The structure and evolution of the ribosomal proteins encoded in the spc operon of the archaeon (Crenarchaeota) Sulfolobus acidocaldarius.

The genes for nine ribosomal proteins, L24, L5, S14, S8, L6, L18, S5, L30, and L15, have been isolated and sequenced from the spc operon in the archaeon (Crenarchaeota) Sulfolobus acidocaldarius, and the putative amino acid sequence of the proteins coded by these genes has been determined. In addition, three other genes in the spc operon, coding for ribosomal proteins S4E, L32E, and L19E (equivalent to rat ribosomal proteins S4, L32, and L19), were sequenced and the structure of the putative proteins was determined. The order of the ribosomal protein genes in the spc operon of the Crenarchaeota kingdom of Archaea is identical to that present in the Euryarchaeota kingdom of Archaea and also identical to that found in bacteria, except for the genes for r-proteins S4E, L32E, and L19E, which are absent in bacteria. Although AUG is the initiation codon in most of the spc genes, GUG (val) and UUG (leu) are also used as initiation codons in S. acidocaldarius. Over 70% of the codons in the Sulfolobus spc operon have A or U in the third position, reflecting the low GC content of Sulfolobus DNA. Phylogenetic analysis indicated that the archaeal r-proteins are a sister group of their eucaryotic counterparts but did not resolve the question of whether the Archaea is monophyletic, as suggested by the L6P, L15P, and L18P trees, or the question of whether the Crenarchaeota is separate from the Euryarchaeota and closer to the Eucarya, as suggested by the S8P, S5P, and L24P trees. In the case of the three Sulfolobus r-proteins that do not have a counterpart in the bacterial ribosome (S4E, L32E, and L19E), the archaeal r-proteins showed substantial identity to their eucaryotic equivalents, but in all cases the archaeal proteins formed a separate group from the eucaryotic proteins.     

Cloning and analysis of the spc ribosomal protein operon of Bacillus subtilis: comparison with the spc operon of Escherichia coli.

A segment of Bacillus subtilis chromosomal DNA homologous to the Escherichia coli spc ribosomal protein operon was isolated using cloned E. coli rplE (L5) DNA as a hybridization probe. DNA sequence analysis of the B. subtilis cloned DNA indicated a high degree of conservation of spc operon ribosomal protein genes between B. subtilis and E. coli. This fragment contains DNA homologous to the promoter-proximal region of the spc operon, including coding sequences for ribosomal proteins L14, L24, L5, S14, and part of S8; the organization of B. subtilis genes in this region is identical to that found in E. coli. A region homologous to the E. coli L16, L29 and S17 genes, the last genes of the S10 operon, was located upstream from the gene for L14, the first gene in the spc operon. Although the ribosomal protein coding sequences showed 40-60% amino acid identity with E. coli sequences, we failed to find sequences which would form a structure resembling the E. coli target site for the S8 translational repressor, located near the beginning of the L5 coding region in E. coli, in this region or elsewhere in the B. subtilis spc DNA.     

Expression of ribosomal protein genes cloned in a hybrid plasmid in Escherichia coli: gene dosage effects on synthesis of ribosomal proteins and ribosomal protein messenger ribonucleic acid.

Using ColE1-TnA hybrid plasmid RSF2124 as the cloning vector, we constructed a hybrid plasmid, pNO1001, which carried seven ribosomal protein (r-protein) genes in the spc operon together with their promoter. The plasmid also carried three r-protein genes which precede the spc operon, but did not carry the bacterial promoter for these genes. Expression of r-protein genes carried by pNO1001 was studied by measuring messenger ribonucleic acid and r-protein synthesis in cells carrying the plasmid. It was found that the messenger ribonucleic acid for all the promoter-distal r-protein genes was synthesized in large excess relative to messenger ribonucleic acid from other chromosomal r-protein genes which are not carried by the plasmid. However, only the two promoter-proximal r-proteins, L14 and L24, were markedly overproduced. The absence of large gene dosage effects on the synthesis of other distal proteins appeared to be due, at least in part, to preferential inactivation and/or degradation of the distal message which codes for these proteins; in addition, some preferential inhibition of translation of the distal message might also have been involved. Overproduced L14 and L24 were found to be degraded in recA+ strains at both 30 and 42 degrees C; in recA strains, the degradation took place at 42 degrees C but was very slow or absent at 30 degrees C. The recA strains carrying pNO1001 failed to form colonies at 30 degrees C, presumably because of overaccumulation of r-proteins. The results suggest that degradation of excess r-proteins is an important physiological process.     

Analysis of the spc ribosomal protein operon of Thermus aquaticus

The gene region of Thermus aquaticus corresponding to the distal portion of the S10 operon and to the 5'-portion of the Escherichia coli spc operon was cloned, using the E. coli gene for the ribosomal protein L5 as hybridization probe. The gene arrangement was found to be identical to E. coli, i.e. S17, L14, L24, L5, S14, S8 and L6. Stop and start regions of contiguous cistrons overlap, except for the S14-S8 intergenic region, whose size (67 bases) even exceeds the corresponding spacer regions in E. coli and Bacillus subtilis. A G + C content of 94% in third positions of codons was found in the ribosomal protein genes of T. aquaticus analyzed here. The stop codon of gene S17 (the last gene of the S10 operon in E. coli) and the start codon of gene L14 (the first gene of the spc operon in E. coli) overlap in T. aquaticus, thus leaving no space to accommodate an intergenic promoter preceding spc-operon-encoded genes in T. aquaticus. A possible promoter, localized within the S17 coding region, yielded only weak resistance (20 micrograms/ml) to chloramphenicol in E. coli and therefore could be largely excluded as the main promoter for spc-operon-encoded genes. We failed to detect a structure resembling the protein S8 translational repressor site, located at the beginning of the L5 gene in E. coli, in the corresponding region or any other region in the cloned T. aquaticus spc DNA.     

Spectinomycin operon ofMicrococcus luteus: Evolutionary implications of organization and novel codon usage

Summary The complete DNA sequence of theMicrococcus luteus spectinomycin (spc) operon and its adjacent regions has been determined. The sequence has revealed the presence of genes that are homologous to those of theEscherichia coli ribosomal and related proteins, L14, L24, L5, S8, L6, L18, S5, L30, L15, and secretion protein Y (secY), and the gene for adenylate kinase (adk). The gene arrangement in the spc operon is essentially the same as that ofE. coli except for the absence in theM. luteus spc operon of the genes for S14 and X protein that exist in theE. coli spc operon.SecY andadk seem to be composed of another operon (adk operon) with at least an open reading frame. The deduced amino acid sequences for these ribosomal proteins are well conserved among the two species (40–65% identity). Reflecting the high genomic guanine and cytosine (GC) content ofM. luteus (74%), the codon usage of the genes is extremely biased toward use of G and C, about 94% of the codon third positions being G or C. Seven codons, AUA, AAA, AGA, UUA, GUA, CUA, and CAA, all of which have A at the codon third positions, are completely absent in theM. luteus genes examined. Out of 11 genes in theM. luteus spc and adk operons, 5 (10) use GUG (UGA) and 6 (1) use AUG (UAA) as an initiation (termination) codon.     

Translational regulation by ribosomal protein S8 in Escherichia coli: structural homology between rRNA binding site and feedback target on mRNA.

It has been previously shown that ribosomal protein synthesis in Escherichia coli is regulated at the level of translation by certain key ribosomal proteins. In the spc operon, S8 regulates the expression of L5 and some of the subsequent genes, while the first two genes (L14 and L24) are regulated independently. We therefore determined the DNA sequence at the junction of the L24 and L5 genes, which corresponds to the putative feedback target for S8. We show that there is a striking homology between the structure of the mRNA for this region and the known binding site for S8 on 16S rRNA. These results support the theory that the regulation of ribosomal protein synthesis is based on competition between rRNA and mRNA for regulatory ribosomal proteins.     

The structure of a ribosomal protein S8/spc operon mRNA complex

In bacteria, translation of all the ribosomal protein cistrons in the spc operon mRNA is repressed by the binding of the product of one of them, S8, to an internal sequence at the 5' end of the L5 cistron. The way in which the first two genes of the spc operon are regulated, retroregulation, is mechanistically distinct from translational repression by S8 of the genes from L5 onward. A 2.8 A resolution crystal structure has been obtained of Escherichia coli S8 bound to this site. Despite sequence differences, the structure of this complex is almost identical to that of the S8/helix 21 complex seen in the small ribosomal subunit, consistent with the hypothesis that autogenous regulation of ribosomal protein synthesis results from conformational similarities between mRNAs and rRNAs. S8 binding must repress the translation of its own mRNA by inhibiting the formation of a ribosomal initiation complex at the start of the L5 cistron.     

Sequencing and analysis of the Thermus thermophilus ribosomal protein gene cluster equivalent to the spectinomycin operon

To assess the organization of the Thermus thermophilus ribosomal protein genes, a fragment of DNA containing the complete S10 region and ten ribosomal protein genes of the spc region was cloned, using an oligonucleotide coding for the N-terminal amino acid (aa) sequence of T. thermophilus S8 protein as hybridization probe. The nucleotide sequence of a 4290 bp region between the rps17 and rpl15 genes was determined. Comparative analysis of this gene cluster showed that the gene arrangement (S17, L14, L24, L5, S14, S8, L6, L18, S5, L30 and L15) is identical to that of eubacteria. However, T. thermophilus ribosomal protein genes corresponding to the Escherichia coli S10 and spc operons are not resolved into two clusters: the stop codon of the rps17 gene (the last gene of the S10 operon in E. coli) and the start codon of the rpl14 gene (the first gene of the spc operon in E. coli) overlap. Most genes, except the rps14-rps8 intergenic spacer (69 bp), are separated by very short (only 3–7 bp) spacer regions or partially overlapped. The deduced aa sequences of T. thermophilus proteins share about 51–100% identities with the sequences of homologous proteins from thermophile Thermus aquaticus and Thermotoga maritima and 27–70% identities with the sequences of their mesophile counterparts.     

Nucleotide sequence of a Lactococcus lactis gene cluster encoding adenylate kinase, initiation factor 1 and ribosomal proteins.

We have previously isolated a putative promoter from the Lactococcus lactis subsp. lactis chromosome. We now report the sequence of the promoter fragment and its extension in the 5'-direction. The region contains several open-reading frames which correspond to ribosomal protein L15, SecY, adenylate kinase, initiation factor 1 and ribosomal proteins B and S13. The order of the genes, rplO (L15), secY, adk, infA, rpmJ (B) and rpsM (S13), is similar to that in the spc and alpha operon region of Bacillus subtilis, with the exception of the map gene, coding for methionine amino peptidase, which is located between adk and infA in B. subtilis. The putative promoter is located between adk and infA.     

Cytoplasmic ribosomal protein genes of the fission yeast Schizosaccharomyces pombe display a unique promoter type: a suggestion for nomenclature of cytoplasmic ribosomal proteins in databases.

We identified 34 new ribosomal protein genes in the Schizosaccharomyces pombe database at the Sanger Centre coding for 30 different ribosomal proteins. All contain the Homol D-box in their promoter. We have shown that Homol D is, in this promoter type, the TATA-analogue. Many promoters contain the Homol E-box, which serves as a proximal activation sequence. Furthermore, comparative sequence analysis revealed a ribosomal protein gene encoding a protein which is the equivalent of the mammalian ribosomal protein L28. The budding yeast Saccharomyces cerevisiae has no L28 equivalent. Over the past 10 years we have isolated and characterized nine ribosomal protein (rp) genes from the fission yeast S.pombe . This endeavor yielded promoters which we have used to investigate the regulation of rp genes. Since eukaryotic ribosomal proteins are remarkably conserved and several rp genes of the budding yeast S.cerevisiae were sequenced in 1985, we probed DNA fragments encoding S.cerevisiae ribosomal proteins with genomic libraries of S.pombe . The deduced amino acid sequence of the different isolated rp genes of fission yeast share between 65 and 85% identical amino acids with their counterparts of budding yeast.     

Characterization of an amber mutation in the structural gene for ribosomal protein L15, which impairs the expression of the protein export gene, secY, in Escherichia coli

We have previously described a temperature-sensitive mutant, ts215, which is defective in protein secretion. Complementation studies indicated that the mutation was located at the distal part of the spc ribosomal protein operon and the gene secY is required for efficient protein secretion. We now report a more complete genetic and biochemical analysis of the ts215 mutant. These studies revealed that the ts215 mutant has an amber mutation in the gene rp10 for ribosomal protein L15, which is located upstream and adjacent to secY. The amber mutation exerts a polar effect on secY causing a defect in protein secretion. These conclusions were supported by the following observations. The mutant strain carries a phi 80 prophage containing a temperature-sensitive suppressor, supFts6. The strain contains decreased amounts of L15 and is suppressible by a temperature-independent nonsense suppressor. In addition, L15 contains an extra tyrosine residue when suppressed by supF. DNA sequence analysis revealed the presence of a single base change in rp10 resulting in an amber codon at the 38th codon of L15. The mutant phenotype is complemented by a plasmid carrying only the secY gene under lac promoter control. The mutant cells complemented by secY can grow and synthesize proteins at normal rates and abundances at 42 degrees C, despite the fact that their ribosomes contain barely detectable levels of L15. These results indicate that ribosomal protein L15 is dispensable for protein synthesis and cell growth. In contrast, the decreased level of expression of the secY gene leads to defective protein secretion and defective cell growth.     

Regulation of ribosomal protein synthesis in Vibrio cholerae

We have investigated the regulation of the S10 and spc ribosomal protein (r-protein) operons in Vibrio cholerae. Both operons are under autogenous control; they are mediated by r-proteins L4 and S8, respectively. Our results suggest that Escherichia coli-like strategies for regulating r-protein synthesis extend beyond the enteric members of the gamma subdivision of proteobacteria.