In vivo stability, maturation and relative differential synthesis rates of individual ribosomal proteins in Escherichia coli B/r

The relative differential synthesis rates² of individual ribosomal proteins (r-proteins) were determined for Escherichia coli B/r growing in succinate medium (growth rate, μ = 0.65 doublings per hour), glucose medium (μ = 1.36) and glucose-amino acids medium (μ = 1.90). These differential synthesis rates were found to increase co-ordinately with increasing bacterial growth rates; this implies that ribosomes from bacteria growing at different rates are homogeneous with respect to their protein composition (i.e. the stoichiometric amounts of the different r-proteins per ribosome are constant and independent of the bacterial growth rate). Following incorporation into ribosomes, the bulk of the r-proteins were found to be as stable as total protein. Only two r-proteins, S6 and S21, were less stable than total protein; their decay half-lives, measured in succinate and glucose-amino acids cultures, were estimated to be approximately 500 minutes. In addition, post-translational modification of proteins S18, L6 and L11 was observed and the possible relations between modification and in vivo ribosome assembly are discussed. Finally, evidence is presented suggesting that the coordinate production of r-proteins may result, in part, from a mechanism that degrades excess r-proteins that are not rapidly incorporated into ribosomal particles.     

Ribosome degradation in growing bacteria

Ribosomes are large ribozymes that synthesize all cellular proteins. As protein synthesis is rate-limiting for bacterial growth and ribosomes can comprise a large portion of the cellular mass, elucidation of ribosomal turnover is important to the understanding of cellular physiology. Although ribosomes are widely believed to be stable in growing cells, this has never been rigorously tested, owing to the lack of a suitable experimental system in commonly used bacterial model organisms. Here, we develop an experimental system to directly measure ribosomal stability in Escherichia coli. We show that (i) ribosomes are stable when cells are grown at a constant rate in the exponential phase; (ii) more than half of the ribosomes made during exponential growth are degraded during slowing of culture growth preceding the entry into stationary phase; and (iii) ribosomes are stable for many hours in the stationary phase. Ribosome degradation occurs in growing cultures that contain almost no dead cells and coincides with a reduction of comparable magnitude in the cellular RNA concentration.     

Decay and synthesis of ribosomal proteins during Dictyostelium discoideum development

Summary Ribosome turnover is a prominent process during cell differentiation in Dictyostelium discoideum. At the end of 24 h of development on filters, the cells contain only 30% of the ribosome content of vegetatively growing cells. We determined the relative rates of synthesis and decay of each of the ribosomal proteins during this period. Approximately 80% of the total vegetative cell ribosomal proteins were degraded during the course of fruiting body construction. Ribosomal RNA and protein degradation apparently occurred coordinately during development. Although all ribosomal proteins decayed during development, some were more stable and a few less stable than the average. In addition, all the ribosomal proteins were synthesized during this period. Most ribosomal proteins were synthesized at the same rate as other cellular proteins, although a number were made at lower or higher rates. It was estimated that about 35% of the ribosomes in developed cells represented those, that were made during cell differentiation. Differential decay and/or synthesis of ribosomal proteins could account for the observed difference in protein content of ribosomes from growing amoebae and late development cells and spores.Paper No. 4 in the series, Studies on Ribosomal Proteins in Dictyostelium discoideum. Paper No. 3 is Ramagopal and Ennis (1982)     

Importance of the 5 S rRNA-binding ribosomal proteins for cell viability and translation in Escherichia coli

A specific complex of 5 S rRNA and several ribosomal proteins is an integral part of ribosomes in all living organisms. Here we studied the importance of Escherichia coli genes rplE, rplR and rplY, encoding 5 S rRNA-binding ribosomal proteins L5, L18 and L25, respectively, for cell growth, viability and translation. Using recombineering to create gene replacements in the E. coli chromosome, it was shown that rplE and rplR are essential for cell viability, whereas cells deleted for rplY are viable, but grow noticeably slower than the parental strain. The slow growth of these L25-defective cells can be stimulated by a plasmid expressing the rplY gene and also by a plasmid bearing the gene for homologous to L25 general stress protein CTC from Bacillus subtilis. The rplY mutant ribosomes are physically normal and contain all ribosomal proteins except L25. The ribosomes from L25-defective and parental cells translate in vitro at the same rate either poly(U) or natural mRNA. The difference observed was that the mutant ribosomes synthesized less natural polypeptide, compared to wild-type ribosomes both in vivo and in vitro. We speculate that the defect is at the ribosome recycling step.     

Turnover as a control of ribonucleic acid accumulation in bacteria undergoing stepdown.

The synthesis of ribosomes was compared in rel+ and rel- strains of Escherichia coli undergoing "stepdown" in growth from glucose medium to one with lactate as principal carbon source. Two strains (CP78 and CP79), isogenic except for rel, showed similar behaviour with respect to (1) the kinetics of labelling total RNA and ribosomes with exogenous uracil, (2) the proportion of newly formed protein that could be bound with nascent rRNA in mature ribosomes, and (3) the rate of induction of enzymically active beta-galactosidase (relative to the rate of ribosome synthesis). It was concluded that, as there was no net accumulation of RNA during stepdown in either strain, rRNA turnover must be occurring at a high rate. The general features of ribosome maturation in rel+ and rel- cells were almost identical with those found in auxotrophic rel+ organisms starved of required amino acids. In both cases, there was a considerable delay in the maturation of new ribosomal particles, owing to a relative shortfall in the rate of synthesis of ribosome-associated proteins. Only about 4-5% of the total protein labelled during stepdown was capable of binding with newly formed rRNA. This compared with 3.5% for rel+ and 0.5% for rel- auxotrophs during amino acid starvation. The turnover rate for newly formed mRNA and rRNA was virtually the same in "stepped-down" rel+ and rel- strains and was similar to that of the same fraction in amino acid-starved rel+ cells. The functional lifetime of mRNA was also identical. It seems that in the rel- strain many of the characteristics typical of the isogenic rel+ strain are displayed under these conditions, at least as regards the speed of ribosome maturation and the induction of beta-galactosidase. Studies on the thermolability of the latter enzyme induced during stepdown indicate that inaccurate translation, which occurs in rel- strains starved for only a few amino acids, is less evident in this situation than in straightforward amino acid deprivation.     

Synthesis of proteins and RNA of the 60S ribosomal subunit in HeLa cells recovering from valine deprivation

The synthesis of ribosomes in HeLa cells was studied during recovery from a 20-hour deprivation for valine. The rates of incorporation of labeled precursors into ribosomal pre-RNA, processed rRNA, total cellular proteins, and proteins of the 60S ribosomal subunit returned to normal or nearly normal levels immediately after restoration of valine to the medium. Specific proteins of the 60S ribosomal subunit, whose apparent net synthesis is reduced more than that of the other proteins of the 60S ribosomal subunit during valine deprivation, were no longer undersynthesized after valine was restored. This rapid recovery suggests that the apparent decrease in the net rate of synthesis of these ribosomal proteins during valine deprivation is effected at the translational or post-translational level. No evidence of significant synchrony in any particular stage of the cell cycle was observed after a 20-hr valine deprivation. Key words: 60S ribosomal subunit; HeLa, cells; valine deprivation.     

Program of early development in the mammal: Changes in absolute rates of synthesis of ribosomal proteins during oogenesis and early embryogenesis in the mouse

The absolute rates of synthesis of specific ribosomal proteins have been determined during growth and meiotic maturation of mouse oocytes, as well as during early embryogenesis in the mouse. These measurements were made possible by the development of a high-resolution twodimensional gel electrophoresis procedure capable of resolving basic proteins with isoelectric points between 9.1 and 10.2. Mouse ribosomal proteins were separated on such gels and observed rates of incorporation of [³⁵S]methionine into each of 12 representative ribosomal proteins were converted into absolute rates of synthesis (femtograms or moles synthesized/hour/oocyte or embryo) by using previously determined values for the absolute rates of total protein synthesis in mouse oocytes and embryos (R. M. Schultz, M. J. LaMarca, and P. M. Wassarman, 1978,Proc. Nat. Acad. Sci. USA,75, 4160;R. M. Schultz, G. E. Letourneau, and P. M. Wassarman, 1979,Develop. Biol.,68, 341–359). Ribosomal proteins were synthesized at all stages of oogenesis and early embryogenesis examined and, while equimolar amounts of ribosomal proteins were found in ribosomes, they were always synthesized in nonequimolar amounts during development. Rates of synthesis of individual ribosomal proteins differed from each other by more than an order of magnitude in some cases. Synthesis of ribosomal proteins accounted for 1.5, 1.5, and 1.1% of total protein synthesis during growth of the oocyte, in the fully grown oocyte, and in the unfertilized egg, respectively. During meiotic maturation of mouse oocytes the absolute rate of synthesis of ribosomal proteins decreased about 40%, from 620 to 370 fg/hr/cell, as compared to a 23% decrease in the rate of total protein synthesis during the same period. On the other hand, during early embryogenesis the absolute rates of synthesis of each of the 12 ribosomal proteins examined increased substantially as compared with those of the unfertilized egg, such that at the eight-cell stage of embryogenesis synthesis of ribosomal proteins (4.17 pg/hr/embryo) accounted for about 8.1% of the total protein synthesis in the embryo. Consequently, while the absolute rate of total protein synthesis increased about 1.5-fold during development from an unfertilized mouse egg to an eight-cell compacted embryo, the absolute rate of ribosomal protein synthesis increased more than 11-fold during the same period. These results seem to reflect the differences reported for the patterns of ribosomal RNA synthesis during early development of mammalian, as compared to nonmammalian, animal species. The results are compared with those obtained using oocytes and embryos fromXenopus laevis.     

Changes in RNA metabolism and accumulation of presumptive messenger RNA during transition from the growing to the quiescent state of cultured mouse fibroblasts.

Mouse fibroblasts maintained in tissue culture regulate total protein and ribosomal RNA synthesis co-ordinately with changes in the cellular growth state. Here we show that changes in the rate of synthesis of nuclear non-polyadenylic acid-containing RNA and the rate of accumulation and breakdown of cytoplasmic ribosomal RNA also accompany the transition from the resting to the growing cellular growth state, while the rate of synthesis of nuclear poly (A)-containing RNA and the rates of accumulation and breakdown of cytoplasmic poly(A) containing RNA (presumptive messenger RNA) are, however, only marginally changed. The small net increase (20% to 30%) in the amount of presumptive mRNA is considerably less than the observed increase in protein synthesis (two to threefold) during this transition. We also isolated and characterized extra-polysomal poly(A)-containing ribonucleoprotein particles from quiescent cultures that were similar to those particles obtained by treatment of polyribosomes with EDTA. These experiments suggest that the early increase in protein synthetic activity when quiescent, cultured cells are induced to grow is partially caused by an increased attachment of pre-existing mRNA molecules to free ribosomes.     

Synthesis and conservation of ribosomal proteins during compensatory renal hypertrophy.

The rate of synthesis of ribosomal proteins was investigated as an index of the rate of production of ribosomes in mouse kidney during the first few days after contralateral nephrectomy. Compensatory renal hypertrophy was not associated with a major increase in the synthetic rate of ribosomal proteins and rRNA. Instead, the ratio of the rate of ribosomal-protein synthesis to that of total protein synthesis remained nearly constant. The conformation of glutaraldehyde-fixed ribosomes and ribosomal subunits was unchanged. During the early stages of compensatory renal hypertrophy the accretion of rRNA is due largely to conservation of ribosomes that would otherwise have been degraded.     

Translational initiation factor expression and ribosomal protein gene expression are repressed coordinately but by different mechanisms in murine lymphosarcoma cells treated with glucocorticoids.

P1798 murine lymphosarcoma cells cease to proliferate upon exposure to 10(-7) M dexamethasone and exhibit a dramatic inhibition of rRNA and ribosomal protein synthesis (O. Meyuhas, E. Thompson, Jr., and R. P. Perry, Mol. Cell Biol. 7:2691-2699, 1987). These workers demonstrated that ribosomal protein synthesis is regulated primarily at the level of translation, since dexamethasone did not alter mRNA levels but shifted the mRNAs from active polysomes into inactive messenger ribonucleoproteins. We have examined the effects of dexamethasone on the biosynthesis of initiation factor proteins in the same cell line. The relative protein synthesis rates of eIF-4A and eIF-2 alpha were inhibited by about 70% by the hormone, a reduction comparable to that for ribosomal proteins. The mRNA levels of eIF-4A, eIF-4D, and eIF-2 alpha also were reduced by 60 to 70%, indicating that synthesis rates are proportional to mRNA concentrations. Analysis of polysome profiles showed that the average number of ribosomes per initiation factor polysome was only slightly reduced by dexamethasone, and little or no mRNA was present in messenger ribonucleoproteins. The results indicate that initiation factor gene expression is coordinately regulated with ribosomal protein synthesis but is controlled primarily by modulating mRNA levels rather than mRNA efficiency.     

Repression of β-actin synthesis and persistence of ribosomal protein synthesis after infection of HeLa cells by herpes simplex virus type 1 infection are under translational control

Synthesis and assembly of ribosomal proteins into mature ribosomes persist late after infection of cells with herpes simplex virus type 1, while synthesis of β-actin is drastically shut off. Since mRNAs encoding ribosomal proteins and β-actin undergo concomitant degradation in infected HeLa cells, we have advanced the hypothesis that translation of the remaining mRNAs is differentially controlled after infection. The behaviour of mRNAs for three ribosomal proteins and for β-actin was investigated during the course of infection. In uninfected cells, β-actin mRNAs are associated with large polyribosomes, while only a part of ribosomal protein mRNAs are present in polyribosomes. In the course of infection, β-actin mRNAs are released from the ribosomes and are sequestered with 40S ribosomal subunits. Simultaneously, ribosomal protein mRNAs become associated with an increased number of ribosomes, even late in infection. In addition, virally induced phosphorylation of ribosomal protein S6 is more efficient in pre-existing ribosomes than in newly assembled ribosomes. These results indicate that in infected cells (i) translation of β-actin mRNA is selectively inhibited at a step necessary for binding the 60S ribosomal subunits; (ii) the rate of initiation of translation of ribosomal protein mRNAs increases after infection; and (iii) it is likely that translation of ribosomal protein mRNAs takes place preferentially on pre-existing ribosomes. Received: 5 February 1997 / Accepted: 28 May 1997     

Protein L5 is crucial for in vivo assembly of the bacterial 50S ribosomal subunit central protuberance

In the present work, ribosomes assembled in bacterial cells in the absence of essential ribosomal protein L5 were obtained. After arresting L5 synthesis, Escherichia coli cells divide a limited number of times. During this time, accumulation of defective large ribosomal subunits occurs. These 45S particles lack most of the central protuberance (CP) components (5S rRNA and proteins L5, L16, L18, L25, L27, L31, L33 and L35) and are not able to associate with the small ribosomal subunit. At the same time, 5S rRNA is found in the cytoplasm in complex with ribosomal proteins L18 and L25 at quantities equal to the amount of ribosomes. Thus, it is the first demonstration that protein L5 plays a key role in formation of the CP during assembly of the large ribosomal subunit in the bacterial cell. A possible model for the CP assembly in vivo is discussed in view of the data obtained.     

Protected nucleotide G2608 in 23S rRNA confers resistance to oxazolidinones in E. coli

The oxazolidinones are a new class of potent antibiotics that are active against a broad spectrum of Gram-positive bacterial pathogens including those resistant to other antibiotics. These drugs specifically inhibit protein biosynthesis whereas DNA and RNA synthesis are not affected. Although biochemical and genetic studies indicate that oxazolidinones target the ribosomal peptidyltransferase center, other investigations suggest that they interact with different regions of ribosomes. Thus, the exact binding site and mechanism of action have remained elusive. Here, we study, by use of base-specific reagents, the effect of the oxazolidinones on the chemical protection footprinting patterns of the 23S rRNA. We report: (i) reproducible protection of G2607 and G2608 of 23S rRNA by a potent oxazolidinone on a ribosome.tRNA.mRNA complex; (ii) no protections were observed on 70S ribosomes devoid of tRNA and mRNA; (iii) EF-G also weakly protected G2607 and G2608; (iv) mutations at G2608 conferred resistance to the oxazolidinones in Escherichia coli cells; and (v) G2607 and G2608 occur near the exit to the peptide tunnel on the 50S subunit. A mechanism for the pleiotropic action of the oxazolidinones is discussed.     

Regulation of synthesis of ribosomal protein in Neurospora crassa

In Neurospora crassa during a nutritional shift-down transition of growth, the synthesis of rRNA is for about 2 h largely inhibited and the rate of protein synthesis is only partially reduced (by about 25 %). During this period the relative rate of synthesis of individual ribosomal proteins, measured irrespectively of their incorporation into ribosomes, is reduced by 70–80%. The ribosomal proteins synthesized during the shift are stable. Thus, the synthesis of ribosomal proteins appears in N. crassa to be coordinately regulated with that of rRNA.