. From these results it was concluded that the integrity of only a part of ribosomal proteins (the sum of their molecular weights not exceeding 1.4 × 10⁵) could be essential for the function of the 30S subunit in the polymerization of phenylalanine. This amount is smaller if the breaks in the RNA chain inactivate the ribosome.     

The binding of ribosomal subunits to endoplasmic reticulum membranes

The binding of ribosomes and ribosomal subunits to endoplasmic reticulum preparations of mouse liver was studied. (1) Membranes prepared from rough endoplasmic reticulum by preincubation with 0.5m-KCl and puromycin bound 60-80% of added 60S subunits and 10-15% of added 40S subunits. Membranes prepared with pyrophosphate and citrate showed less clear specificity for 60S subunits particularly when assayed at low ionic strengths. (2) Ribosomal 40S subunits bound efficiently to membranes only in the presence of 60S subunits. The reconstituted membrane-60S subunit-40S subunit complex was active in synthesis of peptide bonds. (3) No differences in binding to membranes were seen between subunits derived from free and from membrane-bound ribosomes. (4) It is concluded that the binding of ribosomes to membranes does not require that they be translating a messenger RNA, and that the mechanism whereby bound and free ribosomes synthesize different groups of proteins does not depend on two groups of ribosomes that differ in their ability to bind to endoplasmic reticulum.     

Crosslinking of ribosomal proteins to RNA in maize ribosomes by UV-B and its effects on translation

Ultraviolet-B (UV-B) photons can cause substantial cellular damage in biomolecules, as is well established for DNA. Because RNA has the same absorption spectrum for UV as DNA, we have investigated damage to this cellular constituent. In maize (Zea mays) leaves, UV-B radiation damages ribosomes by crosslinking cytosolic ribosomal proteins S14, L23a, and L32, and chloroplast ribosomal protein L29 to RNA. Ribosomal damage accumulated during a day of UV-B exposure correlated with a progressive decrease in new protein production; however, de novo synthesis of some ribosomal proteins is increased after 6 h of UV-B exposure. After 16 h without UV-B, damaged ribosomes were eliminated and translation was restored to normal levels. Ribosomal protein S6 and an S6 kinase are phosphorylated during UV-B exposure; these modifications are associated with selective translation of some ribosomal proteins after ribosome damage in mammalian fibroblast cells and may be an adaptation in maize. Neither photosynthesis nor pigment levels were affected significantly by UV-B, demonstrating that the treatment applied is not lethal and that maize leaf physiology readily recovers.     

The 60S ribosomal subunit is altered in the skeletal muscle of dystrophic hamsters

Polysomes from the skeletal muscle of normal and dystrophic hamsters were dissociated into ribosomal subunits by treatment with puromycin and the subunits from both strains were reassociated in all possible combinations. When their protein synthesis activity was assayed in a poly(U)-directed cell-free system at a low magnesium concentration, the reassociated ribosomes from dystrophic hamsters were less active than the ribosomes from control animals. The ribosomal defect is a property of the 60S subunit and is due to a ribosomal component rather than to abnormal binding of a non-ribosomal protein.     

Active regulator of SIRT1 is required for ribosome biogenesis and function

Active regulator of SIRT1 (AROS) binds and upregulates SIRT1, an NAD⁺-dependent deacetylase. In addition, AROS binds RPS19, a structural ribosomal protein, which also functions in ribosome biogenesis and is implicated in multiple disease states. The significance of AROS in relation to ribosome biogenesis and function is unknown. Using human cells, we now show that AROS localizes to (i) the nucleolus and (ii) cytoplasmic ribosomes. Co-localization with nucleolar proteins was verified by confocal immunofluorescence of endogenous protein and confirmed by AROS depletion using RNAi. AROS association with cytoplasmic ribosomes was analysed by sucrose density fractionation and immunoprecipitation, revealing that AROS selectively associates with 40S ribosomal subunits and also with polysomes. RNAi-mediated depletion of AROS leads to deficient ribosome biogenesis with aberrant precursor ribosomal RNA processing, reduced 40S subunit ribosomal RNA and 40S ribosomal proteins (including RPS19). Together, this results in a reduction in 40S subunits and translating polysomes, correlating with reduced overall cellular protein synthesis. Interestingly, knockdown of AROS also results in a functionally significant increase in eIF2α phosphorylation. Overall, our results identify AROS as a factor with a role in both ribosome biogenesis and ribosomal function.     

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.     

THE SELECTIVE INTERRUPTION OF NUCLEOLAR RNA SYNTHESIS IN HELA CELLS BY CORDYCEPIN

Cordycepin is an analogue of adenosine lacking the 3'-OH. When incorporated into a growing RNA molecule, cordycepin prevents further elongation, thus producing a prematurely terminated RNA molecule. When HeLa cells are exposed to low concentrations of cordycepin, DNA and protein synthesis are unaffected during short exposure periods. The synthesis of completed ribosomal and ribosomal-precursor (45S) RNA is significantly depressed. Partially completed 45S ribosomal precursor molecules accumulate in the nucleolus. 18S ribosomal RNA can be cleaved from these incomplete precursors, while 32S ribosomal precursor cannot be produced from partially snythesized 45S molecules. The synthesis of transfer RNA is also reduced in the presence of cordycepin. The synthesis of the nuclear heterogeneous RNA species is unaffected by the drug while the cytoplasmic heterogeneous RNA is slightly reduced.     

Impact of P-Site tRNA and Antibiotics on Ribosome Mediated Protein Folding: Studies Using the Escherichia coli Ribosome

Background

The ribosome, which acts as a platform for mRNA encoded polypeptide synthesis, is also capable of assisting in folding of polypeptide chains. The peptidyl transferase center (PTC) that catalyzes peptide bond formation resides in the domain V of the 23S rRNA of the bacterial ribosome. Proper positioning of the 3′ –CCA ends of the A- and P-site tRNAs via specific interactions with the nucleotides of the PTC are crucial for peptidyl transferase activity. This RNA domain is also the center for ribosomal chaperoning activity. The unfolded polypeptide chains interact with the specific nucleotides of the PTC and are released in a folding competent form. In vitro transcribed RNA corresponding to this domain (bDV RNA) also displays chaperoning activity.

Results

The present study explores the effects of tRNAs, antibiotics that are A- and P-site PTC substrate analogs (puromycin and blasticidin) and macrolide antibiotics (erythromycin and josamycin) on the chaperoning ability of the E. coli ribosome and bDV RNA. Our studies using mRNA programmed ribosomes show that a tRNA positioned at the P-site effectively inhibits the ribosome''s chaperoning function. We also show that the antibiotic blasticidin (that mimics the interaction between 3′–CCA end of P/P-site tRNA with the PTC) is more effective in inhibiting ribosome and bDV RNA chaperoning ability than either puromycin or the macrolide antibiotics. Mutational studies of the bDV RNA could identify the nucleotides U2585 and G2252 (both of which interact with P-site tRNA) to be important for its chaperoning ability.

Conclusion

Both protein synthesis and their proper folding are crucial for maintenance of a functional cellular proteome. The PTC of the ribosome is attributed with both these abilities. The silencing of the chaperoning ability of the ribosome in the presence of P-site bound tRNA might be a way to segregate these two important functions.     

Crosslinking of Escherichia coli 50S ribosomal subunits with chlorambucilyl oligoprolyl phenylalanyl-tRNA molecular rulers.

A series of P-site probes, chlorambucilyl-(Pro)n-Phe-tRNAPhe, were prepared and reacted with poly(U)-directed Escherichia coli MRE 600 ribosomes. Upon binding of the probes to ribosomes, 90% of the cpm bound were not released following subsequent interaction with puromycin. In the absence of poly(U) or in the presence of poly(C), binding was limited to the amount of cpm bound if ribosomes were incubated in the presence of puromycin before adding modified tRNA and poly(U). AcPhe-tRNAPhe was a competitive inhibitor of chlorambucilyl Phe-tRNAPhe. Binding to 50S subunits was strongly stimulated by poly(U), while binding to 30S subunits was not. Crosslinked 50S proteins were analyzed by two-dimensional gel electrophoresis. Crosslinking with molecular rulers containing zero prolines led to poly(U)-dependent labeling of L1 and L27. With rulers containing five prolines, L6, L25, L28, and the group L18,23,24 were labeled. Analysis of crosslinked ribosomal RNA on sucrose density gradients revealed almost no cpm in the 16S or 23S peaks, but only in the 5S peaks. This was observed with molecular rulers containing either zero or five proline residues.     

The identification of spermine binding sites in 16S rRNA allows interpretation of the spermine effect on ribosomal 30S subunit functions

A photoreactive analogue of spermine, N¹-azidobenzamidino (ABA)-spermine, was covalently attached after irradiation to Escherichia coli 30S ribosomal subunits or naked 16S rRNA. By means of RNase H digestion and primer extension, the cross-linking sites of ABA-spermine in naked 16S rRNA were characterised and compared with those identified in 30S subunits. The 5′ domain, the internal and terminal loops of helix H24, as well as the upper part of helix H44 in naked 16S rRNA, were found to be preferable binding sites for polyamines. Association of 16S rRNA with ribosomal proteins facilitated its interaction with photoprobe, except for 530 stem–loop nt, whose modification by ABA-spermine was abolished. Association of 30S with 50S subunits, poly(U) and AcPhe-tRNA (complex C) further altered the susceptibility of ABA-spermine cross-linking to 16S rRNA. Complex C, modified in its 30S subunit by ABA-spermine, reacted with puromycin similarly to non-photolabelled complex. On the contrary, poly(U)-programmed 70S ribosomes reconstituted from photolabelled 30S subunits and untreated 50S subunits bound AcPhe-tRNA more efficiently than untreated ribosomes, but were less able to recognise and reject near cognate aminoacyl-tRNA. The above can be interpreted in terms of conformational changes in 16S rRNA, induced by the incorporation of ABA-spermine.     

EFFECT OF TEMPERATURE OF ALDEHYDE FIXATION ON THE RADIOAUTOGRAPHIC LOCALIZATION OF RIBONUCLEOPROTEIN IN NUCLEOLI OF HELA CELLS : Inhibition by Puromycin and Actinomycin D

In efforts to clarify the role of the nucleolus and substructures thereof in the assembly or synthesis of protein associated with formation of the complete ribosome, the effect of variation of some conditions of aldehyde fixation on the intranuclear distribution of lysine-³H, arginine-³H, and uridine-³H was studied by differential grain count in radioautographs of PPLO-free HeLa cells. It was found that the nucleolus is a site of rapid assembly or synthesis of a protein, the synthesis of which is inhibited equally by puromycin (200 µg/ml) and by actinomycin D under conditions inhibitory for ribosomal precursor RNA synthesis (P < 0.01). This protein is fixed by phosphate-buffered formalin or glutaraldehyde at pH 7.3, but the label is diminished by fixation in customarily employed acetic ethanol or in formalin at acid pH. Elevation of temperature of formalin or glutaraldehyde fixatives to 37°C consistently reduces the nucleolar protein label, but not the RNA label, by a proportion identical with that incurred by puromycin or actinomycin inhibition. This proportional reduction of nucleolar protein label occurs without evident loss of total grain count and is independent of length of fixation between 30 min and 4 hr, but it is not observed at 23°C. The data support the interpretation that the proportion of nucleolar protein not fixed at 37°C is associated with nucleolar ribosomal RNA but that it is dissociated at 37°C in formalin or glutaraldehyde fixatives, probably on the basis of ionic dissociation of a conjugated ribonucleoprotein.