DnaK-facilitated ribosome assembly in Escherichia coli revisited

Assembly helpers exist for the formation of ribosomal subunits. Such a function has been suggested for the DnaK system of chaperones (DnaK, DnaJ, GrpE). Here we show that 50S and 30S ribosomal subunits from an Escherichia coli dnaK-null mutant (containing a disrupted dnaK gene) grown at 30 degrees C are physically and functionally identical to wild-type ribosomes. Furthermore, ribosomal components derived from mutant 30S and 50S subunits are fully competent for in vitro reconstitution of active ribosomal subunits. On the other hand, the DnaK chaperone system cannot circumvent the necessary heat-dependent activation step for the in vitro reconstitution of fully active 30S ribosomal subunits. It is therefore questionable whether the requirement for DnaK observed during in vivo ribosome assembly above 37 degrees C implicates a direct or indirect role for DnaK in this process.     

Reversion from erythromycin dependence in Escherichia coli: strains altered in ribosomal sub-unit association and ribosome assembly

A mutant of Escherichia coli dependent on erythromycin for growth spontaneously gives erythromycin-independent strains with altered or missing ribosomal proteins. strains with defects in ribosome assembly were sought and obtained from among these revertants. Two organisms in which ribosomal protein L19 is altered and absent respectively have 70S ribosomes whose dissociation into sub-units is particularly sensitive to pressures generated during centrifuging. The mutant that lacks protein L19 also accumulates ribosome precursor particles during exponential growth as do others including mutants that lack proteins S20 or L1. These strains also show unbalanced synthesis of RNA and so will be useful in investigating both the pathways and the regulation of ribosome assembly.     

Abnormal ribosome assembly in a mutant of Escherichia coli.

The mutant strain, 15--28, of Escherichia coli accumulates ribonucleoprotein ('47S') particles that were previously shown [Markey, Sims & Wild (1976) Biochem. J. 158, 451--456] to be an unusual intermediate in the assembly of 50S ribosomal subunits...     

Cold-sensitive ribosome assembly in an Escherichia coli mutant lacking a single methyl group in ribosomal protein L3

Ribosomal protein methylation has been well documented but its function remains unclear. We have examined this phenomenon using an Escherichia coli mutant (prmB2), which fails to methylate glutamine residue number 150 of ribosomal protein L3. This mutant exhibits a cold-sensitive phenotype: its growth rate at 22 degrees C is abnormally low in complete medium. In addition, strains with this mutation accumulate abnormal and unstable ribosomal particles; 50-S and 30-S subunits are formed, but at a lower rate. Once assembled, ribosomes with unmethylated L3 are fully active by several criteria. (a) Protein synthesis in vitro with purified 70-S prmB2 ribosomes is as active as wild-type using either a natural (R17) or an artificial [poly(U)] messenger. (b) The induction of beta-galactosidase in vivo exhibits normal kinetics and the enzyme has a normal rate of thermal denaturation. (c) These ribosomes are standard when exposed in vitro to a low magnesium concentration or increasing molarities of LiCl. Efficient methylation of L3 in vitro requires either unfolded ribosomes or a mixture of ribosomal protein and RNA. We suggest that the L3-specific methyltransferase may qualify as one of the postulated 'assembly factors' of the E. coli ribosome.     

Effects of induction of rRNA overproduction on ribosomal protein synthesis and ribosome subunit assembly in Escherichia coli.

Overproduction of rRNA was artificially induced in Escherichia coli cells to test whether the synthesis of ribosomal protein (r-protein) is normally repressed by feedback regulation. When rRNA was overproduced more than twofold from a hybrid plasmid carrying the rrnB operon fused to the lambda pL promoter (pL-rrnB), synthesis of individual r-proteins increased by an average of about 60%. This demonstrates that the synthesis of r-proteins is repressed under normal conditions. The increase of r-protein production, however, for unknown reasons, was not as great as the increase in rRNA synthesis and resulted in an imbalance between the amounts of rRNA and r-protein synthesis. Therefore, only a small (less than 20%) increase in the synthesis of complete 30S and 50S ribosome subunits was detected, and a considerable fraction of the excess rRNA was degraded. Lack of complete cooperativity in the assembly of ribosome subunits in vivo is discussed as a possible explanation for the absence of a large stimulation of ribosome synthesis observed under these conditions. In addition to the induction of intact rRNA overproduction from the pL-rrnB operon, the effects of unbalanced overproduction of each of the two large rRNAs, 16S rRNA and 23S rRNA, on r-protein synthesis were examined using pL-rrnB derivatives carrying a large deletion in either the 23S rRNA gene or the 16S rRNA gene. Operon-specific derepression after 23S or 16S rRNA overproduction correlated with the overproduction of rRNA containing the target site for the operon-specific repressor r-protein. These results are discussed to explain the apparent coupling of the assembly of one ribosomal subunit with that of the other which was observed in earlier studies on conditionally lethal mutants with defects in ribosome assembly.     

Gratuitous overexpression of genes in Escherichia coli leads to growth inhibition and ribosome destruction.

We attempted to test the idea that the relative abundance of each individual tRNA isoacceptor in Escherichia coli can be altered by varying its cognate codon concentration. In order to change the overall codon composition of the messenger pool, we have expressed in E. coli lacZ with the aid of T7 RNA polymerase so that their respective gene products individually accounted for 30% of the total bacterial protein. Unexpectedly, the maximum expression of either test gene has no specific effect on the relative rates of synthesis of the tRNA species that we studied. Instead, we find that there is a cumulative breakdown of rRNAs, which results in a loss of ribosomes and protein synthetic capacity. After either of the test genes is maximally induced, there is a growing fraction of protein synthesis invested in beta-galactosidase or delta tufB that is matched by a comparable decrease of the fraction of normal protein synthesis. We have also observed enhanced accumulation of two heat shock proteins during overexpression. Finally, after several hours of overexpression of either test protein, the bacteria are no longer viable. These results are relevant to the practical problems of obtaining high expression levels for cloned proteins.     

Processing of precursor ribosomal RNA and the presence of a modified ribosome assembly scheme in Escherichia coli relaxed strain

An electrophoretic system capable of separating 25 S, 23 S, 17.5 S and 16 S ribosomal RNA (rRNA) species was used to study the synthesis and fate of rRNA during amino acid starvation and resupplementation of E. coli relaxed strain KL99. This E. coli relAl strain responded to an amino acid starvation by increasing the rate of synthesis of 25 S and 17.5 S precursor rRNA. When the limiting amino acid was resupplemented, a previously observed 40-fold increase in the cellular guanosine 5'-diphosphate, 3'-diphosphate content [Mol. Gen. Genet. (1983) 192, 5-9] appeared to cause a reduction in new rRNA synthesis. Following amino acid resupplementation, the precursor 25 S and 17.5 S rRNA accumulated during the amino acid starvation were conserved and processed to 23 S and 16 S rRNA species, respectively. This suggests that a modified ribosome assembly scheme involving stable precursor rRNA exists in relAl bacteria during periods of amino acid limitation and resupplementation.     

Methylation of ribosomal proteins during ribosome assembly in Escherichia coli

Summary In Escherichia coli, a number of ribosomal proteins are methylated. The time of methylation of L7 and L11 during ribosome assembly was studied. It was observed that the methylation of L7 could occur in the free protein stage. Both the 32S and 40S ribonucleoprotein intermediates also contained methylated L7 although the extent of methylation in these particles was not as high as in the free L7, the 45S or the 50S particles. Free L11 could also be partially methylated but the bulk of methylation of this protein was found in the 45S and the 50S particles.It was previously reported that the methylation of L7 is inversely proportional to the growth temperature (Chang 1978), we now show that once L7 is methylated at 25°, the methyl group is stable when the culture is shifted to 37°C. However, a partial turnover of the methyl group of L7 is observed when the methylated ribosome is chased at 25°C. On the other hand, the methyl groups of L11 appear to be stable at either 25°C or 37°C. We also observe that the extent of methylation of both L7 and L11 stays nearly constant during the cell growth cycle from early log to stationary phase.     

Mutations in the rpmBG operon of Escherichia coli that affect ribosome assembly.

The rpmBG operon of Escherichia coli codes for ribosomal proteins L28 and L33. Two strains with mutations in the operon are AM81, whose ribosomes lack protein L28, and AM90, whose ribosomes are without protein L33. Neither strain showed major defects in ribosome assembly. However, when the mutations were transferred to other strains of E. coli, ribosome synthesis was greatly perturbed and precursor ribonucleoproteins accumulated. In the new backgrounds, the mutation in rpmB was complemented by synthesis of protein L28 from a plasmid; the rpmG mutation was not complemented by protein L33 because synthesis of protein L28 from the upstream rpmB gene was also greatly reduced. The results suggest that protein L33, in contrast to protein L28, has at best a minor role in ribosome assembly and function.     

Mg2+-induced proton release from Escherichia coli ribosome and ribosomal RNA

Escherichia coli ribosome released protons upon addition of Mg2+. The Mg2+-induced proton release was studied by means of the pH-stat technique. The number of protons released from a 70 S ribosome in the Mg2+ concentration range 1-20 mM was about 30 at pH 7 and 7.6, and increased to about 40 at pH 6.5. The rRNA mixture extracted from 70 S ribosome showed proton release of amount and of pH dependence similar to those of the 70 S ribosome but the ribosomal protein mixture released few. This indicates that rRNA is the main source of the protons released from ribosome. The pH titration of rRNA showed that the pKa values of nucleotide bases were downward shifted upon Mg2+ binding. This pKa shift can account for the proton release. The Scatchard plots of proton release from rRNA and ribosome were concave upward, showing that the Mg2+-binding sites leading to proton release were either heterogeneous or had a negative cooperativity. A model assuming heterogeneous Mg2+-binding sites is shown to be unable to explain the proton release. Electrostatic field effect models are proposed in which Mg2+ modulates the electrostatic field of phosphate groups and the potential change induces a shift of the pKa values of bases that leads to the proton release. These models can explain the main features of the proton release.     

Protein-induced conformational changes in 16 S ribosomal RNA during the initial assembly steps of the Escherichia coli 30 S ribosomal subunit

The mechanism of 16 S ribosomal RNA folding into its compact form in the native 30 S ribosomal subunit of Escherichia coli was studied by scanning transmission electron microscopy and circular dichroism spectroscopy. This approach made it possible to visualize and quantitatively analyze the conformational changes induced in 16 S rRNA under various ionic conditions and to characterize the interactions of ribosomal proteins S4, S8, S15, S20, S17 and S7, the six proteins known to bind to 16 S rRNA in the initial assembly steps. 16 S rRNA and the reconstituted RNA-protein core particles were characterized by their mass, morphology, radii of gyration (RG), and the extent and stability of 16 S rRNA secondary structure. The stepwise binding of S4, S8 and S15 led to a corresponding increase of mass and was accompanied by increased folding of 16 S rRNA in the core particles, as evident from the electron micrographs and from the decrease of RG values from 114 A and 91 A. Although the binding of S20, S17 and S7 continued the trend of mass increase, the RG values of these core particles showed a variable trend. While there was a slight increase in the RG value of the S20 core particles to 94 A, the RG value remained unchanged (94 A) with the further addition of S17. With subsequent addition of S7 to the core particles, the RG values showed an increase to 108 A. Association with S7 led to the formation of a globular mass cluster with a diameter of about 115 A and a mass of about 300 kDa. The rest of the mass (about 330 kDa) remained loosely coiled, giving the core particle a "medusa-like" appearance. Morphology of the 16 S rRNA and 16 S rRNA-protein core particles, even those with all six proteins, does not resemble the native 30 S subunit, contrary to what has been reported by others. The circular dichroism spectra of the 16 S rRNA-protein complexes and of free 16 S rRNA indicate a similarity of RNA secondary structure in the core particles with the first four proteins, S4, S8, S15, S20. The circular dichroism melting profiles of these core particles show only insignificant variations, implying no obvious changes in the distribution or the stability of the helical segments of 16 S rRNA. However, subsequent binding of proteins S17 and S7 affected both the extent and the thermal stability of 16 S rRNA secondary structure.(ABSTRACT TRUNCATED AT 400 WORDS)