RNase catalyzed hydrolysis of ribosomes in several functional states

The RNase A catalyzed hydrolysis of rRNA in ribosomes has been studied for nonwashed 50S and 70S ribosomes, for washed 50S and 70S ribosomes, for runoff 50S ribosomes and for 70S ribosomes in polysomes. The regions available to hydrolysis in the 50S ribosome remain available when the 50S ribosomes become a part of a 70S ribosome or a polysome. The regions available to hydrolysis in the 30S ribosome become unavailable when the 30S ribosome becomes part of a 70S ribosome or a polysome. Removal of tRNA, mRNA and factors from the 50S and 70S ribosome lowers the rate of hydrolysis of one site in the 23S rRNA. This shows that the conformation of one region of the 23S RNA changes for ribosomes in different functional states.     

The distribution of ribosomes between different functional states in livers of fed and starved mice

1. To investigate the role of ribosome function in regulating protein synthesis, the activity, distribution and functional states of ribosomal particles were investigated in livers of mice fed ad libitum or starved overnight. 2. The distribution of protein-synthesizing activity between polyribosomes of different sizes was analysed after incorporation of radioactive leucine, and the quantitative distribution of ribosomes as native subunits, monomers and polyribosomes was analysed after incorporation of orotic acid. Precursors labelled with ³H or ¹⁴C were given separately to fed and starved mice, so that livers from the two groups of animals were processed together. 3. The former experiments showed that starvation has little effect on the distribution of protein-synthesizing activity across polyribosome sedimentation patterns, though the latter experiments showed that the proportion of ribosomes existing as monomers increased from 9.5% to 15.2%, whereas the proportion existing as polyribosomes decreased from 81.4% to 75.6%. Starvation had a negligible effect on the proportion of native subunits, which accounted for 9.1% and 9.2% of the ribosomes in fed and starved mice respectively. 4. The monomeric ribosome fraction was isolated and subjected to ionic conditions which selectively dissociate single ribosomes. Starvation increased the proportion of monomers that dissociated from 59% to 72%, so the monomers that accumulate in livers of starved animals are single ribosomes and not monoribosomes resulting from degradation of polyribosomes. 5. The fate of newly formed ribosomal particles was studied by measuring the specific radioactivity of native subunits, monomers and polyribosomes at different times after injection of radioactively labelled orotic acid. Starvation did not appear to affect equilibration between newly formed particles and polyribosomes, and the radioactivity of polyribosomes in both groups of mice reached about 90% of that in native subunits after 4h. The radioactive labelling of monomers proceeded at a slower rate, especially after starvation. At 4h, the radioactivity of monomers was 64% and 55% that of native subunits in fed and starved mice respectively.     

Binding of tRNA in different functional states to Escherichia coli ribosomes as measured by velocity sedimentation

The binding of initiator and elongator tRNAs to 70-S ribosomes and the 30-S subunits was followed by velocity sedimentation in the analytical ultracentrifuge. fMet-tRNAfMet binds to A-U-G-programmed 30-S subunits, but not to free or misprogrammed particles. Both the formylmethione residue and the initiation factors increase the stability of the 30-S x A-U-G x fMet-tRNAfMet complex. fMet-tRNAfMet is bound only to the P site of the 70-S ribosome even in the absence of A-U-G. Two copies of tRNAPhe or Phe-tRNAPhe are bound to the ribosome with similar affinity. In contrast to a recent report [Rheinberger et al. (1981) Proc. Natl Acad. Sci. USA, 78, 5310-5314], it is shown that three copies of tRNA cannot be bound simultaneously to the ribosome with binding constants higher than 2 x 10(4) M-1. Phe-tRNAPhe when present as the ternary complex Phe-tRNAPhe. EF-Tu x guanosine 5'-[beta,gamma-methylene]triphosphate binds exclusively to the A site. The peptidyl-tRNA analogue, acetylphenylalanine-tRNA, can occupy both ribosomal centers, albeit with a more than tenfold higher affinity for the P site. The thermodynamic data obtained under equilibrium conditions confirm the present view of two tRNA binding sites on the ribosome. The association constants determined are discussed in relation to the mechanism of ribosomal protein synthesis.     

Conformational change of L7/L12 stalk in the different functional states of 50S ribosomes

Conformational change of 50S ribosomes takes place during protein synthesis. The primary change is most likely in the secondary or tertiary structure of rRNA in the L7/L12 stalk region. In order to throw further light on this conformational change, the change in fluorescence of tight couple 50S ribosomes on conversion to loose couple 50S ribosomes containing 5-(iodoacetamido ethyl)-aminonaphthalene-l-sulphonic acid-labelled L7/L12, following the treatment with elongation factor-G and 5′-guanylyl methylene diphosphate was measured. It was enhanced in agreement with the results reported earlier. Further, the quenching of fluorescence of 50S ribosomes containing 5-(iodoacetamido ethyl)-aminonaphthalene-1-sulphonic acid-labelled L7/L12 by acrylamide was studied. The quenching is more in case of loose couples. On conversion of loose couple 50S ribosomes to tight couple ones the quenching becomes less whereas the reverse happens on conversion of tight couple 70S ribosomes to loose couples. These results indicate the conformational change of L7/L12 stalk in the different functional states of 50S ribosomes.     

The location of sulphydryl groups in alpha-crystallin

The microenvironments of the sulphydryl groups in the multimeric protein, alpha-crystallin, were studied by examining: the rate of the reaction of the groups with DTNB; the effect of increasing urea concentrations on their accessibilities; and the quenching of a fluorescent probe. In foetal bovine alpha-crystallin (1 SH/alpha A subunit) both kinetic and quenching studies indicated that over 90% of the sulphydryl groups fell into a single buried class; the remainder was exposed. In the human protein (2 SH/alpha A subunit), half of the groups were buried and the other half exposed. Accessible sulphydryl groups increased gradually as the urea concentration was increased, with complete exposure at about 4.0 M. Sedimentation velocity analyses revealed that no significant dissociation of the aggregates into subunits occurred below 3.5 M urea, at which point over 80% of the sulphydryl groups were exposed. An age-dependent increase (3-35%) was found in the proportion of exposed sulphydryl groups in bovine alpha-crystallin and a decrease in the urea concentration required to expose the remainder. It was concluded that the single cysteine is buried in the newly synthesized protein, but becomes solvent-exposed as a result of age-related conformational changes. Our observations are consistent with a quaternary structure in which all alpha A subunits occupy equivalent sites.     

Ultrastructural characteristics of osteoclasts in different functional states

By electron microscopy and autoradiography, a study was made of osteoclasts in the metaphyseal zones of the femora of 1-7-day old rabbits and rats. It was established that depending on the intensity of resorptive processes osteoclasts noticeably differed in their morphology (shape, size, number of nuclei, degree of development of intracellular organelles, "brush border"), as well as in the level of biosynthetic activity registered by incorporation intensity of 3H-uridine and 3H-methionine in these. According to these indices, osteoclasts were classified as young, mature functionally active, and non-active osteoclasts, as well as perishing ones. The defined morpho-functional states represent successive stages of the life cycle of osteoclasts.     

Direct spectroscopic determination of functional sulphydryl groups on intact cell surfaces by surface-enhanced resonance Raman scattering

Semi-quantitative and direct determination of labelled sulphydryl groups on the surface of intact erythrocytes has been accomplished for the first time with surface-enhanced resonance Raman scattering (SERRS). The method, which involves the use of citrate-reduced silver colloids, is sensitive and selective. A 10^–8 M effective concentration of picomole quantities of sulphydryl groups was determined in the presence of the normally overwhelming signal from haemoglobin. This seminal study suggests that SERRS may be applied to other in situ, site-directed labelling experiments. Correspondence to: W.E. Smith     

Characterization of hibernating ribosomes in mammalian cells

Protein synthesis across kingdoms involves the assembly of 70S (prokaryotes) or 80S (eukaryotes) ribosomes on the mRNAs to be translated. 70S ribosomes are protected from degradation in bacteria during stationary growth or stress conditions by forming dimers that migrate in polysome profiles as 100S complexes. Formation of ribosome dimers in Escherichia coli is mediated by proteins, namely the ribosome modulation factor (RMF), which is induced in the stationary phase of cell growth. It is reported here a similar ribosomal complex of 110S in eukaryotic cells, which forms during nutrient starvation. The dynamic nature of the 110S ribosomal complex (mammalian equivalent of the bacterial 100S) was supported by the rapid conversion into polysomes upon nutrient-refeeding via a mechanism sensitive to inhibitors of translation initiation. Several experiments were used to show that the 110S complex is a dimer of nontranslating ribosomes. Cryo-electron microscopy visualization of the 110S complex revealed that two 80S ribosomes are connected by a flexible, albeit localized, interaction. We conclude that, similarly to bacteria, rat cells contain stress-induced ribosomal dimers. The identification of ribosomal dimers in rat cells will bring new insights in our thinking of the ribosome structure and its function during the cellular response to stress conditions.Key words: ribosome, translation, stress, starvation, polysome     

Characterization of hibernating ribosomes in mammalian cells

Protein synthesis across kingdoms involves the assembly of 70S (prokaryotes) or 80S (eukaryotes) ribosomes on the mRNAs to be translated. 70S ribosomes are protected from degradation in bacteria during stationary growth or stress conditions by forming dimers that migrate in polysome profiles as 100S complexes. Formation of ribosome dimers in Escherichia coli is mediated by proteins, namely the ribosome modulation factor (RMF), which is induced in the stationary phase of cell growth. It is reported here a similar ribosomal complex of 110S in eukaryotic cells, which forms during nutrient starvation. The dynamic nature of the 110S ribosomal complex (mammalian equivalent of the bacterial 100S) was supported by the rapid conversion into polysomes upon nutrient-refeeding via a mechanism sensitive to inhibitors of translation initiation. Several experiments were used to show that the 110S complex is a dimer of nontranslating ribosomes. Cryo-electron microscopy visualization of the 110S complex revealed that two 80S ribosomes are connected by a flexible, albeit localized, interaction. We conclude that, similarly to bacteria, rat cells contain stress-induced ribosomal dimers. The identification of ribosomal dimers in rat cells will bring new insights in our thinking of the ribosome structure and its function during the cellular response to stress conditions.