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A R Subramanian

Publications and source records attributed to A R Subramanian.

At least 37 records · Page 2Linked to original sources
PubMed1992

Effect of E. coli ribosomal protein S1 on the fidelity of the translational elongation step: reading and misreading of poly(U) and poly(dT).

Ribosomal protein S1 was selectively removed from E. coli ribosomes by affinity chromatography and the effect of added S1 on the translation of poly(dT) [which is read as poly(U) in the presence of neomycin] and on the misreading of poly(U) and poly(dT) were examined. S1 enhances the translation of poly(dT) at low template concentration, which is similar to the effect of S1 on poly(U) translation. The misreading of poly(dT) by E. coli ribosomes is at a lower level than is the case with poly(U). This low misreading is the same for "S1-dependent" and "S1-independent" modes of translation. On the other hand, the misreading of poly(U) is significantly reduced when S1 is present. These results thus indicate that S1 not only facilitates the binding of mRNA to the ribosome as already known, but also plays a role in the correct codon-dependent selection of aminoacyl-tRNA.

Bacterial Proteins↗
PubMed1991

Expression and functional assembly into bacterial ribosomes of a nuclear-encoded chloroplast ribosomal protein with a long NH2-terminal extension.

Chloroplast ribosomal protein L13 is encoded in the plant nucleus and is considerably larger than its eubacterial homologue by having NH2- and COOH-terminal extensions with no homology to any known sequences (Phua et al., J Biol. Chem. 264, 1968-1971, 1989). We made two gene constructs of L13 cDNA using the polymerase chain reaction (PCR) and expressed them in Escherichia coli. Analysis of the ribosomes and polysomes from these cells, using an antiserum specific to chloroplast L13, shows that the expressed proteins are incorporated, in the presence of the homologous E. coli L13, into functional ribosomes which participate in protein synthesis (i.e. polysomes). Evidence is obtained that the large NH2-terminal extension probably lies on the surface of these 'mosaic ribosomes.' This first report of the assembly into E. coli ribosomes of nuclear-coded chloroplast ribosomal protein with terminal extensions thus suggest an extraordinary conservation in the function of eubacterial type ribosomal proteins, despite the many changes in protein structure during their evolution inside a eukaryotic system.

Amino Acid Sequence↗
PubMed1991

Chloroplast ribosomal protein L15, like L1, L13 and L21, is significantly larger than its E. coli homologue.

The purification and identification by peptide sequence and immunological data of the spinach chloroplast homologue of E. coli L15 is presented. A significant increase in its mass over the E. coli counterpart is shown and is accounted for, in part, by a sequenced 18-residue N-terminal extension. A still larger C-terminal extension or internal insertion(s) is inferred. The migration position of the L15 in a 2D gel pattern of spinach chloroplast 50S subunit proteins is shown. Lack of sequence identity with the known chloroplast genomic data confirms the nuclear coding of this protein, and the N-terminal sequence given here provides the transit peptide cleavage site of the cytoplasmic precursor.

Amino Acid Sequence↗
PubMed1991

A heptapeptide repeat contributes to the unusual length of chloroplast ribosomal protein S18. Nucleotide sequence and map position of the rpl33-rps18 gene cluster in maize.

The rpl33-rps18 gene cluster of the maize chloroplast genome has been mapped and sequenced. The derived amino acid sequence of the S18 protein shows a 7-fold repeat of a hydrophilic heptapeptide domain, S K Q P F R K, in the N-terminal region. Such a sequence is absent in the E. coli S18 and in the chloroplast S18 of the lower plant liverwort. In tobacco and rice chloroplast S18 it is present 2 and 6 times, respectively. Thus a long N-terminal repeat (resembling in composition the large C-terminal heptapeptide repeat in the eukaryotic pol II) appears to be characteristic of monocot cereal S18.

Amino Acid Sequence↗
PubMed1991

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.

Amino Acid Sequence↗
PubMed1991

Construction of a chloroplast DNA library from rye (Secale cereale), a cereal plant of temperature-inducible chloroplast ribosome deficiency.

The chloroplast genomes of flowering plants are circular DNA molecules, 120 to 160 kilobase pairs long, encoding the rRNA, all tRNAs, and 21 r-proteins of the chloroplast translational apparatus as well as key protein components of the photosynthetic and carbon reduction cycle reactions. In this paper we describe some characteristics of the rye chloroplast (plastid) genome and the construction and characterization of a clone library of 93% of its DNA in a plasmid and a cosmid vector. The size of rye chloroplast DNA is estimated at 135 kbp, similar to that for wheat and rice but slightly smaller than the estimate for maize (139 kbp). Chloroplast ribosome deficiency is induced in rye seedlings by germination and growth at 32 degrees-34 degrees C; therefore these clones would be useful for analyzing the regulation of chloroplast ribosome synthesis in higher plants, a process that requires coordinate expression of genes located in the nucleus and the chloroplast.

Chloroplasts↗
PubMed1990

Enhanced translational utilization of chloroplast ribosomal protein mRNAs from two AUG codons shown by site-directed mutation.

The cDNA for protein L12 of the chloroplast ribosome contains two ATG codons, both in consensus initiator context, at the beginning of its transit peptide coding region [Giese, K., & Subramanian, A. R. (1989) Biochemistry 28, 3525-3529]. Due to the location in the transit peptide, translational start from either ATG codon would yield the same mature protein after transport into chloroplasts and N-terminal cleavage. To test whether this arrangement, also found in the cDNAs of several other chloroplast ribosomal proteins, could enhance the utilization of mRNA, we constructed a chimeric gene containing the 5' part of the L12 cDNA fused to the neomycin phosphotransferase gene. A frameshift that would prematurely terminate the translation from the first AUG codon was introduced into the construct by site-directed mutagenesis. Transcription-translation of the fusion gene in vitro and its expression in vivo in spinach protoplasts showed that protein synthesis occurs from both initiation codons: at 70-80% of the total level from the first and 20-30% from the second. The results thus show that (1) eukaryotic ribosomes can initiate to a significant level from a downstream AUG codon and (2) the occurrence of two in-frame initiation codons enhances translational efficiency.

Amino Acid Sequence↗
PubMed1990

Ribosomal protein L35: identification in spinach chloroplasts and isolation of a cDNA clone encoding its cytoplasmic precursor.

We describe the isolation of spinach chloroplast ribosomal protein L35 and characterization of a cDNA clone encoding its cytoplasmic precursor. This protein was only recently identified in ribosomes, but the sequences of four L35 genes have now been reported and confirm its presence in eubacteria, chloroplasts, and cyanelles. Using N-terminal sequence data, oligonucleotides were designed and a cDNA library was screened. The nucleotide sequence of the cDNA clones shows that the spinach L35 protein is encoded as a precursor of 159 residues, comprising a mature protein of 73 residues and a transit peptide of 86 residues. The cleavage site for forming the mature protein is deduced to be Thr-Val-Phe-Ala decreases Ala-Lys-Gly-Tyr. The L35 protein in the photosynthetic organelle of the protozoan Cyanophora paradoxa is encoded in the organelle DNA [Bryant & Stirewalt (1990) FEBS Lett. 259, 273-280]. The corresponding gene has not been found in the chloroplast DNA of a lower plant (liverwort) and two higher plants. Our results demonstrate that the L35 protein in a higher plant (spinach) is encoded in the nucleus. This finding, in light of the endosymbiont hypothesis, suggests an organelle to nucleus transfer of the L35 gene at the evolutionary beginnings of land plants.

Amino Acid Sequence↗
PubMed1990

Ribosomal protein S1 of Escherichia coli is the effector for the regulation of its own synthesis.

To facilitate the study of the regulation of the rpsA gene, a translational fusion between the rpsA gene and the lacZ gene was constructed. Synthesis of the fusion protein was repressed about 10-fold when rpsA was supplied in trans on a multicopy plasmid. This repression is similar to the post-transcriptional regulation previously found for the wild type rpsA gene. Addition of purified protein S1 to a coupled in vitro transcription-translation system caused a specific reduction in the synthesis of the rpsA-lacZ fusion protein. Addition of various subdomain fragments of protein S1 to the coupled in vitro system showed that the N-terminal fragment, possessing the ribosome binding domain of protein S1, was able to repress the synthesis of the rpsA-lacZ fusion protein. In contrast, fragments from the C-terminal region, containing the nucleic acid binding domain of protein S1, were inactive in this repression. Induction of truncated rpsA genes, coding for either the N-terminal 101 or 329 amino acids caused a reduction in the synthesis of the chromosomally encoded protein S1, thus confirming in vivo that the N-terminal part of protein S1 represses rpsA expression.

Amino Acid Sequence↗
PubMed1990

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.

Amino Acid Sequence↗
PubMed1990

Cloning and characterization of the genes for ribosomal proteins L10 and L12 from Synechocystis Sp. PCC 6803: comparison of gene clustering pattern and protein sequence homology between cyanobacteria and chloroplasts.

The endosymbiont theory proposes that chloroplasts have originated from ancestral cyanobacteria through a process of engulfment and subsequent symbiotic adaptation. The molecular data for testing this theory have mainly been the nucleotide sequence of rRNAs and of photosystem component genes. In order to provide additional data in this area, we have isolated genomic clones of Synechocystis DNA containing the ribosomal protein gene cluster rplJL. The nucleotide sequence of this cluster and flanking regions was determined and the derived amino acid sequences were compared to the available homologous sequences from other eubacteria and chloroplasts. In Escherichia coli these two genes are part of a larger cluster, i.e., rplKAJL-rpoBC. In Synechocystis, the genes for the RNA polymerase subunit (rpoBC) are shown to be widely separated from the r-protein genes. The Synechocystis gene arrangement is similar to that in the chloroplast system, where the rpoBC1C2 and rplKAJL clusters are separated and located in two cell compartments, the chloroplast and the nucleus, respectively.

Amino Acid Sequence↗
PubMed1990

Identification of a plastid-specific ribosomal protein in the 30 S subunit of chloroplast ribosomes and isolation of the cDNA clone encoding its cytoplasmic precursor.

We describe the isolation and characterization of a chloroplast ribosomal protein and a clone of its cDNA. This protein has no homology to any Escherichia coli ribosomal protein or to any known proteins. Due to this novel finding we propose it be called PSrp-1, i.e. a plastid-specific ribosomal protein. The precursor form of PSrp-1, deduced from the cDNA sequence, is 302-amino acid residues long. The mature PSrp-1, identified by amino-terminal sequencing, is a protein of 236 residues. The NH2-terminal 66 amino acids form the transit peptide that targets PSrp-1 into the chloroplast. We show that PSrp-1 is a protein of the chloroplast 30 S ribosomal subunit by Western blotting and sequencing the excised protein after two-dimensional gel electrophoresis. The possible evolutionary origin of PSrp-1 from the nucleated host cell of the endosymbiont theory is discussed.

Amino Acid Sequence↗
PubMed1989

A new rearrangement of angiosperm chloroplast DNA in rye (Secale cereale) involving translocation and duplication of the ribosomal rpS15 gene.

The mapping and nucleotide sequencing of the rpS15 gene in the rye chloroplast DNA has shown that it is located in the inverted repeat (IR) and thus has two copies/genome. This is in contrast to tobacco and liverwort chloroplasts where rpS15 occurs as single-copy gene localized in the small single copy region (SSC). The direction of transcription of both gene copies in rye is toward SSC; that in tobacco and liverwort is toward IR-II. Further sequence data have revealed that the 3' end of each rye rpS15 gene copy is only 352 base pairs away from the corresponding IR.SSC junction and that this rearrangement event in rye involves also a 3' downstream-encoded and highly conserved chloroplast gene designated ORF393 in tobacco and ORF392 in liverwort. The latter in rye starts in both the IRs, but continues to full length into the SSC only from the IR-II. The direction of transcription of the nontruncated gene is fixed toward IR-I, being thus the inverse of ORF393/392 in tobacco and liverwort. Northern blot analysis has shown that the rearranged rpS15 gene is actively transcribed in rye chloroplasts and etioplasts and that its transcription pattern is different from that recently reported for tobacco rpS15.

Amino Acid Sequence↗
PubMed1989

Chloroplast ribosomal protein L12 is encoded in the nucleus: construction and identification of its cDNA clones and nucleotide sequence including the transit peptide.

An architectural feature found in all classes of ribosomes is a thin, 10-nm-long protuberance in the large subunit, generated by multiple copies of r-protein L12. The primary structure of spinach chloroplast r-protein L12 is known [Bartsch, M., Kimura, M., & Subramanian, A. R. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 6871-6875], but the location of its gene, whether in the organelle or in the nucleus, has not been determined. Therefore, we synthesized four oligodeoxynucleotides based on the amino acid sequence data and used them to probe a spinach cDNA library we constructed in lambda gt11 vector. cDNA inserts from four of the hybridizing recombinant clones were characterized and sequenced. The data showed that they are reverse transcripts of varying length, all derived from a single poly(A+) RNA species. The longest cDNA molecule is 900 base pairs (bp) long and includes a 5' noncoding sequence followed by two neighboring AUG codons both in the consensus, eukaryotic initiator context, a 56-codon-long transit peptide sequence (starting from the first AUG codon), the amino acid sequence of mature L12 protein, and a 238 bp long 3' downstream noncoding sequence including a polyadenylation signal and the start of the poly(A) tail. The transit peptide sequence has an unusual amino acid composition similar to that of other known chloroplast transit peptides. Northern blot analysis of the poly(A+) RNA isolated from spinach seedlings and probed with the cDNA insert revealed the occurrence of a strong, broad, 950-nucleotide-long band of the corresponding poly(A+)-containing mRNA species.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1989

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.

Amino Acid Sequence↗