Mutants of Escherichia coli lacking ribosomal protein L1

Two independently isolated mutants of Escherichia coli, RD19 and MV17-10, that appeared to lack protein L1 on their ribosomes, as determined by two-dimensional gels, were subjected to a battery of immunological tests to find if L1 was indeed lacking. The tests involved Ouchterlony double diffusion, modified immunoelectrophoresis, dimer formation on sucrose gradients, and affinity chromatography. By all these criteria, protein L1 was missing from the ribosome in these mutants. Nor was any L1 cross-reacting material detectable in the supernatant. There was, however, a specific two- to fivefold increase in concentrations of protein L11 in the supernatants of the mutants, which was evidence that protein L1 acts as a feedback inhibitor of expression of the operon coding for the genes for proteins L11 and L1.Electron micrographs of ribosomes obtained from these mutants were indistinguishable from those of wild-type strains. 50 S ribosomal subunits from mutants RD19 and MV17-10 were reconstituted with purified L1 from wild-type and investigated by immunoelectron microscopy. The three-dimensional location of ribosomal protein L1 on the surface of the large subunit was determined. L1 is located on the wider lateral protuberance of the 50 S subunit. The position of protein L1 in 50 S subunits reconstituted from mutants RD19 and MV17-10 was indistinguishable from the position in subunits from wild-type.     

Structural properties of ribosomal protein L11 from Escherichia coli

Protein L11 has been isolated from the large subunit of the E. coli ribosome under non-denaturing conditions and studied by proton magnetic resonance spectroscopy, limited proteolysis, and fluorescence and UV spectroscopy. The protein consists of two domains, a tightly-folded N-terminal part and a C-terminal half with an extended and loosely folded conformation. It is likely that the N-terminal domain is located on the surface of the subunit whereas the C-terminal part is buried within the ribosomal structure. The two tyrosines in the N-terminal region behave as solvent-exposed residues, in good agreement with iodination studies on L11 in situ. It appears probable that the central region of L11, in which the protease cleavages occur, plays an important part in structural and functional aspects.     

The binding site for ribosomal protein L11 within 23 S ribosomal RNA of Escherichia coli

Ribosomal protein L11 of Escherichia coli was bound to 23 S rRNA and the resultant complex was digested with ribonuclease T1. A single RNA fragment, protected by protein L11, was isolated from such digests and was shown to rebind specifically to protein L11. The nucleotide sequence of this RNA fragment was examined by two-dimensional fingerprinting of ribonuclease digests. It proved to be 61 residues long and the constituent oligonucleotides could be fitted perfectly between residues 1052 and 1112 of the nucleotide sequence of E. coli 23 S rRNA.     

Isoaspartate in ribosomal protein S11 of Escherichia coli

Isoaspartyl sites, in which an aspartic acid residue is linked to its C-flanking neighbor via its beta-carboxyl side chain, are generally assumed to be an abnormal modification arising as proteins age. The enzyme protein L-isoaspartate methyltransferase (PIMT), present in many bacteria, plants, and animals, catalyzes the conversion of isoaspartate to normal alpha-linked aspartyl bonds and is thought to serve an important repair function in cells. Having introduced a plasmid into Escherichia coli that allows high-level expression of rat PIMT, we explored the possibility that the rat enzyme reduces isoaspartate levels in E. coli proteins, a result predicted by the repair hypothesis. The present study demonstrates that this is indeed the case; E. coli cells expressing rat PIMT had significantly lower isoaspartate levels than control cells, especially in stationary phase. Moreover, the distribution of isoaspartate-containing proteins in E. coli differed dramatically between logarithmic- and stationary-phase cultures. In stationary-phase cells, a number of proteins in the molecular mass range of 66 to 14 kDa contained isoaspartate, whereas in logarithmic-phase cells, nearly all of the detectable isoaspartate resided in a single 14-kDa protein which we identified as ribosomal protein S11. The near stoichiometric levels of isoaspartate in S11, estimated at 0.5 mol of isoaspartate per mol of S11, suggests that this unusual modification may be important for S11 function.     

On the biological role of ribosomal protein BM-L11 of Bacillus megaterium, homologous with Escherichia coli ribosomal protein L11.

Ribosomes from a thiostrepton-resistant mutant of Bacillus megaterium lack a protein, BM-L11, which is homologous with Escherichia coli ribosomal protein L11. Such ribosomes retain partial activity in cell-free synthesis of polyphenylalanine and can be restored to full activity by reconstitution with protein BM-L11. Examination of individual steps involved in polypeptide chain elongation suggested a role for protein BM-L11, and by inference for E. coli protein L11, in promoting the ribosomal GTP hydrolysis dependent upon elongation factor EF G. Evidently, however, protein BM-L11 is not indispensable for ribosomal function.     

Requirement for the Escherichia coli ribosomal protein L11 in peptide chain termination.

Subparticles of the Escherichia coli 50 S ribosome subunit containing varying amounts of the protein L11 have been prepared. These core particles have been used to form 70 S couples containing f[³H]Met-tRNA as a substrate for the peptidyl hydrolysis reaction of in vitro termination. Studies with antibodies against L11 suggested previously that the protein was involved in this event. The peptidyl transferase of the 50 S subunit core particles containing no more than 6% of the normal complement of L11 was fully active. The 70 S couples formed from 50 S cores lacking L11 showed some decrease in their ability to bind fMet-tRNA. Ribosomes lacking the proteins $\text{L}\text{7}\text{L}\text{12}$ retained about 50% of their activity for the peptidyl-tRNA hydrolysis event of in vitro termination. Cores lacking both $\text{L}\text{7}\text{L}\text{12}$ and L11 were almost as active as those lacking only $\text{L}\text{7}\text{L}\text{12}$. L11 is, therefore, not absolutely required for peptidyl-tRNA hydrolysis at termination in vitro. The ribosome subparticles lacking L11 have been reconstituted with $\text{L}\text{7}\text{L}\text{12}$. Despite the absence of L11, they regained significant activity for the codon-directed in vitro termination reaction.     

Shape of protein L11 from the 50S ribosomal subunit of Escherichia coli.

Protein L11 from the 50S ribosomal subunit of Escherichia coli A19 was purified by a method using nondenaturing conditions. Its shape in solution was studied by hydrodynamic and low-angle x-ray scattering experiments. The results from both methods are in good agreement. In buffers similar to the ribosomal reconstitution buffer, the protein is monomeric at concentrations up to 3 mg/mL and has a molecular weight of 16 000-17 000. The protein molecule resembles a prolate ellipsoid with an axial ratio of 5-6:1 a radius of gyration of 34 A, and a maximal length of 150 A. From the low-angle x-ray diffraction data, a more refined model of the protein molecule has been constructed consisting of two ellipsoids joined by their long axes.     

Methylated amino acids in ribosomal proteins from Escherichia coli treated with ethionine and from a mutant lacking methylation of protein L11.

In the present study, the nature, proportions and distribution of methylated amino acids in ribosomal proteins from Escherichia coli grown in the presence of ethionine and from mutant prm 1 were studied. The undermethylated ribosomes had been labeled by addition in vitro or in vivo of radioactive methyl groups from S-adenosylmethionine or from methionine. The following compounds were identified : N alpha-mono-, di- and trimethylalanines, N epsilon-mono-, di- and trimethyllysines, methylamine and N alpha-trimethylalanyllysine. Except for the latter compound and N-alpha-dimethylalanine, all other derivatives had been previously identified in the literature. It is shown that the dipeptide had been in the past mistaken for N epsilon-monomethyllysine, and arises through incomplete hydrolysis in 24 hrs of the N-terminal peptide bond of protein L11. The results of the present study are discussed in the light of previous work on ribosomal protein methylation by the authors and other workers in the field.     

Characterization of Escherichia coli 50S ribosomal protein L31

The published C-terminal sequence of Escherichia coli 50S ribosomal protein L31, ellipsisRFNK (Brosius, J. (1978) Biochemistry 17, 501-508), differs from that predicted by the gene sequence, ellipsisRFNKRFNIPGSK (GenBank accession no. X78541). This discrepancy might be due to post-translational processing of the protein. To examine this possibility, we have isolated L31 from E. coli strain MRE600 and sequenced the C-terminal tryptic peptide. We find the sequence to be FBIPGSK. Size comparisons of L31 from several E. coli strains demonstrate that all are identical in size to the protein isolated from MRE600 and larger than the previously described protein, indicating that ellipsisRFNKRFNIPGSK represents the true C-terminus of L31. In addition, we show that the failure to identify L31 in many ribosome preparations is probably due to the protein's loose association with the ribosome and its ability to form various intramolecular disulfide bonds, leading to L31 forms with distinct mobilities in gels.     

Primary structure of Escherichia coli ribosomal protein L31.

Protein L31 from the 50S ribosomal subunit of Escherichia coli was manually sequenced by the dansyl-Edman method. Owing to the availability of only small quantities of purified L31, sequencing methods were scaled down such that the entire primary structure could be determined with 700 microgram of protein. The techniques employed are described in detail. The protein consists of a single chain of 62 amino acids, with a calculated molecular weight of 6967. Four half-cystine residues were identified at positions 16, 18, 37, and 40. Evidence is presented that suggests that these residues form two disulfide bridges in the protein, as isolated.     

Mutational alterations of translational coupling in the L11 ribosomal protein operon of Escherichia coli.

The L11 operon in Escherichia coli consists of the genes coding for ribosomal proteins L11 and L1. It is known that translation of L1 does not take place unless the preceding L11 cistron is translated, that is, the two cistrons are translationally coupled, and this is the basis of coregulation of the translation of the two cistrons by a single repressor, L1. Several mutational analyses were carried out to define the region responsible for coupling L1 translation with L11 translation. First, by introducing several amber mutations into the L11 gene by a site-directed mutagenesis technique, it was shown that translation by ribosomes down to a position 21 nucleotides upstream, but not to a position 45 nucleotides upstream, from the end of the L11 cistron allowed the initiation of L11 translation. Second, deletion analysis indicated that a region located 23 to 20 nucleotides from the end of the L11 gene was involved in preventing independent initiation from L1 translation. Third, five different mutations obtained by screening for activation of the masked L1 initiation site were found to be clustered in a small region immediately upstream from the Shine-Dalgarno sequence of L1, and all of them were G-to-A transitions. These results, together with some additional experiments with oligonucleotide-directed mutagenesis, defined the region involved in the coupling and suggest that some special feature of this region, probably different from simple masking of the initiation site by base pairing, is responsible for translational coupling. The present results also suggest that there might be specific differences in the primary nucleotide sequence that distinguish independent translational initiation sites from translationally coupled (i.e., masked) initiation sites.     

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.     

A nuclear mutation conferring thiostrepton resistance in Chlamydomonas reinhardtii affects a chloroplast ribosomal protein related to Escherichia coli ribosomal protein L11

We have isolated a nuclear mutant (tsp-1) of Chlamydomonas reinhardtii which is resistant to thiostrepton, an antibiotic that blocks bacterial protein synthesis. The tsp-1 mutant grows slowly in the presence or absence of thiostrepton, and its chloroplast ribosomes, although resistant to the drug, are less active than chloroplast ribosomes from the wild type. Chloroplast ribosomal protein L-23 was not detected on stained gels or immunoblots of total large subunit proteins from tsp-1 probed with antibody to the wild-type L-23 protein from C. reinhardtii. Immunoprecipitation of proteins from pulse-labeled cells showed that tsp-1 synthesizes small amounts of L-23 and that the mutant protein is stable during a 90 min chase. Therefore the tsp-1 phenotype is best explained by assuming that the mutant protein synthesized is unable to assemble into the large subunit of the chloroplast ribosome and hence is degraded over time. L-23 antibodies cross-react with Escherichia coli r-protein L11, which is known to be a component of the GTPase center of the 50S ribosomal subunit. Thiostrepton-resistant mutants of Bacillus megaterium and B. subtilis lack L11, show reduced ribosome activity, and have slow growth rates. Similarities between the thiostreptonresistant mutants of bacteria and C. reinhardtii and the immunological relatedness of Chlamydomonas L-23 to E. coli L11 suggest that L-23 is functionally homologous to the bacterial r-protein L11.     

Regulation of ribosomal protein synthesis in an Escherichia coli mutant missing ribosomal protein L1.

In an Escherichia coli B strain missing ribosomal protein L1, the synthesis rate of L11 is 50% greater than that of other ribosomal proteins. This finding is in agreement with the previous conclusion that L1 regulates synthesis of itself and L11 and indicates that this regulation is important for maintaining the balanced synthesis of ribosomal proteins under physiological conditions.     

The ribosomal protein L24 of Escherichia coli is an assembly protein.

Incubation of 50 S subunits with 4.2 M LiCl leads to 4.2c cores and the complementary split protein fraction SP4.2, the latter containing quantitatively L24. L24 was removed from the split fraction by means of CM-cellulose chromatography. Partial and total reconstitution experiments performed with this protein preparation in the absence and presence of L24 demonstrate the crucial role of L24 in the early stage of assembly. However, this protein is dispensable for the subsequent steps of the in vitro assembly. 50 S subunits lacking L24 are fully active in the translation of artificial (poly(U)) and natural (R17 RNA) mRNA, indicating that L24 is not involved in any function of protein synthesis of the mature ribosome. It is therefore a mere assembly protein.     

Escherichia coli ribosomal protein L10 is rapidly degraded when synthesized in excess of ribosomal protein L7/L12.

In Escherichia coli the genes encoding ribosomal proteins L10 and L7/12, rplJ and rplL, respectively, are cotranscribed and subject to translational coupling. Synthesis of both proteins is coordinately regulated at the translational level by binding of L10 or a complex of L10 and L7/L12 to a single target in the mRNA leader region upstream of rplJ. Unexpectedly, small deletions that inactivated the ribosome-binding site of the rplL gene carried on multicopy plasmids exerted a negative effect on expression of the upstream rplJ gene. This effect could be overcome by overproduction of L7/L12 in trans from another plasmid. This apparent stimulation resulted from stabilization of the overproduced L10 protein by L7/L12, presumably because free L10, in contrast to L10 complexed with L7/L12, is subject to rapid proteolytic decay. The contribution of this decay mechanism to the regulation of the rplJL operon is evaluated.     

The tetrameric form of ribosomal protein L7/L12 from Escherichia coli

A tetrameric form of the ribosomal protein L7/L12 has been prepared and its structure studied by using hydrodynamic methods, photon correlation spectroscopy, and small angle x-ray scattering. The tetrameric nature of the protein preparation is confirmed by three independent determinations of its molecular weight, with analysis of accurate sedimentation equilibrium data giving the most reliable estimate. The species has a Stokes radius of 4.0 +/- 0.1 nm and an absolute frictional ratio of 1.7. Taken together, the hydrodynamic measurements suggest the possibility of a flat structure, and this is consistent with the x-ray scattering results. The molecule has a radius of gyration of 3.6 +/- 0.05 nm and a maximum dimension of 11-12 nm. A geometric model consisting of four elongated monomers, arranged in a plane, is proposed.