A rice (Oryza sativa L.) cDNA encodes a protein sequence homologous to the eukaryotic ribosomal 5S RNA-binding protein

A rice (Oryza sativa L.) cDNA clone coding for the cytoplasmic ribosomal protein L5, which associates with 5 S rRNA for ribosome assembly, was cloned and its nucleotide sequence was determined. The primary structure of rice L5, deduced from the nucleotide sequence, contains 294 amino acids and has intriguing features some of which are also conserved in other eucaryotic homologues. These include: four clusters of basic amino acids, one of which may serve as a nucleolar localization signal; three repeated amino acid sequences; the conservation of glycine residues. This protein was identified as the nuclear-encoded cytoplasmic ribosomal protein L5 of rice by sequence similarity to other eucaryotic ribosomal 5 S RNA-binding proteins of rat, chicken, Xenopus laevis, and Saccharomyces cerevisiae. Rice L5 shares 51 to 62% amino acid sequence identity with the homologues. A group of ribosomal proteins from archaebacteria including Methanococcus vanniellii L18 and Halobacterium cutirubrum L13, which are known to be associated with 5 S rRNA, also related to rice L5 and the other eucaryotic counterparts, suggesting an evolutionary relationship in these ribosomal 5 S RNA-binding proteins.     

Identification of candidate SNPs for drug induced toxicity from differentially expressed genes in associated tissues

The full-length cDNA sequence (1158 bp) encoding a ribosomal L5 protein, designated as TaL5, was firstly isolated from common wheat (Triticum aestivum L.) using the rapid amplification of cDNA ends method (RACE). The open reading frame (ORF) of TaL5 gene was 906 bp, and its deduced amino acid sequence (301 residues) shared high similarity to those of other higher plant L5 proteins. TaL5 protein contained a putative 5S binding region (74 amino acids). TaL5 DNA sequence was further cloned, and sequence analysis showed that it contained 7 introns and 8 exons. Predicated using TargetP software, TaL5 protein was putatively located in mitochondria and contains a transit peptide of 12 amino acids. During grain filling period, temporal expression pattern of TaL5 gene was approximately consistent with the rates of starch accumulation in grains. Additionally, TaL5 gene was dramatically induced by salt, drought and freezing stresses, exogenous abscisic acid (ABA) and salicylic acid (SA) in wheat seedlings. These implied that TaL5 gene could function in growth, development and abiotic stresses in wheat plants.     

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.     

Structures of complexes of 5S RNA with ribosomal proteins L5, L18 and L25 from Escherichia coli: identification of kethoxal-reactive sites on the 5S RNA.

The topography of Escherichia coli 5S RNA has been examined in the presence of ribosomal proteins L5, L18 and L25 and their different combinations, by comparing the kethoxal modification characteristics of the various RNA-protein complexes with those of the free A-conformer of 5S RNA (Noller &; Garrett, 1979, accompanying paper).Two of the four most reactive guanines, G₁₃ and G₄₁, are unaffected by the protein, in accord with the finding that these are the only two guanines that are accessible in the 50S subunit (Noller &; Herr, 1974). The other two very reactive guanines, G₂₄ and G₆₉, are strongly protected by protein L18, either in the presence or absence of proteins L5 and L25. Protein binding studies with kethoxal-modified 5S RNA provide evidence that one or both of these two guanines are directly involved in the protein-RNA interactions, and this conclusion is supported by the occurrence of guanines in these two positions in all the other sequenced prokaryotic 5S RNAs.The group of less reactive guanines, G₁₆, G₂₃, G₄₄, G₈₆ and G₁₀₇, are protected to some extent by each of the proteins L5, L18 and L25; the strongest effect is with L18. We suggest that this is attributable to a small increase in the conformational homogeneity of the 5S RNA and that L18, in particular, induces some tightening of the RNA structure.Only one guanine, G₆₉, is rendered more accessible by the proteins. This effect is produced by protein L25, which is known to cause some destructuring of the 5S RNA (Bear et al., 1977). There was no other evidence for any destructuring of the 5S RNA. In particular, the sequence 72 to 83, which is complementary to a sequence in 23S RNA (Herr &; Noller, 1975), is not modified. However, in contrast to an earlier report (Erdmann et al., 1973), the conserved sequence G₄₄-A-A-C, which has been implicated in tRNA binding, was not rendered more accessible by the proteins.     

Effect of 50 S subunit proteins L5, L18 and L25 on the fluorescence of 5 S RNA-bound ethidium bromide

Among the three Escherichia coli 50 S subunit proteins L5, L18 and L25, which have an affinity for 5 S RNA, only protein L18 exerts a strong effect on the fluorescence of 5 S RNA-ethidium bromide complexes, without changing the quantum yield of the fluorescence. Proteins L5 and L25, although they have little effect on the fluorescence, have a strong stabilizing influence on the 5 S RNA-L18 complex. The results are discussed in terms of the secondary and tertiary structures of 5 S RNA in relation to ribosomal protein binding.     

Protein L5 is crucial for in vivo assembly of the bacterial 50S ribosomal subunit central protuberance

In the present work, ribosomes assembled in bacterial cells in the absence of essential ribosomal protein L5 were obtained. After arresting L5 synthesis, Escherichia coli cells divide a limited number of times. During this time, accumulation of defective large ribosomal subunits occurs. These 45S particles lack most of the central protuberance (CP) components (5S rRNA and proteins L5, L16, L18, L25, L27, L31, L33 and L35) and are not able to associate with the small ribosomal subunit. At the same time, 5S rRNA is found in the cytoplasm in complex with ribosomal proteins L18 and L25 at quantities equal to the amount of ribosomes. Thus, it is the first demonstration that protein L5 plays a key role in formation of the CP during assembly of the large ribosomal subunit in the bacterial cell. A possible model for the CP assembly in vivo is discussed in view of the data obtained.     

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.     

Crystal structures of mutant ribosomal proteins L1

Nine mutant ribosomal proteins L1 from the bacterium Thermus thermophilus and archaeon Methanococcus jannaschii were obtained and their crystal structures were determined and analyzed. The structure of the S179C TthL1 mutant, determined earlier, was also analyzed. In half of the proteins studied, point mutations of the amino acid residues exposed on the protein surface essentially changed the spatial structure of the protein. This proves that a correct study of biological processes with the help of site-directed mutagenesis requires a preliminary determination or, at least, modeling of the structures of mutant proteins. A detailed comparison of the structures of the L1 mutants and the corresponding wild-type L1 proteins demonstrated that the side chain of a mutated amino acid residue tends to adopt a location similar to that of the side chain of the corresponding residue in the wild-type protein. This observation assists in modeling the structure of mutant proteins.     

球毛壳菌60S核糖体蛋白L10a基因克隆与特性分析

用粗糙脉孢菌（Neurospora crassa）XP_322380和赤霉菌（Gibberella zeag）PH-1（EAA76971）的60S核糖体蛋白L10a基因（60S ribosomal protein L10a,RPL10a）蛋白序列对球毛壳菌（Chaetomium globosum）ESTs序列数据库进行tBlastn检索,获得了球毛壳菌RPL10a cDNA序列。cDNA序列长765bp,开放阅读框654bp,编码217个氨基酸组成的多肽,蛋白分了量为23．9kD。BlastP分析表明该基因氨基酸序列与粗糙脉胞菌相似最高为89％;与玉蜀黍黑粉菌（Ustilago maydis）相似性最低为78％。cDNA序列及推测的氨基酸序列在GenBank登录（登录号分别为AY669070,AAT74578）。     

Nuclear-encoded chloroplast ribosomal protein L27 of Nicotiana tabacum: cDNA sequence and analysis of mRNA and genes.

A tobacco (Nicotiana tabacum cv. Petite Havana) leaf cDNA library was constructed in the expression vector lambda gt11. Immunological and nucleic acid hybridization screening yielded several cDNAs encoding an M(r) 19,641 precursor to an M(r) 14,420 mature protein which is homologous to Escherichia coli ribosomal protein L27. One cDNA (L27-1; 882 nucleotides long) contains 104 bp of 5'-noncoding sequence, 51 codons for a transit peptide, 128 codons for the predicted mature L27 polypeptide, and 241 bp of 3'-noncoding sequence, including the poly(A)29 tail. A beta-galactosidase-L27 fusion protein was bound to nitrocellulose filters, expressed, and used as an affinity matrix to purify monospecific antibody to L27 protein from an antiserum of rabbits immunized with 50S chloroplast ribosomal proteins. Using this monospecific antibody, protein L27 was identified among HPLC-purified tobacco chloroplast ribosome 50S subunit proteins. The predicted amino terminus of the mature L27 protein was confirmed by partial sequencing of the HPLC-purified L27 protein. The mature L27 protein has 66%, 61%, 56%, and 48% amino acid sequence identity with the L27-type ribosomal proteins of Bacillus subtilis, E. coli, Bacillus stearo-thermophilus, and yeast mitochondria (MRP7), respectively, in the homologous overlapping regions. The transit peptide of tobacco chloroplast ribosomal protein L27 has 41% amino acid sequence similarity with the MRP7 mitochondrial targeting sequence. Tobacco chloroplast L27 protein also has a 40 amino acid long carboxyl-terminal extension (compared to its bacterial counterparts) which is similar to the corresponding portion of yeast MRP7.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular cloning of ribosomal protein L26 (RPL26) cDNA from Ailuropoda melanoleuca and its potential value in phylogenetic study

The cDNA fragment of ribosomal protein L26 (RPL26) was cloned from Ailuropoda melanoleuca using RT-PCR method. The cDNA fragment is composed of 475 bp, containing an open reading frame of 145 amino acids. Alignment analyses indicated that the nucleotide sequence and the deduced amino acid sequence showed high identity to other known RPL26 sequences from vertebrates and invertebrates. The cDNA sequence was used to construct phylogenetic trees with other known vertebrate and invertebrate RPL26 sequences, and the obtained trees demonstrated similar topology with the classical systematics, indicating the potential value of RPL26 gene in phylogenetic analysis.     

Ribosomal proteins as biomarkers for bacterial identification by mass spectrometry in the clinical microbiology laboratory

Whole-cell matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) is a rapid method for identification of microorganisms that is increasingly used in microbiology laboratories. This identification is based on the comparison of the tested isolate mass spectrum with reference databases. Using Neisseria meningitidis as a model organism, we showed that in one of the available databases, the Andromas database, 10 of the 13 species-specific biomarkers correspond to ribosomal proteins. Remarkably, one biomarker, ribosomal protein L32, was subject to inter-strain variability. The analysis of the ribosomal protein patterns of 100 isolates for which whole genome sequences were available, confirmed the presence of inter-strain variability in the molecular weight of 29 ribosomal proteins, thus establishing a correlation between the sequence type (ST) and/or clonal complex (CC) of each strain and its ribosomal protein pattern. Since the molecular weight of three of the variable ribosomal proteins (L30, L31 and L32) was included in the spectral window observed by MALDI-TOF MS in clinical microbiology, i.e., 3640–12000 m/z, we were able by analyzing the molecular weight of these three ribosomal proteins to classify each strain in one of six subgroups, each of these subgroups corresponding to specific STs and/or CCs. Their detection by MALDI-TOF allows therefore a quick typing of N. meningitidis isolates.     

A ribosomal protein from Thermus thermophilus is homologous to a general shock protein

The gene encoding the ribosomal protein from Thermus thermophilus, TL5, which binds to the 5S rRNA, has been cloned and sequenced. The codon usage shows a clear preference for G/C rich codons that is characteristic for many genes in thermophilic bacteria. The deduced amino acid sequence consists of 206 residues. The sequence of TL5 shows a strong similarity to a general shock protein from Bacillus subtilis, named CTC. The protein CTC is homologous in its N-terminal part to the 5S rRNA binding protein, L25, from E coli. An alignment of the TL5, CTC and L25 sequences displays a number of residues that are totally conserved. No clear sequence similarity was found between TL5 and other proteins which are known to bind to 5S rRNA. The evolutionary relationship of a heat shock protein in mesophiles and a ribosomal protein in thermophilic bacteria as well as a possible role of TL5 in the ribosome are discussed.     

A competition mechanism regulates the translation of the Escherichia coli operon encoding ribosomal proteins L35 and L20

Escherichia coli ribosomal protein (r-protein) L20 is essential for the assembly of the 50S ribosomal subunit and is also a translational regulator of its own rpmI-rplT operon, encoding r-proteins L35 and L20 in that order. L20 directly represses the translation of the first cistron and, through translational coupling, that of its own gene. The translational operator of the operon is 450 nt in length and includes a long-range pseudoknot interaction between two RNA sequences separated by 280 nt. L20 has the potential to bind both to this pseudoknot and to an irregular hairpin, although only one site is occupied at a time during regulation. This work shows that the rpmI-rplT operon is regulated by competition between L20 and the ribosome for binding to mRNA in vitro and in vivo. Detailed studies on the regulatory mechanisms of r-protein synthesis have only been performed on the rpsO gene, regulated by r-protein S15, and on the alpha operon, regulated by S4. Both are thought to be controlled by a trapping mechanism, whereby the 30S ribosomal subunit, the mRNA, and the initiator tRNA are blocked as a nonfunctional preternary complex. This alternative mode of regulation of the rpmI-rplT operon raises the possibility that control is kinetically and not thermodynamically limited in this case. We show that the pseudoknot, which is known to be essential for L20 binding and regulation, also enhances 30S binding to mRNA as if this structure is specifically recognised by the ribosome.     

The primary structure of chicken ribosomal protein L5.

The nucleotide sequence of a cDNA for chicken ribosomal protein L5, which is considered to associate with 5S rRNA, was determined. The cDNA is 975 bp long. The deduced protein has 297 amino acids and has a molecular mass of 34,090 Da. A comparative analysis of the amino acid sequences of chicken L5 and its homologous proteins revealed an extremely conserved region which contains a cluster of basic amino acids.