Organization of genes for ribosomal proteins S7 and S12, elongation factors EF-Tu and EF-G in the cyanobacterium Spirulina platensis

The gene encoding ribosomal proteins S12 and probably S7 as well as protein synthesis elongation factors Tu (EF-Tu) and G (EF-G) of Spirulina platensis have been identified and cloned. Gene expression was determined for ribosomal protein S12 by genetic complementation of the appropriate Escherichia coli mutant, whereas for the EF-Tu gene it was determined by production of the protein in E. coli minicells. On the basis of these experiments we suggest the following gene order in the S. platensis chromosome: S12, S7, EF-G, EF-Tu.     

Identification of genes for elongation factor Ts and ribosomal protein S2 in E. coli

The structural gene for elongation factor EF-TS (tsf) and that for ribosomal protein S2 (rpsB) have been identified in E. coli. Both genes are carried by λ transducing phages that have been isolated as dapD^?polC⁺ transducing phages. Synthesis of both S2 and EF-Ts was demonstrated in ultraviolet light-irradiated E. coli cells infected with these phages. Experiments were also done using other transducing phages that carry dapD⁺ but not polC⁺. The data indicate that both the tsf and rpsB genes map near dapD at about 4 min on the E. coli genetic map. This location is different from the two chromosomal locations, the str-spc region and the rif region, where many ribosomal protein genes, the genes for RNA polymerase components, as well as other elongation factor genes (fus, tufA, and tufB) are located.     

Elongation factor Ts from the Antarctic eubacterium Pseudoalteromonas haloplanktis TAC 125: biochemical characterization and cloning of the encoding gene

The elongation factor Ts was isolated from the psychrophilic Antarctic eubacterium Pseudoalteromonas haloplanktis TAC 125 strain (PhEF-Ts), and its functional properties were studied. At 0 degrees C PhEF-Ts enhanced the [(3)H]GDP/GDP exchange rate on the preformed PhEF-Tu.[(3)H]GDP complex by 2 orders of magnitude even at very low Tu:Ts ratio, by lowering the energy of activation of the exchange reaction. PhEF-Ts is a monomeric protein, and in solution it forms a stable dimeric complex with PhEF-Tu. The PhEF-Ts encoding gene was cloned and sequenced. Its structural organization was similar to that of Escherichia coli because it showed at its 5' end the gene encoding the ribosomal protein S2. The translated amino acid sequence had a calculated molecular weight of 30762, and showed a high sequence identity with E. coli (68%) and Thermus thermophilus (44%) EF-Ts. The PhEF-Ts primary structure contains well-preserved almost all the amino acid residues interacting at the interfaces of the E. coli EF-Ts.EF-Tu complex. Finally, the high concentration of PhEF-Ts in this psychrophilic eubacterium might represent an adaptive tool to ensure an efficient nucleotide exchange even at low temperature.     

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.     

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.     

Restoration by ribosomal protein S1 of the defective translation in a temperature-sensitive mutant of Escherichia coli K-12: characterization and genetic studies.

A temperature-sensitive mutant of Escherichia coli was isolated that had a temperature-sensitive defect in ribosomal-wash protein(s) required for translation in vitro of E. coli endogenous messenger ribonucleic acid. It was found that 30S ribosomal protein S1 rescued the defect in the ribosomal-wash protein(s) of the mutant and that the complete restoration to the wild-type level was attained when 1 mol of protein S1 was added to 1 mol of 70S ribosome. The mutation, tss, causing such a defect was mapped at 21 min and was closely linked to the pyrD locus, the region of which was entirely different from that of the other genes coding for the many ribosomal proteins of E. coli. These results indicate that the gene specified by this mutation is involved in the function of the 30S ribosomal protein S1.     

Sequence and functional similarity between a yeast ribosomal protein and the Escherichia coli S5 ram protein.

The accurate and efficient translation of proteins is of fundamental importance to both bacteria and higher organisms. Most of our knowledge about the control of translational fidelity comes from studies of Escherichia coli. In particular, ram (ribosomal ambiguity) mutations in structural genes of E. coli ribosomal proteins S4 and S5 have been shown to increase translational error frequencies. We describe the first sequence of a ribosomal protein gene that affects translational ambiguity in a eucaryote. We show that the yeast omnipotent suppressor SUP44 encodes the yeast ribosomal protein S4. The gene exists as a single copy without an intron. The SUP44 protein is 26% identical (54% similar) to the well-characterized E. coli S5 ram protein. SUP44 is also 59% identical (78% similar) to mouse protein LLrep3, whose function was previously unknown (D.L. Heller, K.M. Gianda, and L. Leinwand, Mol. Cell. Biol. 8:2797-2803, 1988). The SUP44 suppressor mutation occurs near a region of the protein that corresponds to the known positions of alterations in E. coli S5 ram mutations. This is the first ribosomal protein whose function and sequence have been shown to be conserved between procaryotes and eucaryotes.     

Nucleotide sequences of Bacillus stearothermophilus ribosomal protein genes: part of the ribosomal S10 operon

Restriction fragments from Bacillus stearothermophilus chromosomal DNA were cross-hybridized with the Escherichia coli ribosomal protein L2 gene rplB. A 2-kb EcoRI fragment which showed cross-hybridization was cloned into the M13 phage and sequenced by the dideoxy chain-terminating method. Comparison of the deduced amino-acid sequences with the corresponding sequences of E. coli ribosomal proteins showed that this fragment contains the region encoding the C-terminus of L2, the genes encoding S19, L22, S3 as well as the N-terminus of L16. Thus the organization of this gene cluster is the same as that in the S10 operon of E. coli. The deduced sequences of proteins L22 and S3, which have not been determined so far, were found to have 52% or 55% amino-acid identity, respectively, with those of the corresponding proteins in E. coli. The deduced B. stearothermophilus S19 protein sequence was in accordance with the reinvestigated protein sequence (H. Hirano, personal communication).     

The rpsD gene, encoding ribosomal protein S4, is autogenously regulated in Bacillus subtilis.

Although the mechanisms for regulation of ribosomal protein gene expression have been established for gram-negative bacteria such as Escherichia coli, the regulation of these genes in gram-positive bacteria such as Bacillus subtilis has not yet been characterized. In this study, the B. subtilis rpsD gene, encoding ribosomal protein S4, was found to be subject to autogenous control. In E. coli, rpsD is located in the alpha operon, and S4 acts as the translational regulator for alpha operon expression, binding to a target site in the alpha operon mRNA. The target site for repression of B. subtilis rpsD by protein S4 was localized by deletion and oligonucleotide-directed mutagenesis to the leader region of the monocistronic rpsD gene. The B. subtilis rpsD leader exhibits little sequence homology to the E. coli alpha operon leader but may be able to form a pseudoknotlike structure similar to that found in E. coli.     

Cloning, sequencing, and expression in Escherichia coli of the gene encoding a 45-kilodalton protein, elongation factor Tu, from Chlamydia trachomatis serovar F.

The gene encoding a 45-kDa protein (45K) of Chlamydia trachomatis serovar F was cloned, sequenced, and overexpressed in Escherichia coli. Alignment of the deduced peptide sequence with E. coli elongation factor Tu (EF-Tu) demonstrated 69% identity. The 45K was recognized by a Chlamydia genus-specific monoclonal antibody GP-45 and cross-reacted with a monospecific polyclonal antibody to E. coli EF-Tu. Purified recombinant 45K has the capability to bind GDP, and the binding was enhanced in the presence of E. coli elongation factor Ts (EF-Ts). The GDP binding was specifically inhibited by the monoclonal antibody GP-45. These data suggest that the 45K is a chlamydial EF-Tu, and it forms a functional complex with E. coli EF-Ts protein.     

5S rRNA binding proteins from the hyperthermophilic archaeon, Pyrococcus furiosus

A determination was made of the nucleotide sequence of the 2719 bp region of a ribosomal protein gene cluster (PfeL32-PfeL19-PfL18-PfS5-PfL30) containing a 5S rRNA binding protein L18 homolog of hyperthermophilic archaea Pyrococcus furiosus. The organization of the archaeal ribosomal protein gene cluster is similar to that in the spc-operon of Escherichia coli (L6-L18-S5-L30-L15) but has two additional genes, namely those encoding PfeL32 and PfeL19, which were identified as extra proteins that are apparently not present in bacterial E. coli. Using an inducible expression system, P. furiosus mature PfL18 protein and a mutant PfL18 with the basic N-terminal amino acid region deleted were produced in large amounts in E. coli and Northwestern analysis showed the N-terminal region of PfL18, including the conserved arginine-rich region, to have a significant role in 5S rRNA-PfL18 interaction.     

Plant cytosolic ribosomal protein S11 and chloroplast ribosomal protein CS17. Their primary structures and evolutionary relationships

We have isolated cDNA clones specific for Arabidopsis thaliana cytosolic ribosomal protein S11 and plastid ribosomal protein CS17, both of which are encoded in the nuclear genome, through the use of the corresponding soybean and pea cDNAs as probes, respectively. The nucleotide sequences of all four cDNAs were determined. The amino acid sequences derived from these cDNA sequences show that the soybean and A. thaliana S11 cDNAs encode proteins that are homologous to rat ribosomal protein S11 and that the pea and A. thaliana CS17 cDNAs encode proteins that are homologous to Escherichia coli ribosomal protein S17. The plant S11 cytosolic ribosomal proteins also show significant sequence similarity to both E. coli ribosomal protein S17 and plastid CS17 indicating that these are all related proteins. Comparison of A. thaliana CS17 with A. thaliana S11 and with E. coli S17 suggests that CS17 is more related to S17 than it is to S11. These results support the idea that the gene encoding CS17 was derived from a prokaryotic endosymbiont and not from a duplication of the eukaryotic S11 gene.     

Structural and functional analyses of a yeast mitochondrial ribosomal protein homologous to ribosomal protein S15 of Escherichia coli.

We have purified a small subunit mitochondrial ribosomal protein, MRPS28p, from the yeast, Saccharomyces cerevisiae. Sequence from the amino terminus of MRPS28p was used to design a degenerate oligonucleotide that was complementary to the MRPS28 gene. The MRPS28 gene was isolated and its sequence determined. The MRPS28 sequence encodes a 28 kDa protein that has a region of homology with ribosomal protein S15 of E. coli. This region spans the entire length of the E. coli protein, but as MRPS28p is larger, includes only the portion of the MRPS28p sequence from amino acids 150 to 238. Based on this homology, we predict that MRPS28p, like E. coli S15, interacts directly with small subunit rRNA and functions as an early protein in ribosome assembly. Cells carrying a disrupted chromosomal copy of MRPS28 are unable to respire and spontaneously lose portions of their mitochondrial genomes at a high frequency. These phenotypes are consistent with an essential role for MRPS28p in the assembly and/or function of the mitochondrial ribosome.     

An archaebacterial gene from Methanococcus vannielii encoding a protein homologous to the ribosomal protein L10 family

An open reading frame upstream of the Methanococcus vannielii L12 gene has been detected. The beginning of this open reading frame agrees with the N-terminal region of a protein (MvaL10) which has been isolated from the 50 S ribosomal subunit of M. vannielii and sequenced. The length of this gene is 1008 nucleotides, coding for 336 amino acids. Excellent sequence similarities were found to the L10-like ribosomal proteins from Halobacterium halobium and man. The N-terminal part of the MvaL10 protein shows significant sequence similarities to the E. coli L10 protein. MvaL10 is more than twice as long as E. coli L10 but is of length similar to those of the homologous halobacterial and human proteins. Interestingly, the C-terminal region of MvaL10 shows exceptionally high similarity to the C-terminal sequence of the MvaL12 protein. This is not the case for the E. coli proteins but was also observed for the human, Halobacterium and Sulfolobus proteins.     

Cotransduction of strA and Ribosomal Protein Cistrons in Escherichia coli-Salmonella typhimurium Hybrids

Genetic mapping of ribosomal protein cistrons of Salmonella typhimurium and Escherichia coli was performed by phage P1 mediated, generalized transduction. From an E. coli hybrid strain which carried a S. typhimuirum F' factor, an E. coli strain was constructed which had integrated S. typhimurium genetic material including the region of the strA locus. Salmonella genetic material from this hybrid was transduced into E. coli recipients. The ribosomal protein electrophoretic patterns of these hybrid transductants were correlated with the presence of markers contributed by each parent.The results of these studies indicate that cistrons for at least three characteristic S. typhimurium and two E. coli 30S ribosomal proteins are closely linked to the strA locus on the genetic maps of both organisms. At least one cistron coding for a 50S ribosomal protein is also closely linked to this locus on both maps. These findings support the concept that cistrons coding for the ribosomal proteins are clustered in one area of the genome. Mutations to spectinomycin and streptomycin resistance are closely linked in S. typhimurium and are located at strA.     

Antibiotic Resistance Mutations in the Chloroplast 16s and 23s Rrna Genes of Chlamydomonas Reinhardtii: Correlation of Genetic and Physical Maps of the Chloroplast Genome

Mutants resistant to streptomycin, spectinomycin, neamine/kanamycin and erythromycin define eight genetic loci in a linear linkage group corresponding to about 21 kb of the circular chloroplast genome of Chlamydomonas reinhardtii. With one exception, all of these mutants represent single base-pair changes in conserved regions of the genes encoding the 16S and 23S chloroplast ribosomal RNAs. Streptomycin resistance can result from changes at the bases equivalent to Escherichia coli 13, 523, and 912-915 in the 16S gene, or from mutations in the rps12 gene encoding chloroplast ribosomal protein S12. In the 912-915 region of the 16S gene, three mutations were identified that resulted in different levels of streptomycin resistance in vitro. Although the three regions of the 16S rRNA mutable to streptomycin resistance are widely separated in the primary sequence, studies by other laboratories of RNA secondary structure and protein cross-linking suggest that all three regions are involved in a common ribosomal neighborhood that interacts with ribosomal proteins S4, S5 and S12. Three different changes within a conserved region of the 16S gene, equivalent to E. coli bases 1191-1193, confer varying levels of spectinomycin resistance, while resistance to neamine and kanamycin results from mutations in the 16S gene at bases equivalent to E. coli 1408 and 1409. Five mutations in two genetically distinct erythromycin resistance loci map in the 23S rDNA of C. reinhardtii, at positions equivalent to E. coli 2057-2058 and 2611, corresponding to the rib3 and rib2 loci of yeast mitochondria respectively. Although all five mutants are highly resistant to erythromycin, they differ in levels of cross-resistance to lincomycin and clindamycin. The order and spacing of all these mutations in the physical map are entirely consistent with our genetic map of the same loci and thereby validate the zygote clone method of analysis used to generate this map. These results are discussed in comparison with other published maps of chloroplast genes based on analysis by different methods using many of the same mutants.     

Nucleotide sequence of the ribosomal RNA gene of Physarum polycephalum: intron 2 and its flanking regions of the 26S rRNA gene.

The 26S ribosomal RNA gene of Physarum polycephalum is interrupted by two introns, and we have previously determined the sequence of one of them (intron 1) (Nomiyama et al. Proc.Natl.Acad.Sci.USA 78, 1376-1380, 1981). In this study we sequenced the second intron (intron 2) of about 0.5 kb length and its flanking regions, and found that one nucleotide at each junction is identical in intron 1 and intron 2, though the junction regions share no other sequence homology. Comparison of the flanking exon sequences to E. coli 23S rRNA sequences shows that conserved sequences are interspersed with tracts having little homology. In particular, the region encompassing the intron 2 interruption site is highly conserved. The E. coli ribosomal protein L1 binding region is also conserved.