Eubacterial Ribosomal Proteins Research Articles

Binding of the human ribosomal protein S13 to the central domain of 18S rRNA

Human ribosomal protein S13 is a structural element of the small subunit of ribosome. It is a homologue of eubacterial ribosomal protein S15, and, besides, it possesses an extended N-terminal region, characteristic of the S15p family in eukaryotes and archaea. In the present study, we investigated binding of recombinant ribosomal protein S13 and its mutants containing deletions or substitutions of amino acid residues in different regions with an RNA transcript corresponding to a fragment of the central domain of 18S rRNA. We found that replacement of ultra-conservative residues H101 and D108 as well as deletions of either 29 C-terminal or 27 N-terminal residues substantially reduced affinity of the protein to the RNA transcript. Deletion of 54 C-terminal or 80 N-terminal residues completely deprived the protein of binding capacity. Using a footprinting assay, we identified sites in the RNA transcript changing their accessibilities to action of hydroxyl radicals under binding of either full-length protein S13 or its mutant lacking 27 N-terminal residues. It is shown that these sites are located mainly in helix H22 of the 18S rRNA and in the region of its junction with helix H20 and are consistent predominantly with contacts of the rRNA with the conserved part of the protein. We concluded that binding of ribosomal protein S13 to 18S rRNA is provided mainly by conserved motifs of the protein corresponding to those motifs in its eubacterial homologue that are involved in the interaction with 16S rRNA in the 30S subunit. Role of the N-terminal region of the protein in its binding to the central domain of 18S rRNA is discussed.

A region in the C-terminal domain of ribosomal protein SA required for binding of SA to the human 40S ribosomal subunit

The human ribosomal protein SA, known also as a precursor of the cell-surface laminin receptor, LAMR, is a protein of the 40S ribosomal subunit. It is homologous to eubacterial ribosomal protein S2p, but has a eukaryote-specific C-terminal domain (CTD) that is responsible in LAMR for the binding of laminin as well as prions and several viruses. Using serial deletions in the SA CTD, we showed that region between amino acids 236–262 is required for binding of the protein to 40S ribosomal subunits. All SA mutants containing this region protected nucleotides in hairpin 40 (which is not bound to any protein in the eubacterial 30S ribosomal subunit) of the 18S rRNA from hydroxyl radical attack. Comparison of our data with the cryo-EM models of the mammalian 40S ribosomal subunit allowed us to locate the SA CTD in the spatial structure of the 40S subunit.

B. subtilis Ribosomal Proteins: Structural Homology and Post-Translational Modifications

Ribosomal proteins of the model gram-positive bacterium B. subtilis 168 were extensively characterized in a proteomic study. Mass spectra of the 52 proteins expected to be constitutive components of the 70S ribosome were recorded. Peptide MS/MS analysis with an average sequence coverage of 85% supported the identification of these proteins and facilitated the unambiguous assignment of post-translational modifications, including the methylation of S7, L11, and L16 and the N-terminal acetylation of S9. In addition, the high degree of structural homology between B. subtilis and other eubacterial ribosomal proteins was demonstrated through chemical labeling with S-methylthioacetimidate. One striking difference from previous characterizations of bacterial ribosomal proteins is that dozens of protein masses were found to be in error and not easily accounted for by post-translational modifications. This, in turn, led us to discover an inordinate number of sequencing errors in the reference genome of B. subtilis 168. We have found that these errors have been corrected in a recently revised version of the genome.

RibAlign: a software tool and database for eubacterial phylogeny based on concatenated ribosomal protein subunits

BackgroundUntil today, analysis of 16S ribosomal RNA (rRNA) sequences has been the de-facto gold standard for the assessment of phylogenetic relationships among prokaryotes. However, the branching order of the individual phlya is not well-resolved in 16S rRNA-based trees. In search of an improvement, new phylogenetic methods have been developed alongside with the growing availability of complete genome sequences. Unfortunately, only a few genes in prokaryotic genomes qualify as universal phylogenetic markers and almost all of them have a lower information content than the 16S rRNA gene. Therefore, emphasis has been placed on methods that are based on multiple genes or even entire genomes. The concatenation of ribosomal protein sequences is one method which has been ascribed an improved resolution. Since there is neither a comprehensive database for ribosomal protein sequences nor a tool that assists in sequence retrieval and generation of respective input files for phylogenetic reconstruction programs, RibAlign has been developed to fill this gap.ResultsRibAlign serves two purposes: First, it provides a fast and scalable database that has been specifically adapted to eubacterial ribosomal protein sequences and second, it provides sophisticated import and export capabilities. This includes semi-automatic extraction of ribosomal protein sequences from whole-genome GenBank and FASTA files as well as exporting aligned, concatenated and filtered sequence files that can directly be used in conjunction with the PHYLIP and MrBayes phylogenetic reconstruction programs.ConclusionUp to now, phylogeny based on concatenated ribosomal protein sequences is hampered by the limited set of sequenced genomes and high computational requirements. However, hundreds of full and draft genome sequencing projects are on the way, and advances in cluster-computing and algorithms make phylogenetic reconstructions feasible even with large alignments of concatenated marker genes. RibAlign is a first step in this direction and may be particularly interesting to scientists involved in whole genome sequencing of representatives of new or sparsely studied eubacterial phyla. RibAlign is available at

MrpL36p, a highly diverged L31 ribosomal protein homolog with additional functional domains in Saccharomyces cerevisiae mitochondria.

Translation in mitochondria utilizes a large complement of ribosomal proteins. Many mitochondrial ribosomal components are clearly homologous to eubacterial ribosomal proteins, but others appear unique to the mitochondrial system. A handful of mitochondrial ribosomal proteins appear to be eubacterial in origin but to have evolved additional functional domains. MrpL36p is an essential mitochondrial ribosomal large-subunit component in Saccharomyces cerevisiae. Increased dosage of MRPL36 also has been shown to suppress certain types of translation defects encoded within the mitochondrial COX2 mRNA. A central domain of MrpL36p that is similar to eubacterial ribosomal large-subunit protein L31 is sufficient for general mitochondrial translation but not suppression, and proteins bearing this domain sediment with the ribosomal large subunit in sucrose gradients. In contrast, proteins lacking the L31 domain, but retaining a novel N-terminal sequence and a C-terminal sequence with weak similarity to the Escherichia coli signal recognition particle component Ffh, are sufficient for dosage suppression and do not sediment with the large subunit of the ribosome. Interestingly, the activity of MrpL36p as a dosage suppressor exhibits gene and allele specificity. We propose that MrpL36p represents a highly diverged L31 homolog with derived domains functioning in mRNA selection in yeast mitochondria.

Cloning, Sequencing, and Characterization of Ribosomal Protein and RNA Polymerase Genes from the Region Analogous to the α-Operon of Escherichia coli in Halophilic Archaea, Halobacterium halobium

A determination was made of the nucleotide sequence of the 3215-bp region of a ribosomal protein gene cluster (HS13, HS4, HS11, and HeL18), RNA polymerase (RNA poly D), and tRNA genes (tRNAser and tRNAarg) of halophilic Archaea Halobacterium halobium, which is analogous to the α-operon of Escherichia coli (tRNAser-HS13-HS4-HS11-RNA poly D-tRNAarg-HeL18). The seven-gene string was preceded by a pseudoknot-like structure similar to the proposed S4 ribosomal protein binding site of the α-operon mRNA leader in E. coli. Using an inducible expression system H. halobium HS4 was produced in large amounts in E. coli, and immunoblot analysis showed the S4 to constitute a 21-kDa polypeptide component of the ribosome. Analysis of the deduced amino acids sequence revealed that the HS13, HS4, and HS11 sequences including the RNA polymerase subunit are more similar to their eukaryotic than to their bacterial counterparts. HeL18, located downstream of the gene cluster analogous to the E. coli α-operon (S13-S11-S4-RNA poly D-L17), was similar to both the eukaryotic (eL18) and eubacterial ribosomal protein L15 located in the spc-operon, but not to L17 positioned as the terminal gene of the bacterial α-operon.

Structure and function of MRP20 and MRP49, the nuclear genes for two proteins of the 54 S subunit of the yeast mitochondrial ribosome.

MRP20 and MRP49 are proteins of the large subunit of the mitochondrial ribosome in Saccharomyces cerevisiae. Their genes were identified through immunological screening of a genomic library in the expression vector lambda gt11. Nucleotide sequencing revealed that MRP49 is tightly linked to TPK3 and encodes a 16-kDa, basic protein with no significant relatedness to any other known protein. MRP20 specifies a 263-amino-acid polypeptide with sequence similarity to members of the L23 family of ribosomal proteins. The levels of the mRNAs and proteins for both MRP20 and MRP49 were regulated in response to carbon source. In [rho0] strains lacking mitochondrial rRNA, the levels of the two proteins were reduced severalfold, presumably because the unassembled proteins are unstable. Null mutants of MRP20 converted to [rho-] or [rho0], a characteristic phenotype of mutations in essential genes for mitochondrial translation. Inactivation of MRP49 caused a cold-sensitive respiration-deficient phenotype, indicating that MRP49 is not an essential ribosomal protein. The mrp49 mutants were defective in the assembly of stable 54 S ribosomal subunits at the nonpermissive temperature. With the results presented here, there are now published sequences for 14 yeast mitochondrial ribosomal proteins, only five of which bear discernable relationships to eubacterial ribosomal proteins.

Mutants lacking individual ribosomal proteins as a tool to investigate ribosomal properties

We have isolated and charact rized mutants which lack one or two sixteen of the proteins of the Escherichia coli ribosome. The mutation responsible in each case mapped close to, and probably in, the corresponding gene. A conditional lethal phenotype and a variable degree of impairment in growth was observed. The missing protein was readily restored to the organelle if E coli or other eubacterial ribosomal proteins were added to a suspension of the mutant particles. The mutants have been used to investigate the role of individual proteins in ribosome function and assembly. They have also aided in the topographic prinpointing of proteins on the surface of the organelle.

Evidence for an additional archaebacterial gene cluster in Halobacterium marismortui encoding ribosomal proteins HL46e and HL30

A small and extremely basic ribosomal protein (HL46e) has been purified from Halobacterium marismortui using reversed-phase high-performance liquid chromatography (HPLC). The amino acid sequence of the protein was determined by automated N-terminal and internal sequence analysis. Comparison of this sequence with other ribosomal protein sequences from eubacteria, archaebacteria and eukaryotes revealed a strong homology to SL46e from Sulfolobus solfataricus, YeaL46 from yeast and RL39 from rat. No significant sequence similarity was found to any eubacterial ribosomal protein so far known. Using a specific oligonucleotide probe the HL46e gene was identified, cloned and the nucleotide sequence including the 5′- and 3′-flanking regions were analysed. The HL46e gene is followed by the gene coding for HL30. A putative halobacterial promotor sequence with the motive ‘TTTAAA’ has been localized 32 bp upstream of the HL46e gene and a putative terminator sequence localized downstream from the HL30 gene. An equivalent to this HL46e/HL30 operon is apparently not present in Escherichia coli.

Organization and structure of the Methanococcus transcriptional unit homologous to the Escherichia coli “spectinomycin operon” : Implications for the evolutionary relationship of 70 S and 80 S ribosomes

By means of an immunological approach and a subsequent chromosome-walking strategy a chromosomal region encoding ribosomal proteins in the archaebacterium Methanococcus vannielii was cloned. The determination of the nucleotide sequence of the 7.8 × 10 3 base DNA fragment revealed the existence of 14 putative ribosomal protein genes and two unidentified open reading frames. They are organized in a transcriptional unit that is very similar to the Escherichia coli “spectinomycin operon” in respect of both gene composition and gene order. The Methanococcus transcriptional unit contains, in addition to those genes whose products have a homologue in the E. coli operon, three genes whose products share sequence similarity with eukaryotic 80 S but not with eubacterial ribosomal proteins. The Methanococcus ribosomal proteins almost exclusively exhibit a higher sequence similarity to eukaryotic 80 S ribosomal proteins than to those of eubacteria and many of them have a size intermediate between those of their eukaryotic and eubacterial homologues. These results are discussed in terms of a hypothesis that implies that the recent eubacterial ribosome developed by a “minimization” process from a more complex organelle and that the archaebacterial ribosome has maintained features of this ancestor.

A small basic ribosomal protein in Sulfolobus solfataricus equivalent to L46 in yeast: Structure of the protein and its gene

The structure of the gene for a small, very basic ribosomal protein in Sulfolobus solfataricus has been determined and the structure of the protein coded by this gene (L46e) has been confirmed by partial amino acid sequencing. The protein shows substantial sequence homology to the eukaryotic ribosomal proteins L39 in rat and L46 in yeast. There is no sequence homology to any of the eubacterial ribosomal proteins suggesting that this protein is absent in the eubacterial ribosome.

Ribosomal protein gene cluster of Halobacterium halobium: nucleotide sequence of the genes coding for S3 and L29 equivalent ribosomal proteins

A 1643 base pair fragment encoding the S3 and L29 equivalent ribosomal proteins has been sequenced from the archaebacterium Halobacterium halobium. The incomplete open reading frame present upstream from the S3 gene encodes a protein homologous to the eubacterial ribosomal protein L22. The initiation codons of the S3 and L29 genes overlap with the termination codons of the upstream genes. A tight physical organization suggests that these genes are transcribed as a polycistronic operon. Peculiarities of the protein structure and gene organization are discussed.

The primary structures of ribosomal proteins L16, L23 and L33 from the archaebacterium Halobacterium marismortui

The complete amino acid sequences of ribosomal proteins L16, L23 and L33 from the archaebacterium Halobacterium marismortui were determined. The sequences were established by manual sequencing of peptides produced with several proteases as well as by cleavage with dilute HCl. Proteins L16, L23 and L33 consist of 119, 154 and 69 amino acid residues, and their molecular masses are 13538, 16812 and 7620 Da, respectively. The comparison of their sequences with those of ribosomal proteins from other organisms revealed that L23 and L33 are related to eubacterial ribosomal proteins from Escherichia coli and Bacillus stearothermophilus, while protein L16 was found to be homologous to a eukaryotic ribosomal protein from yeast. These results provide information about the special phylogenetic position of archaebacteria.

Primary structures of three highly acidic ribosomal proteins S6, S12 and S15 from the archaebacterium Halobacterium marismortui

The amino acid sequences of three extremely acidic ribosomal proteins, S6, S12, and S15, from Halobacterium marismortui have been determined. The sequences were obtained by the sequence analysis of peptides derived by enzymatic digestion with trypsin. Stapylococcus aureus protease and chymotrypsin, as well as by cleavage with dilute HCl. The proteins, S6, S12 and S15, consist of 116, 147 and 102 amino acid residues, and have molecular masses of 12251, 16440 and 11747 Da, respectively. Comparison of the amino acid sequences of these proteins with ribosomal protein sequences of other organisms revealed that halobacterial protein S12 has homology with the eukaryotic protein S16A from Saccharomyces cerevisiae, while S15 is significantly related to the Xenopus laevis S19 protein. No homology was found between these halobacterial proteins and any eubacterial ribosomal proteins.

The complete amino acid sequence of ribosomal protein H-S11 from the archaebacterium Halobacterium marismortui

The complete amino acid sequence of ribosomal protein H-S11 from the extremely halophilic archaebacterium Halobacterium marismortui is presented. This has been achieved by the sequence analysis of peptides derived by enzymatic digestions with trypsin, pepsin and Staphylococcus aureus protease, as well as by chemical cleavage with cyanogen bromide and o-iodosobenzoic acid. The protein consists of 155 amino acid residues and has a molecular mass of 17545 Da. Comparison of this sequence with other ribosomal proteins by the computer programmes RELATE and ALIGN showed that the C-terminal two-thirds of H-S11 is homologous to the eubacterial ribosomal protein S15 and that the N-terminal one-third of H-S11 is possibly related to the N-terminal region of the eubacterial ribosomal protein S8. To explain this finding, possible genetic events during evolution, e.g. fusion or splitting of genes, are discussed.