Thermus thermophilus L11 methyltransferase, PrmA, is dispensable for growth and preferentially modifies free ribosomal protein L11 prior to ribosome assembly

The ribosomal protein L11 in bacteria is posttranslationally trimethylated at multiple amino acid positions by the L11 methyltransferase PrmA, the product of the prmA gene. The role of L11 methylation in ribosome function or assembly has yet to be determined, although the deletion of Escherichia coli prmA has no apparent phenotype. We have constructed a mutant of the extreme thermophile Thermus thermophilus in which the prmA gene has been disrupted with the htk gene encoding a heat-stable kanamycin adenyltransferase. This mutant shows no growth defects, indicating that T. thermophilus PrmA, like its E. coli homolog, is dispensable. Ribosomes prepared from this mutant contain unmethylated L11, as determined by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), and are effective substrates for in vitro methylation by cloned and purified T. thermophilus PrmA. MALDI-TOF MS also revealed that T. thermophilus L11 contains a total of 12 methyl groups, in contrast to the 9 methyl groups found in E. coli L11. Finally, we found that, as with the E. coli methyltransferase, the ribosomal protein L11 dissociated from ribosomes is a more efficient substrate for in vitro methylation by PrmA than intact 70S ribosomes, suggesting that methylation in vivo occurs on free L11 prior to its incorporation into ribosomes.     

Mutants of Escherichia coli lacking ribosomal protein L11

Three mutants with ribosomes apparently lacking Protein L11, AM68, AM76, and AM77, were investigated using a variety of immunological techniques to determine whether L11 was indeed lacking. Ouchterlony double diffusion, modified immunoelectrophoresis, and dimer formation on sucrose gradients all gave results indicating Protein L11 was missing from the ribosome in these mutants. Electron micrographs of ribosomes of the mutants were indistinguishable from those of wild type. Ribosomes of AM68, AM76, and AM77, did not bind the antibiotic thiostrepton, but binding was recovered upon reconstitution with wild type Protein L11.     

Genetics of ribosomal protein methylation in Escherichia coli. I. A mutant deficient in methylation of protein L11.

Summary Several thousand mutagenized clones of Escherichia coli were screened for methyl group incorporation into protein in crude extracts, in order to isolate mutants lacking the full complement of methyl groups in ribosomal proteins. One mutant isolated by this method and designated prm-1 incorporated 6–7 methyl groups per ribosome upon incubation of its ribosomes with a partially purified enzyme preparation from E. coli wild-type. The methyl groups were located exclusively in the 50S particle and for the most part (85%) in protein L11. Three methylated amino acids were detected: -N-trimethyllysine, -N-monomethyllysine, and an uncharacterized amino acid. These accounted respectively for 4.6, 1.3 and 0.9 methyl groups per ribosome. These results indicate that protein L11 in wild-type contains a stoichiometric amount of these methylated amino acids which are absent in mutant prm-1. Since this mutant is fully viable, its methylation deficiency does not result in a major defect in ribosome assembly or functioning.     

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.     

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.     

Ribosomal protein L30 is dispensable in the yeast Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, L30 is one of many ribosomal proteins that is encoded by two functional genes. We have cloned and sequenced RPL30B, which shows strong homology to RPL30A. Use of mRNA as a template for a polymerase chain reaction demonstrated that RPL30B contains an intron in its 5' untranslated region. This intron has an unusual 5' splice site, C/GUAUGU. The genomic copies of RPL30A and RPL30B were disrupted by homologous recombination. Growth rates, primer extension, and two-dimensional ribosomal protein analyses of these disruption mutants suggested that RPL30A is responsible for the majority of L30 production. Surprisingly, meiosis of a diploid strain carrying one disrupted RPL30A and one disrupted RPL30B yielded four viable spores. Ribosomes from haploid cells carrying both disrupted genes had no detectable L30, yet such cells grew with a doubling time only 30% longer than that of wild-type cells. Furthermore, depletion of L30 did not alter the ratio of 60S to 40S ribosomal subunits, suggesting that there is no serious effect on the assembly of 60S subunits. Polysome profiles, however, suggest that the absence of L30 leads to the formation of stalled translation initiation complexes.     

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.     

Multiple-site trimethylation of ribosomal protein L11 by the PrmA methyltransferase

Ribosomal protein L11 is a universally conserved component of the large subunit, and plays a significant role during initiation, elongation, and termination of protein synthesis. In Escherichia coli, the lysine methyltransferase PrmA trimethylates the N-terminal alpha-amino group and the epsilon-amino groups of Lys3 and Lys39. Here, we report four PrmA-L11 complex structures in different orientations with respect to the PrmA active site. Two structures capture the L11 N-terminal alpha-amino group in the active site in a trimethylated post-catalytic state and in a dimethylated state with bound S-adenosyl-L-homocysteine. Two other structures show L11 in a catalytic orientation to modify Lys39 and in a noncatalytic orientation. The comparison of complex structures in different orientations with a minimal substrate recognition complex shows that the binding mode remains conserved in all L11 orientations, and that substrate orientation is brought about by the unusual interdomain flexibility of PrmA.     

The N-terminal extension of Escherichia coli ribosomal protein L20 is important for ribosome assembly, but dispensable for translational feedback control

The Escherichia coli autoregulatory ribosomal protein L20 consists of two structurally distinct domains. The C-terminal domain is globular and sits on the surface of the large ribosomal subunit whereas the N-terminal domain has an extended shape and penetrates deep into the RNA-rich core of the subunit. Many other ribosomal proteins have analogous internal or terminal extensions. However, the biological functions of these extended domains remain obscure. Here we show that the N-terminal tail of L20 is important for ribosome assembly in vivo. Indeed, a truncated version of L20 without its N-terminal tail is unable to complement the deletion of rplT, the gene encoding L20. In addition, this L20 truncation confers a lethal-dominant phenotype, suggesting that the N-terminal domain is essential for cell growth because it could be required for ribosome assembly. Supporting this hypothesis, partial deletions of the N-terminal tail of the protein are shown to cause a slow-growth phenotype due to altered ribosome assembly in vivo as large amounts of intermediate 40S ribosomal particles accumulate. In addition to being a ribosomal protein, L20 also acts as an autogenous repressor. Using L20 truncations, we also show that the N-terminal tail of L20 is dispensable for autogenous control.     

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.     

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.     

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.     

Ribosomal assembly deficiency in an Escherichia coli thermosensitive mutant having an altered L24 ribosomal protein.

Localized P1 mutagenesis was used to screen for conditionally lethal mutations in ribosomal protein genes. One such mutation, 2859mis, has been mapped inside the ribosomal protein gene cluster at 72 minutes on the Escherichia coli chromosome and cotransduces at 98% with rpsE (S5). The 2869mis mutation leads to thermosensitivity and impaired assembly in vivo of 50 S ribosomal particles at 42 °C. The strain carrying the mutation has an altered L24 ribosomal protein which at 42 °C shows weaker affinity for 23 S RNA than the wild-type protein. The mutational alteration involves a replacement of glycine by aspartic acid in protein L24 from the mutant. We conclude therefore that the 2859mis mutation affects the structural gene for protein L24 (rplX).     

Characterization of the smtA gene encoding an S-adenosylmethionine-dependent methyltransferase of Escherichia coli

Abstract The mukB operon is located at 21 min on the Escherichia coli chromosome and seems to consist of four genes, orf30 ( smtA ), mukF , mukE , and mukB . Based on sequence similarity, the promoter-proximal gene, orf30 ( smtA ), could encode an S-adenosylmethionine-dependent methyltransferase. The smtA gene is not essential for cell growth and its expression is positively regulated by H-NS, an Escherichia coli histone-like 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.     

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.     

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.