Topography of 5.8 S rRNA in rat liver ribosomes. Identification of diethyl pyrocarbonate-reactive sites

The topography of 5.8 rRNA in rat liver ribosomes has been examined by comparing diethyl pyrocarbonate-reactive sites in free 5.8 S RNA, the 5.8 S-28 rRNA complex, 60 S subunits, and whole ribosomes. The ribosomal components were treated with diethyl pyrocarbonate under salt and temperature conditions which allow cell-free protein synthesis; the 5.8 S rRNA was extracted, labeled in vitro, chemically cleaved with aniline, and the fragments were analyzed by rapid gel-sequencing techniques. Differences in the cleavage patterns of free and 28 S or ribosome-associated 5.8 S rRNA suggest that conformational changes occur when this molecule is assembled into ribosomes. In whole ribosomes, the reactive sites were largely restricted to the "AU-rich" stem and an increased reactivity at some of the nucleotides suggested that a major change occurs in this region when the RNA interacts with ribosomal proteins. The reactivity was generally much less restricted in 60 S subunits but increased reactivity in some residues was also observed. The results further indicate that in rat ribosomes, the two -G-A-A-C- sequences, putative binding sites for tRNA, are accessible in 60 S subunits but not in whole ribosomes and suggest that part of the molecule may be located in the ribosomal interface. When compared to 5 S rRNA, the free 5.8 S RNA molecule appears to be generally more reactive with diethyl pyrocarbonate and the cleavage patterns suggest that the 5 S RNA molecule is completely restricted or buried in whole ribosomes.     

Role of the 5.8S rRNA in ribosome translocation.

Studies on the inhibition of protein synthesis by specific anti 5.8S rRNA oligonucleotides have suggested that this RNA plays an important role in eukaryotic ribosome function. Mutations in the 5. 8S rRNA can inhibit cell growth and compromise protein synthesis in vitro . Polyribosomes from cells expressing these mutant 5.8S rRNAs are elevated in size and ribosome-associated tRNA. Cell free extracts from these cells also are more sensitive to antibiotics which act on the 60S ribosomal subunit by inhibiting elongation. The extracts are especially sensitive to cycloheximide and diphtheria toxin which act specifically to inhibit translocation. Studies of ribosomal proteins show no reproducible changes in the core proteins, but reveal reduced levels of elongation factors 1 and 2 only in ribosomes which contain large amounts of mutant 5.8S rRNA. Polyribosomes from cells which are severely inhibited, but contain little mutant 5.8S rRNA, do not show the same reductions in the elongation factors, an observation which underlines the specific nature of the change. Taken together the results demonstrate a defined and critical function for the 5.8S rRNA, suggesting that this RNA plays a role in ribosome translocation.     

The conserved 5 S rRNA complement to tRNA is not required for translation of natural mRNA

We have tested a putative base-paired interaction between the conserved GT psi C sequence of tRNA and the conserved GAAC47 sequence of 5 S ribosomal RNA by in vitro protein synthesis using ribosomes containing deletions in this region of 5 S rRNA. Ribosomes reconstituted with 5 S rRNA possessing a single break between residues 41 and 42, deletion of residues 42-46, or deletion of residues 42-52 were tested for their ability to translate phage MS2 RNA. Initiator tRNA binding, aminoacyl-tRNA binding, ppGpp synthesis, and miscoding were also tested. All of the measured functions could be carried out by ribosomes carrying the deleted 5 S rRNAs. The sizes and relative amounts of the polypeptides synthesized by MS2 RNA-programmed ribosomes were identical whether or not the 5 S RNA contained deletions. Aminoacyl-tRNA binding and miscoding were essentially unaffected. Significant reduction in ApUpG (but not poly(A,U,G) or MS2 RNA)-directed fMet-tRNA binding and ppGpp synthesis were observed, particularly in the case of the larger (residues 42-52) deletion. We conclude that if tRNA and 5 S rRNA interact in this fashion, it is not an obligatory step in protein synthesis.     

TRANSFER RNA AND THE REGULATION OF PROTEIN SYNTHESIS IN RAT CEREBRAL CORTEX DURING NEURAL DEVELOPMENT

Protein synthesis was measured in ribosomal systems derived from the cerebral cortex of 5-and 35-day-old rats. Under optimal conditions incorporation of radioactive leucine per mg ribosomal protein was four times higher with ribosomes from the younger animals than with ribosomes from the 35-day-old rats. This suggests that a decrease in the rate of protein synthesis occurs during neural development. Both ribosomes and the pH enzyme fraction from the cerebral cortex of 35-day-old rats had lower activities than preparations from the younger rats. Cerebral cortical ribosomes from 35-day-old animals had a lower polyribosome content than similar preparations from 5-day-old rats. A three-fold higher requirement for the pH 5 enzyme fraction was observed with the ribosomal system from 5-day-old rats, an observation which correlated with the yields of pH 5 enzyme and ribosomal protein from the younger tissue. The nature of the changes in the composition of the pH 5 enzyme fraction was investigated. Methylated albumin kiesselguhr (MAK) and Sephadex G-75 column chromatography showed that RNA from the pH 5 enzyme fraction was heterogeneous, containing tRNA, rRNA, and a small molecular weight RNA. This latter RNA, perhaps a degradation product of rRNA, comprised the greatest portion of RNA from the pH 5 enzyme fraction of cerebral cortex. The data obtained with MAK chromatography were used to estimate the total tRNA content of the cerebral cortex, with no age-related differences being observed. Since evidence of RNA degradation was seen, tRNA was also isolated by phenol extraction of whole cerebral cortex in the presence of bentonite. Purification of tRNA by NaCl and isopropanol fractionation gave preparations with no detectable rRNA or small molecular weight RNA. With this purification method, the tRNA yield was greater than estimated by the MAK method, demonstrating that losses of tRNA occurred during the cell fractionation steps. With the purification method 1.6 times more tRNA was obtained from the cerebral cortex of 5-day-old animals than from the older tissue. This higher level of tRNA in the younger, more active tissue appeared to involve all tRNA species, since in vitro aminoacyiation studies revealed nearly identical acceptance values for 18 individual amino acids. These results suggest that the rate of protein synthesis in cerebral cortex is regulated in part by the total amount of tRNA present to translate the higher level of polysome-bound mRNA.     

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.     

Polyamine stimulation of ribosomal synthesis and activity in a polyamine-dependent mutant of Escherichia coli

A polyamine-dependent mutant of Escherichia coli KK101 was isolated by treatment of E. coli MA261 with N-methyl-N'-nitro-N-nitrosoguanidine. In the absence of putrescine, doubling time of the mutant was 496 min. The mutation was accompanied by a change in the nature of the 30 S ribosomal subunits. Addition of putrescine to the mutant stimulated the synthesis of proteins and subsequently, this led to stimulation of RNA and DNA synthesis. Under these conditions, we determined which proteins were preferentially synthesized. Putrescine stimulated the synthesis of ribosomal protein S1 markedly, but stimulated ribosomal proteins S4, L20, and X1, and RNA polymerase slightly. The amounts of initiation factors 2 and 3 synthesized were not influenced significantly by putrescine. The preferential stimulation of the synthesis of ribosomal protein S1 occurred as early as 20 min after the addition of putrescine, while stimulation of the synthesis of the other ribosomal proteins and RNA polymerase appeared at 40 min. The stimulation of the synthesis of ribosomal RNA also occurred at 40 min after addition of putrescine. Our results indicate that putrescine can stimulate both the synthesis and the activity of ribosomes. The increase in the activity of ribosomes was achieved by the association of S1 protein to S1-depleted ribosomes. The early stimulation of ribosomal protein S1 synthesis after addition of putrescine may be important for stimulation of cell growth by polyamines.     

On the accessibility and selection of the initiator site of mRNA in protein synthesis.

The specificity of the cell-free system of Escherichia coli for mRNA was examined, and the "accessibility" of some natural and synthetic RNAs to the ribosomes was determined by measurement of AcPhe-tRNA and fMet-tRNA binding, AcPhe-puromycin and fMet-puromycin formation, and polypeptide synthesis. The E. coli system effectively initiates the translation of various synthetic RNAs with AcPhe-tRNA or fMet-tRNA under conditions optimal for the translation of viral RNA. Poly(A,G,U) is accessible to the ribosomes according to all of the above criteria. Poly(A,C,G,U), 23 S rRNA, R17 RNA, and MS2 RNA, on the other hand, show limited accessibility when tested for initiator tRNA binding, or for AcPhe-puromycin and fMet-puromycin formation. MS2 and R17 RNA, but not poly(A,C,G,U) and 23 S rRNA, show accessibility when measured by polypeptide synthesis. The results suggest that, except at initiator sites of natural mRNA, an RNA containing about equal amounts of all four bases is inaccessible to E. coli ribosomes for polypeptide synthesis. Rate constants obtained for fMet-tRNA binding with MS2 RNA, poly(A,G,U), and poly(C,G,U) indicate that the ribosomes do not have any special affinity for the viral RNA. Thus, the selection of the initiator site in protein synthesis may be critically determined more by the accessibility of the initiator codon than by ribosomal recognition of the site.     

A structural model for the large subunit of the mammalian mitochondrial ribosome

Protein translation is essential for all forms of life and is conducted by a macromolecular complex, the ribosome. Evolutionary changes in protein and RNA sequences can affect the 3D organization of structural features in ribosomes in different species. The most dramatic changes occur in animal mitochondria, whose genomes have been reduced and altered significantly. The RNA component of the mitochondrial ribosome (mitoribosome) is reduced in size, with a compensatory increase in protein content. Until recently, it was unclear how these changes affect the 3D structure of the mitoribosome. Here, we present a structural model of the large subunit of the mammalian mitoribosome developed by combining molecular modeling techniques with cryo-electron microscopic data at 12.1A resolution. The model contains 93% of the mitochondrial rRNA sequence and 16 mitochondrial ribosomal proteins in the large subunit of the mitoribosome. Despite the smaller mitochondrial rRNA, the spatial positions of RNA domains known to be involved directly in protein synthesis are essentially the same as in bacterial and archaeal ribosomes. However, the dramatic reduction in rRNA content necessitates evolution of unique structural features to maintain connectivity between RNA domains. The smaller rRNA sequence also limits the likelihood of tRNA binding at the E-site of the mitoribosome, and correlates with the reduced size of D-loops and T-loops in some animal mitochondrial tRNAs, suggesting co-evolution of mitochondrial rRNA and tRNA structures.     

Enhancement of ribosomal ribonucleic acid synthesis by deoxyribonucleic acid gyrase activity in Escherichia coli.

The effect of the deoxyribonucleic acid (DNA) gyrase inhibitors coumermycin A1, novobiocin, and oxolinic acid on ribonucleic acid (RNA) synthesis in Escherichia coli was studied in vivo and in vitro. Preferential inhibition of ribosomal RNA (rRNA) synthesis was observed. No effect of oxolinic acid and coumermycin on rRNA synthesis was seen in mutants having a DNA gyrase which is resistant to these inhibitors. In a temperature-sensitive DNA gyrase mutant rRNA synthesis was decreased at nonpermissive temperatures. Thus, a functional DNA gyrase is required for rRNA synthesis. Purified DNA gyrase had no effect on rRNA synthesis in a purified system. However, DNA gyrase does show preferential stimulation of rRNA synthesis in a system supplemented with other proteins. Apparently, DNA gyrase stimulation of rRNA synthesis requires another protein.     

Amino acid regulation of synthesis of ribonucleic acid and protein in the liver of rats

Weanling (23-day-old) rats were fed on either a low-protein diet (6% casein) or a diet containing an adequate amount of protein (18% casein) for 28 days. Hepatic cells from animals fed on the deficient diet were characterized by markedly lower concentrations of protein and RNA in all cellular fractions as compared with cells from control rats. The bound rRNA fraction was decreased to the greatest degree, whereas the free ribosomal concentrations were only slightly less than in control animals. A good correlation was observed between the rate of hepatic protein synthesis in vivo and the cellular protein content of the liver. Rates of protein synthesis both in vivo and in vitro were directly correlated with the hepatic concentration of individual free amino acids that are essential for protein synthesis. The decreased protein-synthetic ability of the ribosomes from the liver of protein-deprived rats was related to a decrease in the number of active ribosomes and heavy polyribosomes. The lower ribosomal content of the hepatocytes was correlated with the decreased concentration of essential free amino acids. In the protein-deprived rats, the rate of accumulation of newly synthesized cytoplasmic rRNA was markedly decreased compared with control animals. From these results it was concluded that amino acids regulate protein synthesis (1) by affecting the number of ribosomes that actively synthesize protein and (2) by inhibiting the rate of synthesis of new ribosomes. Both of these processes may involve the synthesis of proteins with a rapid rate of turnover.     

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.     

Structure of the ribosome-associated 5.8 S ribosomal RNA

The structure of the 5.8 S ribosomal RNA in rat liver ribosomes was probed by comparing dimethyl sulfate-reactive sites in whole ribosomes, 60 S subunits, the 5.8 S-28 S rRNA complex and the free 5.8 S rRNA under conditions of salt and temperature that permit protein synthesis in vitro. Differences in reactive sites between the free and both the 28 S rRNA and 60 S subunit-associated 5.8 S rRNA show that significant conformational changes occur when the molecule interacts with its cognate 28 S rRNA and as the complex is further integrated into the ribosomal structure. These results indicate that, as previously suggested by phylogenetic comparisons of the secondary structure, only the "G + C-rich" stem may remain unaltered and a universal structure is probably present only in the whole ribosome or 60 S subunit. Further comparisons with the ribosome-associated molecule indicate that while the 5.8 S rRNA may be partly localized in the ribosomal interface, four cytidylic acid residues, C56, C100, C127 and C128, remain reactive even in whole ribosomes. In contrast, the cytidylic acid residues in the 5 S rRNA are not accessible in either the 60 S subunit or the intact ribosome. The nature of the structural rearrangements and potential sites of interaction with the 28 S rRNA and ribosomal proteins are discussed.     

Protein synthesis inhibitors and catalytic RNA. Effect of puromycin on tRNA precursor processing by the RNA component of Escherichia coli RNase P

RNase P and ribosomes must interact with similar substrate molecules, tRNA precursors in the case of RNase P and aminoacyl-, peptidyl- or free tRNAs in the case of ribosomes. In order to compare the substrate recognition mechanisms between ribosomes and RNase P, protein synthesis inhibitors have been assayed for their effect on the catalytic activity of the RNA component of Escherichia coli RNase P (M1 RNA). Puromycin has an inhibitory effect that could be related to similar substrate recognition mechanisms by rRNA in the ribosome and by M1 RNA in RNase P.     

Inhibition ofCandida utilis by cycloheximide and acetoxycycloheximide

Cycloheximide and acetoxycyloheximide were similar in their effect on the synthesis of RNA and protein by exponentially growing cells ofCandida utilis. Both antibiotics inhibited the synthesis of protein more than that of RNA. During inhibition there was preferential synthesis of ribosomal protein and some completed ribosomes were formed. However, the synthesis of ribosomal RNA was reduced more than that of transfer RNA. The actions of these drugs onCandida utilis are compared with the effects of other antibiotics whose primary effect on bacteria is to inhibit protein synthesis.     

Relationships Among Deoxyribonucleic Acid, Ribonucleic Acid, and Specific Transfer Ribonucleic Acids in Escherichia coli 15T− at Various Growth Rates

The levels of macromolecules in Escherichia coli 15T(-) growing in broth, glucose, succinate, and acetate media were determined to compare relationships among deoxyribonucleic acid (DNA), ribosomal ribonucleic acid (rRNA), transfer RNA (tRNA), and protein in cells at different growth rates. DNA and protein increased in relative amounts with decreasing growth rate; relative amounts of rRNA and tRNA decreased, tRNA making up a slightly larger proportion of RNA. For several amino acid-specific tRNAs studied, acceptor capacities per unit of DNA increased with increasing growth rate. The syntheses of tRNA and rRNA are regulated by similar, yet different, mechanisms. Chromatographic examination on columns of benzoylated diethylaminoethyl-cellulose of isoaccepting tRNAs for arginine, leucine, lysine, methionine, phenylalanine, serine, and valine did not reveal differences in the isoaccepting profiles for rapidly (broth culture) and slowly growing (acetate culture) cells. Therefore, isoacceptors for individual amino acids appear to be regulated as a group. Lower efficiencies of ribosomal function in protein synthesis can be explained, in part, by a low ratio of tRNA to the number of ribosomes available and by a decreasing concentration of tRNA with decreasing growth rate. Data on the tRNAs specific for seven amino acids indicate that the decreasing concentration of tRNA is a general event rather than a severe limitation of any one tRNA or isoaccepting tRNA.     

Interaction of unfolded tRNA with the 3''-terminal region of E. coli 16S ribosomal RNA.

Fragments of tRNA possessing a free TpsiC-loop or a free D-loop form stable complexes with the colicin fragment (1494-1542) of 16S ribosomal RNA from E. coli. The colicin fragment does not bind to tRNA in which the T-loop and the D-loop are involved in tertiary interactions. Colicin cleavage of the 16S rRNA from E. coli is inhibited by aminoacyl-tRNA or tRNA fragments, indicating that a similar interaction may take place on the intact 70S ribosomes. The oligonucleotide d(G-T-T-C-G-A)homologous to the conserved sequence G-T-psi-C-Pu-(m1)A in the TpsiC-region of many elongator tRNAs binds to the conserved sequence U-C-G-mU-A-A-C (1495-1501) of the 16S rRNA. It is suggested that the 3'-end of the 16S rRNA may provide the part of the binding site for the elongator tRNAs on bacterial ribosomes.