Bacterial Ribosomal Protein Research Articles

A Leuconostoc lactis protein with homology to ribosomal protein S1 shares common epitopes and common DNA binding properties with a mammalian DNA binding nuclear factor.

A mouse testis cDNA expression library (Clontech) was screened with a synthetic oligonucleotide ligand containing CT-rich motifs derived from the rat skeletal muscle actin gene promoter. These motifs bind nuclear proteins, and seem to be involved in the regulation of the gene. Analysis of isolated clones, which expressed proteins that specifically bind the oligonucleotide, indicated that they were derived from a single gene. This gene was identified as a contaminant of bacterial origin (Leuconostoc lactis). The cloned gene from L. lactis encodes a protein with significant homology to bacterial ribosomal protein S1, which we designated LrpS1-L. Band shift analysis and competition experiments indicated that both the bacterial protein and a mouse nuclear protein specifically bind to the same CT-rich motif of the skeletal muscle actin promoter. Furthermore, antibodies against the recombinant bacterial protein interfered with the formation of complex between the CT-rich element and the mouse nuclear protein. These results indicate that the bacterial LrpS1-L protein and the mammalian protein bind the same CT-rich motif and share common antigenic epitopes.

A Delayed-early Response Nuclear Gene Encoding MRPL12, the Mitochondrial Homologue to the Bacterial Translational Regulator L7/L12 Protein

We have characterized a new delayed-early response mRNA encoding a 21-kDa product (MRPL12) that accumulates during the G1 phase of growth-stimulated cells. MRPL12 is the mammalian homologue to chloroplastic and bacterial L12 ribosomal proteins. Immunofluorescence microscopy and cell fractionation indicate a predominant mitochondrial localization in various mammalian cell lines. The NH2-terminal 49 amino acids are necessary and sufficient to target the protein within the mitochondria and are probably cleaved off during import. MRPL12 proteins associated in vitro and cofractionate with ribosomal structures, as is the case for prokaryotic L12 proteins. Expression of a dominant inhibitory truncated protein leads to a severe reduction in cell growth by inhibiting mitochondrial ATP production. MRPL12 is the first mammalian mitochondrial ribosomal protein to be characterized.

Proteins P1, P2, and P0, components of the eukaryotic ribosome stalk. New structural and functional aspects.

The eukaryoic ribosomal stalk is thought to consist of the phosphoproteins P1 and P2, which form a complex with protein PO. This complex interacts at the GTPase domain in the large subunit rRNA, overlapping the binding site of the protein L11-like eukaryotic counterpart (Saccharomyces cerevisiae protein L15 and mammalian protein L12). An unusual pool of the dephosphorylated forms of proteins P1 and P2 is detected in eukaryotic cytoplasm, and an exchange between the proteins in the pool and on the ribosome takes place during translation. Quadruply disrupted yeast strains, carrying four inactive acidic protein genes and, therefore, containing ribosomes totally depleted of acidic proteins, are viable but grow with a doubling time threefold higher than wild-type cells. The in vitro translation systems derived from these stains are active but the two-dimensional gel electrophoresis pattern of proteins expressed in vivo and in vitro is partially different. These results indicate that the P1 and P2 proteins are not essential for ribosome activity but are able to affect the translation of some specific mRNAs. Protein PO is analogous to bacterial ribosomal protein L10 but carries an additional carboxyl domain showing a high sequence homology to the acidic proteins P1 and P2, including the terminal peptide DDDMGFGLFD. Successive deletions of the PO carboxyl domain show that removal of the last 21 amino acids from the PO carboxyl domain only slightly affects the ribosome activity in a wild-type genetic background; however, the same deletion is lethal in a quadruple disruptant deprived of acidic P1/P2 proteins. Additional deletions affect the interaction of PO with the P1 and P2 proteins and with the rRNA. The experimental data available support the implication of the eukaryotic stalk components in some regulatory process that modulates the ribosomal activity.

Cloning, sequencing and expression of Pseudomonas testosteroni gene encoding 3α-hydroxysteroid dehydrogenase

We describe the cloning, sequencing and expression of the 3α-hydroxysteroid dehydrogenase (3α-HSD) gene of Pseudomonas testosteroni. A genomic library of P. testosteroni total DNA constructed from SauIIIA digests ligated to an λgt11 vector was probed with a polyclonal antibody raised against purified enzyme. Subclones derived from a recombinant phage containing a 1746 bp insert were sequenced and found to contain an open reading frame of 696 bp that corresponds to a protein of 231 amino acid residues. A search for homologous proteins was performed. No similarity was observed when comparing 3α-HSD with known members of the short-chain dehydrogenase family. However a small proteic fragment (80 amino acids) shows homology with the N-terminal sequence of bacterial L7 L12 ribosomal proteins.

Identification of the yeast nuclear gene for the mitochondrial homologue of bacterial ribosomal protein L16.

An open reading frame encoding a member of the L16 family of ribosomal proteins is adjacent to the URA7 gene on the left arm of chromosome II in Saccharomyces cerevisiae. The predicted L16-like polypeptide is basic (pl 11.12), contains 232 amino acids (26.52 kDa) and has 36% amino acid sequence identity to E. coli L16. Immunoblot analysis with polyclonal antibodies to the L16-like polypeptide showed specific cross-reaction with a 22,000 Mr mitochondrial polypeptide that co-sediments with the large subunit of the mitochondrial ribosome in sucrose density gradients. The levels of the L16 mRNA and protein varied in response to carbon source. In [rho degree] cells lacking mitochondrial rRNA, the L16 mRNA accumulated at normal levels, but the protein was barely detectable, indicating RNA-dependent accumulation of the L16 protein. Gene disruption experiments demonstrated that the yeast mitochondrial L16 is an essential ribosomal protein in vivo.

Brucella ribosomal protein L7/L12 is a major component in the antigenicity of brucellin INRA for delayed-type hypersensitivity in brucella-sensitized guinea pigs.

A delayed-type hypersensitivity (DTH) reaction in the course of brucellosis in humans and animals can be revealed by the brucellin INRA (Brucellergen) skin test. Brucellergen is composed of more than 20 proteins of different molecular weights. A 12-kDa protein eliciting DTH in Brucella melitensis Rev1-sensitized guinea pigs was found to be a significant component for the allergenic properties of Brucellergen. Sequencing of the gene encoding this protein identified it as the L7/L12 ribosomal protein. The L7/L12 gene of B. melitensis was amplified by PCR and subcloned in the Escherichia coli pQE30 plasmid. The resulting recombinant protein did not produce a DTH reaction in sensitized animals. It was used to raise specific antibodies in a rabbit. Affinity chromatography with these antibodies was used to isolate a single protein from Brucellergen and from B. melitensis cytosol preparations which produced a DTH reaction in guinea pigs sensitized with B. melitensis Rev1. N-terminal amino acid sequencing of the protein confirmed that it was the L7/L12 ribosomal protein. This is the first complete report on the involvement of a defined bacterial ribosomal protein in the DTH response of animals infected with intracellularly multiplying bacteria.

Crystallization and preliminary X-ray diffraction studies of bacterial ribosomal protein L14.

Based on amino-acid sequence homology, it is predicted that ribosomal protein L14 is a member of a recently identified family of structurally related RNA-binding proteins. To verify this, the gene for Bacillus stearothermophilus L14 has been cloned, and the protein has been purified and crystallized. The crystals are in space group C2 with cell dimensions a = 67.0, b = 32.7, c = 49.4 A, and beta = 101.8 degrees, and there is one molecule in the asymmetric unit (V(m) = 2.0 A(3) Da(-1)). They are of high quality, and a native data set has been collected to a resolution of 1.6 A, with an R(merge) of 5.3%.

Nitrofurantoin: mechanism of action and implications for resistance development in common uropathogens.

Nitrofurantoin is an effective urinary tract antibacterial to which no clinically significant resistance development has occurred. We have previously shown that nitrofurantoin susceptibility in bacteria correlates with the presence of bacterial nitroreductases which convert nitrofurantoin to highly reactive electrophilic intermediates. These intermediates were shown to attack bacterial ribosomal proteins non-specifically, causing complete inhibition of protein synthesis. In the present study, we confirm previous reports that low concentrations of nitrofurantoin specifically inhibit inducible enzyme synthesis in bacteria, and show that this inhibition occurs at levels equivalent to the MICs of nitrofurantoin for several bacterial species. Our previous studies had shown that nitrofurantoin at different concentrations interacts with bacterial ribosomal proteins in qualitatively the same fashion; we now report that quantitative differences are seen in the labelling observed at different nitrofurantoin concentrations and discuss these differences as they may relate to the inhibition of inducible enzyme synthesis. In addition, we have now demonstrated the existence of a novel mechanism of action for nitrofurantoin which does not require the production of reactive nitrofurantoin metabolites by bacterial reductases. The lack of clinically significant bacterial resistance development to nitrofurantoin is likely due to the combination of nitrofurantoin's multiple sites of attack and multiple mechanisms of action.

The S12 ribosomal protein of Podospora anserina belongs to the S19 bacterial family and controls the mitochondrial genome integrity through cytoplasmic translation.

In the filamentous fungus Podospora anserina, two nuclear genes are involved in the premature death syndrome associated with a site-specific deletion of the mitochondrial DNA: a mutant allele of the AS1 gene, encoding the cytoplasmic ribosomal protein S12, and an uncharacterized gene closely linked to the mating-type locus. We describe here the cloning and the sequencing of the wild-type and two mutant alleles of the AS1 gene. The P. anserina S12 protein belongs to the bacterial S19 ribosomal protein family and shows 72% identity with the S15 human ribosomal protein. Transformation experiments have shown that the AS1-4 mutation itself is responsible for the premature death phenotype and that it corresponds to a Gly to Asp change in the highly conserved COOH-terminal part of the protein. Use of antibodies directed against S12 did not permit detection of the mutant ribosomal protein inside the mitochondria. However, cross-reactions were observed with at least one mitochondrial ribosomal protein displaying a higher molecular weight than S12. The mitochondrial protein does not seem to be a by-product of the AS1 gene but is more likely the mitochondrial homologue of S12. These results strongly suggest that the mutant S12 protein acts indirectly to promote the mitochondrial deletion, via the cytoplasmic translation.

Recombinant hybrid toxin with dual enzymatic activities. Potential use in preparing highly effective immunotoxins.

Bacterial toxins and ribosomal inhibitory proteins isolated from plants are used to prepare tumor-specific cytotoxic conjugates. The ability of these conjugates to kill tumor cells depends on binding, internalization, translocation to cytoplasm, and translation inhibition. Modulation of any one of these processes can improve cytotoxicity. Since bacterial and plant toxins act at a distinct step in translation, a combination of their activities could be more effective. Therefore, a chimeric protein was prepared by genetically fusing the coding region of the ricin A chain (RTA) and the fragment A of diphtheria toxin (DTA). The hybrid protein (RTA-DTA) expressed in bacteria retained the N-glycosidase activity of the RTA and ADP-ribosylation activity of the DTA. The hybrid toxin was more potent than the ricin A chain (11-fold) and the diphtheria toxin (50-fold) in inhibiting cell-free translation. Immunotoxin made with the hybrid toxin was about 100- and 1000-fold more effective than RTA or DTA conjugate, respectively, in inhibiting tumor cell growth in vitro. These results indicate that the hybrid toxin with dual activities could be useful in preparing potent immunotoxins with better anti-tumor cell activity.

Translational initiation factors IF-1 and eIF-2α share an RNA-binding motif with prokaryotic ribosomal protein S1 and polynucleotide phosphorylase

Initiation of translation is a complicated process involving numerous accessory factors whose functions remain incompletely understood. Bacterial ribosomal protein S1 is known to contain a repeated sequence motif (S1-RM), also found in polynucleotide phosphorylase, that is thought to be involved in binding to RNA. Using the technique of profile analysis, the S1-RM can also be found in bacterial and chloroplast translation initiation factor IF-1 sequences, and in the sequences of eukaryotic translation initiation factor eIF-2α chains. The significance of the similarity of the sequences is very high suggesting that the occurrence of the S1-RM in these diverse proteins represents homology. The similarity of S1 to IF-1 further suggests that S1 has evolved from an IF-1 like ancestor, and therefore that the two proteins have a similar or competitive function. The most obvious common function of the proteins containing the S1-RM seems to be RNA binding, suggesting that IF-1 and eIF-2α may bind to RNA.

Recognition of a tetranucleotide loop of signal recognition particle RNA by protein SRP19.

The interaction of protein SRP19 with the RNA component of human signal recognition particle (SRP) was studied by site-directed mutagenesis of the SRP RNA. The effects of nucleotide changes in the tetranucleotide loop (tetraloop) of helix 6 showed that SRP19 recognizes a tetraloop in a sequence-specific manner. Adenosine 149 at the third position of the tetraloop was essential for binding. In contrast, changes of the base at the second position had no effect. Mutations that disrupt or compensate individual SRP RNA helices were generated to investigate the importance of base pairing and to identify other binding sites. Considerable base pairing was essential in helix 6. Another SRP19-binding site was located in the distal part of helix 8. The primary sequences of the tetraloop-binding protein SR19 and of bacterial ribosomal protein S15 are shown to be similar.

NusG, a new Escherichia coli elongation factor involved in transcriptional antitermination by the N protein of phage lambda.

We have reconstituted biologically relevant transcriptional antitermination in vitro by the phage lambda N protein. This required the isolation of NusG, a newly identified Escherichia coli transcription elongation factor. NusG is encoded by an E. coli gene, formerly called U and now called nusG, in which a mutation affects antitermination by N in vivo. Efficient antitermination by N in our reconstituted system depends on the bacterial proteins NusG, NusA, NusB, and ribosomal protein S10 (which functions without ribosomes in transcriptional antitermination). In reactions containing E. coli S100 extract, NusG is stoichiometrically bound to lambda N-modified transcription elongation complexes. We used RNA polymerase affinity chromatography to show that NusG binds to the core component of E. coli RNA polymerase. This binding is weak, and the stable association of NusG with lambda elongation complexes additionally requires at least N, NusA, and the boxA component of an N utilization site. In reactions containing bacterial S100 extract, NusG and NusB are also present in elongation complexes transcribing E. coli boxA-containing rDNA.

The murine MHC encodes a mammalian homolog of bacterial ribosomal protein S13.

The mouse major histocompatibility complex (MHC) contains many genes in addition to the classical immune response genes. We have screened overlapping cosmid clones covering 170 kb of the H-2K region for genes expressed in embryonal carcinoma (EC) cells. The Ke-3 gene (Abe et al. 1988) found in this region was further studied by Southern, Northern, and sequence analysis. It is an expressed, intron-containing locus encoding a mouse homolog of the bacterial ribosomal protein S13. This is the first non-organelle S13 homolog identified in metazoans, and its genomic location has been determined precisely.

Identification of an amino acid-regulated mRNA from rat liver as the mammalian equivalent of bacterial ribosomal protein L22.

Amino acid deprivation of rat hepatoma cells induced the levels of a 612-base pair mRNA termed ASI (Shay, N. F., Nick, H. S., and Kilberg, M. S. (1990) J. Biol. Chem. 265, 17844-17848). The ASI mRNA was present at levels equal to or greater than actin in every rat tissue tested. The corresponding full-length cDNA was cloned, and the present report demonstrates that the deduced 184-residue amino acid sequence shares greater than 30% identity to a number of bacterial and chloroplast L22 ribosomal proteins, including those from Escherichia coli and Halobacterium halobium. A monospecific anti-peptide antibody was produced that upon immunochemical analysis of subcellular fractions of rat liver recognized a band in the microsomal fraction and, more specifically, reacted with a single polypeptide in the ribosomal large subunit fraction. The antibody did not react with any proteins of the mitochondrial large subunit, but did recognize a protein in human liver homogenate at the same relative mobility (23 kDa) as that observed for rat liver.

Requirement of the RNA helicase-like protein PRP22 for release of messenger RNA from spliceosomes

The product of the yeast PRP22 gene acts late in the splicing of yeast pre-messenger RNA, mediating the release of the spliced mRNA from the spliceosome. The predicted PRP22 protein sequence shares extensive homology with that of PRP2 and PRP16 proteins, which are also involved in nuclear pre-mRNA splicing. The homologous region contains sequence elements characteristic of several demonstrated or putative ATP-dependent RNA helicases. A putative RNA-binding motif originally identified in bacterial ribosomal protein S1 and Escherichia coli polynucleotide phosphorylase has also been found in PRP22.

Reversed-phase chromatography of Escherichia coli ribosomal proteins : Correlation of retention time with chain length and hydrophobicity

The elution times of 52 bacterial ribosomal proteins from a C 4 reversed-phase column have been predicted. The prediction is based upon the use of the hydrophobicity coefficients for the protein amino acid content as defined by Guo [ J. Chromatogr., 359 (1986) 499–518]. A strong positive correlation was observed when the difference between predicted and observed protein retention time was plotted against the product of net hydrophobicity and natural log of protein chain length. Observations with this class of related proteins strengthens and extends the observations of Mant [ J. Chromatogr., 476 (1989) 363–375]. Observed deviations from predicted chromatographic behavior can be explained for several proteins which elute as dimers or which have modified amino acid residues.

The 26S rRNA binding ribosomal protein equivalent to bacterial protein L11 is encoded by unspliced duplicated genes in Saccharomyces cerevisiae

Transformant phages expressing L15, a yeast ribosomal protein which binds to 26S rRNA and interacts with the acidic ribosomal proteins, were isolated by screening a yeast cDNA expression library in lambda gt11 with specific monoclonal antibodies. Using yeast DNA HindIII fragments that hybridize with the cDNA insert from the L15-expressing clones, minilibraries were prepared in pUC18, which were afterward screened with the same cDNA probe. In this way, plasmids carrying two different types of genomic DNA inserts were obtained. The inserts were subcloned and sequenced and we found a similar coding sequence in both cases flanked by 5' and 3' regions with very low homology. Sequences homologous to the consensus TUF-binding UAS boxes are present in the 5' flanking regions of both genes. Southern analysis revealed the presence of two copies of the L15 gene in the Saccharomyces cerevisiae genome, which are located in different chromosomes. The encoded amino acid sequence corresponds, as expected, to protein L15 and shows a high similarity to bacterial ribosomal protein L11.

Detection of methylated asparagine and glutamine residues in polypeptides

A residue of γ- N-methylasparagine (γ-NMA) is found at position β-72 of many phycobiliproteins. δ- N-Methylglutamine is present in some bacterial ribosomal proteins. γ-NMA was synthesized by reacting the ω-methyl ester of aspartate with methylamine and δ- N-methylglutamine by reaction of pyroglutamate with methylamine. These derivatives and the ω-methyl esters of aspartate and glutamate were characterized by melting point, by thin-layer chromatography, by amino acid analysis, by NMR spectroscopy, and after conversion to the phenylthiohydantoin (PTH) derivative. The γ-NMA residues in peptides from allophycocyanin, C-phycocyanin, and B-phycoerythrin were stable under the conditions of automated sequential gas-liquid phase Edman degradation. On HPLC, PTH-γ-NMA coeluted with PTH-serine and was accompanied by a minor component eluting just prior to dimethylphenylthiourea. Similar results were obtained on manual derivatization of synthetic γ-NMA to prepare the PTH derivative. The PTH-δ- N-methylglutamine standard eluted near the position of dimethylphenylthiourea under the usual conditions employed for the identification of PTH-amino acid derivatives in automated protein sequencing.

Protein blotting followed by microsequencing

The use of new membranes such as activated or derivatized glass fibers as well as synthetic membranes, which are compatible with the hazardous sequencing reagents, are described. Precautions to be taken in order to prevent N-terminal blockage of the proteins during electrophoresis and blotting are described, as well as the conditions for protein detection after blotting and protein treatment for in situ amino acid analysis, fragmentation and microsequencing. For a number of standard proteins and bacterial ribosomal proteins microsequence analysis is reported for two commercially available sequencers (Applied Biosystems and Knauer).