Nuclear-encoded tobacco chloroplast ribosomal protein L24. Protein identification, sequence analysis of cDNAs encoding its cytoplasmic precursor, and mRNA and genomic DNA analysis.

Using a Nicotiana tabacum leaf cDNA library in the expression vector lambda gt11, two cDNAs encoding the full-length precursor polypeptide (M(r) 20,696) of tobacco chloroplast ribosomal protein L24 were identified and sequenced. These cDNAs encode a mature protein of 146 amino acids (M(r) 16,418) with a transit peptide of 41 amino acids (M(r) 4,278). The mature tobacco L24 protein has 78, 65, 45, and 35% sequence identity with ribosomal proteins L24 of pea, spinach, Bacillus subtilis, and Escherichia coli, respectively. The transit peptide of tobacco L24 is 54 and 57% identical with that of L24 chloroplast ribosomal proteins of pea and spinach, respectively. An expressed beta-galactosidase:L24 fusion protein, bound to nitrocellulose filters, was used as affinity matrix to purify monospecific antibody to L24 protein. Using this monospecific antibody protein L24 was identified among high performance liquid chromatography (HPLC)-purified tobacco chloroplast ribosome 50 S subunit proteins. The predicted amino terminus of the mature L24 protein was confirmed by partial sequencing of the HPLC-purified L24 protein. Northern blot analysis revealed a single mRNA band (0.85-0.90 kilobase) corresponding in size to full-length L24 cDNA. The presence of multiple genes for L24 is suggested by Southern blot hybridization and characterization of two cDNAs for L24 which only differ in their 3'-noncoding sequences.     

Nuclear-encoded chloroplast ribosomal protein L12 of Nicotiana tabacum: characterization of mature protein and isolation and sequence analysis of cDNA clones encoding its cytoplasmic precursor.

Poly(A)+ mRNA isolated from Nicotiana tabacum (cv. Petite Havana) leaves was used to prepare a cDNA library in the expression vector lambda gt11. Recombinant phage containing cDNAs coding for chloroplast ribosomal protein L12 were identified and sequenced. Mature tobacco L12 protein has 44% amino acid identity with ribosomal protein L7/L12 of Escherichia coli. The longest L12 cDNA (733 nucleotides) codes for a 13,823 molecular weight polypeptide with a transit peptide of 53 amino acids and a mature protein of 133 amino acids. The transit peptide and mature protein share 43% and 79% amino acid identity, respectively, with corresponding regions of spinach chloroplast ribosomal protein L12. The predicted amino terminus of the mature protein was confirmed by partial sequence analysis of HPLC-purified tobacco chloroplast ribosomal protein L12. A single L12 mRNA of about 0.8 kb was detected by hybridization of L12 cDNA to poly(A)+ and total leaf RNA. Hybridization patterns of restriction fragments of tobacco genomic DNA probed with the L12 cDNA suggested the existence of more than one gene for ribosomal protein L12. Characterization of a second cDNA with an identical L12 coding sequence but a different 3'-noncoding sequence provided evidence that at least two L12 genes are expressed in tobacco.     

cDNA cloning and sequencing of tobacco chloroplast ribosomal protein L12

Tobacco chloroplast ribosomal protein L12 was isolated as a ssDNA-cellulose-binding protein from a chloroplast soluble protein fraction. Based on the N-terminal amino acid sequence of chloroplast L12, a cDNA clone was isolated and characterized. The precursor protein deduced from the DNA sequence consists of a transit peptide of 53 amino acid residues and a mature L12 protein of 133 amino acid residues. The chloroplast L12 protein was synthesized with a reticulocyte lysate and subjected to nucleic acid-binding assays. L12 synthesized in vitro does not bind to ssDNA, dsDNA nor ribonucleotide homopolymers, but it binds to cellulose matrix.     

The primary structure of ribosomal protein L2 from Bacillus stearothermophilus

The complete amino acid sequence of ribosomal protein L2 from the moderate thermophile Bacillus stearothermophilus has been determined. This has been achieved by the sequence analysis of peptides derived by enzymatic digestion with Staphylococcus aureus protease, trypsin and chymotrypsin, as well as by chemical cleavage with o-iodosobenzoic acid. The protein contains 275 amino acid residues and has a calculated molecular mass of 30201 Da. Comparison of this sequence with sequences of the corresponding proteins from Escherichia coli and from spinach and tobacco chloroplasts reveals that 60% of the residues of protein L2 from B. stearothermophilus are identical to those of the protein from E. coli and 45% are identical to those found in the two chloroplast proteins. There are extended regions of totally conserved sequence at positions 54-58 (GGGHK), 81-86 (EYDPNR), and 224-230 (MNPVDHP) in all four proteins.     

The nuclear:organelle distribution of chloroplast ribosomal proteins genes. Features of a cDNA clone encoding the cytoplasmic precursor of L11.

The majority of chloroplast ribosomal proteins are encoded in the nuclear genome. In order to characterize these proteins through their mRNA, we have previously constructed a spinach cDNA expression library and raised antisera to several spinach chloroplast ribosomal proteins. Here we describe the immuno isolation of cDNA clones encoding protein L11 and its chloroplast-targeting presequence. The cytoplasmic precursor form of L11 is 224 amino acid residues long (Mr 23,662); the mature L11 and the transit sequence are predicted to be of approximately 159 and approximately 65 residues, respectively. The predicted chloroplast L11 is significantly longer than the E coli L11, but similar (in size) to archaebacterial and yeast cytoplasmic L11. In sequence it is closer to E coli L11 (54% identity) than to the archaebacterial (32%) or yeast (23%) proteins. These results and the conservation of the contexts of the 3 methyl modified residues found in E coli L11 are discussed in the light of the endosymbiont theory and nuclear relocation of the rp/KAJL gene cluster.     

A putative gene of tobacco chloroplast coding for ribosomal protein similar to E. coli ribosomal protein S19.

A partial sequence of a cloned 3.2 Md BamHI fragment from tobacco chloroplast DNA revealed the occurrence of a putative gene for ribosomal protein. The putative gene is located on the left margin of the large single-copy region in the chloroplast DNA. The coding region contains 276 bp (92 codons). The amino acid sequence deduced from the DNA sequence shows 55% homology with that of E. coli S19 (91 amino acid residues).     

Nucleotide sequence and linkage map position of the secX gene in maize chloroplast and evidence that it encodes a protein belonging to the 50S ribosomal subunit

The nucleotide sequence of the segment of maize chloroplast DNA lying between the map coordinate positions 32.59 and 32.98 Kb and containing the secX gene has been determined. The derived amino acid sequence of maize chloroplast secX is 95%, 87% and 62% identical to the corresponding derived amino acid sequences from two plant chloroplasts and Escherichia coli, respectively. It is also 70% identical to the experimentally determined amino acid sequence of a protein isolated from Bacillus stearothermophilus ribosomes. Separation of the 50S ribosomal subunit proteins of E. coli by reversed phase HPLC gave a peak which contained pure secX protein, as determined by N-terminal amino acid sequencing. Spinach chloroplast 50S subunit proteins separated by HPLC also gave a peak corresponding to pure secX protein. From these results we conclude that the secX gene in E. coli and in plant chloroplasts encodes a small (37-38 amino acid residues) ribosomal protein belonging to the 50S subunit. The same conclusion has been reached recently by A. Wada with respect to E. coli secX. In agreement with Wada, we name the secX protein L36. Its chloroplast gene is designated rpL36.     

Chloroplast ribosomal protein L32 is encoded in the chloroplast genome

The 50 S subunit of chloroplast ribosomes was prepared from tobacco leaves. The proteins were fractionated and the N-terminal amino acid sequence of a 14 kDa protein was determined. This sequence matches the N-terminal sequence deduced from ORF55 located between ndhF and trnL on the small single-copy region of tobacco chloroplast DNA. The deduced protein shows homology to E. coli and B. stearothermophilus L32 proteins, and it has been named as CL32 and ORF55 as rpl32. The tobacco chloroplast genome therefore contains 21 different ribosomal protein genes.     

Cloning and characterization of the chloroplast elongation factor EF-Tu cDNA of Oryza sativa L

From the rice leaf cDNA library, we have cloned a cDNA encoding rice chloroplast translational elongation factor EF-Tu (tufA). The rice tufA cDNA clone contains 1678 nucleotides and codes for a 467 amino acid protein including a putative chloroplast transit peptide of 59 amino acid residues. The predicted molecular mass of the mature protein is approximately 45 kDa. This cDNA clone contains the 61 nucleotides of the 5' untranslated region (UTR) and the 213 nucleotides of 3' UTR. Amino acid sequence identity of the rice tufA with the mature chloroplast EF-Tu proteins of tobacco, pea, arabidopsis, and soybean ranges from 83% to 86%. The deduced polypeptide of the rice tufA cDNA contains GTP binding domains in its N-terminal region and chloroplast EF-Tu signature regions in the C-terminal region. The rice tufA appears to exist as a single copy gene, although its homologues of maize and oat exist as multiple copy genes. The rice tufA gene is located in chromosome 1 and is more highly expressed in the leaf than in root tissue.     

Sequence of the gene for ribosomal protein L23 from the archaebacterium Methanococcus vannielii

The N-terminal sequence of HPLC-purified protein L23 from the Methanococcus vannielii ribosome has been determined by automated liquid-phase Edman degradation. Using the N-terminal amino acid sequence, an oligonucleotide probe complementary to the 5'-end of the gene was synthesized. The 26-mer oligonucleotide, containing two inosines, was used for hybridization with digested M. vannielii chromosomal DNA. The hybridizing band from HpaII-digested genomic DNA was ligated into pUC18 to yield plasmid pMvaZ1 containing the entire gene of protein L23. The nucleotide sequence complemented the partial amino acid sequence, and the gene codes for a protein of 9824 Da. The amino acid sequence of protein L23 form M. vannielii was compared to that of ribosomal proteins from other archaebacteria as well as from eubacteria and eukaryotes. The number of identical amino acids is highest when the M. vannielii protein is compared to the homologous protein from yeast and lowest vs that from tobacco chloroplasts. Interestingly, the secondary structures of the proteins as predicted by computer programs are more conserved than the primary structures.     

Complete amino acid sequences of the ribosomal proteins L25, L29 and L31 from the archaebacterium Halobacterium marismortui

Ribosomal proteins were extracted from 50S ribosomal subunits of the archaebacterium Halobacterium marismortui by decreasing the concentration of Mg2+ and K+, and the proteins were separated and purified by ion-exchange column chromatography on DEAE-cellulose. Ten proteins were purified to homogeneity and three of these proteins were subjected to sequence analysis. The complete amino acid sequences of the ribosomal proteins L25, L29 and L31 were established by analyses of the peptides obtained by enzymatic digestion with trypsin, Staphylococcus aureus protease, chymotrypsin and lysylendopeptidase. Proteins L25, L29 and L31 consist of 84, 115 and 95 amino acid residues with the molecular masses of 9472 Da, 12293 Da and 10418 Da respectively. A comparison of their sequences with those of other large-ribosomal-subunit proteins from other organisms revealed that protein L25 from H. marismortui is homologous to protein L23 from Escherichia coli (34.6%), Bacillus stearothermophilus (41.8%), and tobacco chloroplasts (16.3%) as well as to protein L25 from yeast (38.0%). Proteins L29 and L31 do not appear to be homologous to any other ribosomal proteins whose structures are so far known.     

Chloroplast ribosomal protein L13 is encoded in the nucleus and is considerably larger than its bacterial homologue. Construction, immunoisolation, and nucleotide sequence (including transit peptide) its cDNA clone from an angiosperm

Chloroplast ribosomes of higher plants are of the prokaryotic ribosome motif but, unlike in bacteria, their ribosomal protein (r-protein) genes are distributed between the organelle and the nucleus. In order to isolate some of the nuclear-encoded r-protein genes, we have raised antibodies to several spinach chloroplast r-proteins and constructed spinach cDNA expression libraries in lambdagt11. Screening the libraries with one of the antisera yielded three cDNA clones for r-protein L13, an early 50 S subunit assembly protein essential for RI50 formation. The cDNA clone encodes, beginning with a Met codon in the consensus plant initiator context, a polypeptide of 250 amino acid residues. The NH2-terminal 60 residues bear the characteristic features of a chloroplast transit peptide. The putative mature L13 protein, which has common immunoepitopes with Escherichia coli L13, is 34% longer than the E. coli homologue. It has 56% sequence identity with E. coli L13 in the homologous region, but no identity to any known protein in the extra stretch. There are two neighboring ATG codons in the 5' region and two putative plant polyadenylation signals in the 3'-untranslated region of the cDNA. Their possible effect to increase translational efficiency is discussed, and the importance of encoding a RI50 protein in the nuclear genome for possible nuclear control of chloroplast protein synthesis is noted.     

Nucleotide sequence and linkage map position of the genes for ribosomal proteins L14 and S8 in the maize chloroplast genome

The nucleotide sequence of a 1287-base-pair segment of the maize (Zea mays) chloroplast DNA, encoding chloroplast ribosomal proteins L14, S8 and the C-terminal part of L16, has been determined using the dideoxy-chain-termination method. These data from a monocot plant are compared to the corresponding data from a dicot and a lower plant and from two bacteria. The deduced amino acid sequence of maize chloroplast L14 shows 80%, 81%, 51% and 52% and that of S8 shows 75%, 58%, 39% and 38% sequence identity, respectively, to the corresponding sequences of Nicotiana tabacum, Marchantia polymorpha, Bacillus stearothermophilus and Escherichia coli. The starting map coordinates of rpL14 and rpS8 in the physical map of the maize chloroplast DNA [Larrinua, I. M., Muskavitch, K. M. T., Gubbins, E. J. and Bogorad, L. (1983) Plant Mol. Biol. 2, 129-140] are 31.330 and 31.841. The gene order is rpL16-spacer-rpL14-spacer-rpS8. Shine-Dalgarno sequences (GGA and AGGAGG) and computer-derived stem-loop structures of dyad symmetry are present in the spacers and the 3' downstream region of rpS8, respectively, but a chloroplast promoter-like sequence could not be detected suggesting that the latter might be located further upstream in this ribosomal protein gene cluster in maize chloroplast DNA.     

A ribosomal protein is encoded in the chloroplast DNA in a lower plant but in the nucleus in angiosperms. Isolation of the spinach L21 protein and cDNA clone with transit and an unusual repeat sequence

The distribution of chloroplast ribosomal protein genes between the organelle DNA and the nuclear DNA is highly conserved in land plants, but a notable exception is rpl21. This gene has been found in the completely sequenced chloroplast genome of a lower plant but not in that of two higher plants. We describe the purification and characterization of the spinach chloroplast ribosomal protein L21 and the isolation and nucleotide sequence of a cDNA clone that encodes its cytoplasmic precursor. The mature protein, identified by NH2-terminal sequencing, has 201 residues (Mr 22,766) and is thus substantially larger than either its Escherichia coli (103 residues) or the lower plant homologue (116 residues). The extra length is in peptide extensions at both amino and carboxyl termini. The COOH-terminal extension is unusual in that it comprises seven Ala-Glu repeats, a feature not found in any other ribosomal proteins described so far. The cDNA clone also encodes a 55-residue long transit peptide (with a high proportion of the polar residues, threonine and serine), to target the L21 protein into chloroplasts. The identification of rpl21 as a nuclear gene in a higher plant (spinach) and chloroplast gene in a lower plant (liverwort) suggests an organelle-to-nucleus gene relocation during the evolution of the former.     

The primary structure of rat ribosomal protein L7. The presence near the amino terminus of L7 of five tandem repeats of a sequence of 12 amino acids

The covalent structure of rat ribosomal protein L7 was determined in part from the sequence of nucleotides in a recombinant cDNA and in part from the sequence of amino acids in portions of the protein. The complementary analyses supplemented and confirmed each other. Ribosomal protein L7 contains 258 amino acids and has a molecular weight of 30,040. The protein has an unusual and striking structural feature near the NH2 terminus: five tandem repeats of a sequence of 12 residues. Rat L7 appears to be related to ribosomal protein L7 from the moderate halophile Vibrio costicola and perhaps to L30 from Bacillus stearothermophilus, to L7 from the moderate halophile NRCC 41227, and to L22 from Nicotinia tobaccum chloroplast. In addition, there is a sequence of 24 amino acids in rat protein L7 that may be related to segments of the same number of residues in Escherichia coli ribosomal proteins S10, S15, L9, and L22.     

Protein-rRNA binding features and their structural and functional implications in ribosomes as determined by cross-linking studies.

We have investigated protein-rRNA cross-links formed in 30S and 50S ribosomal subunits of Escherichia coli and Bacillus stearothermophilus at the molecular level using UV and 2-iminothiolane as cross-linking agents. We identified amino acids cross-linked to rRNA for 13 ribosomal proteins from these organisms, namely derived from S3, S4, S7, S14, S17, L2, L4, L6, L14, L27, L28, L29 and L36. Several other peptide stretches cross-linked to rRNA have been sequenced in which no direct cross-linked amino acid could be detected. The cross-linked amino acids are positioned within loop domains carrying RNA binding features such as conserved basic and aromatic residues. One of the cross-linked peptides in ribosomal protein S3 shows a common primary sequence motif--the KH motif--directly involved in interaction with rRNA, and the cross-linked amino acid in ribosomal protein L36 lies within the zinc finger-like motif of this protein. The cross-linked amino acids in ribosomal proteins S17 and L6 prove the proposed RNA interacting site derived from three-dimensional models. A comparison of our structural data with mutations in ribosomal proteins that lead to antibiotic resistance, and with those from protein-antibiotic cross-linking experiments, reveals functional implications for ribosomal proteins that interact with rRNA.