RNA-binding characteristics of the chloroplast S1-like ribosomal protein CS1

The chloroplast ribosomal protein CS1, the homolog of the bacterial ribosomal protein S1, is believed to be involved in the process of ribosome binding to mRNA during translation. Since translation control is an important step in chloroplast gene expression, and in order to study initiation complex formation, we studied the RNA-binding properties of CS1 protein. We found that most of the CS1 protein in spinach chloroplast co-purified with the 30S ribosomal subunit. The relative binding affinity of RNA to CS1 was determined using the UV-crosslinking competition assay. CS1 protein binds the ribohomopolymer poly(U) with a relatively high binding affinity. Very low binding affinities were obtained for the other ribohomopolymers, poly(G), poly(A) and poly(C). In addition, no specific binding of CS1, either in the 30S complex or as a recombinant purified protein, was obtained to the 5′-untranslated region of the mRNA in comparison to the other parts. RNA-binding experiments, in which the N- and C-termini of the protein were analyzed, revealed that the RNA-binding site is located in the C-terminus half of the protein. These results suggest that CS1 does not direct the 30S complex to the initiation codon of the translation site by specific binding to the 5′-untranslated region. In bacteria, specific binding is derived by base pairing between 16S rRNA and the Shine–Dalagarno sequences. In the chloroplast, nuclear encoded and gene-specific translation factors may be involved in the determination of specific binding of the 30S subunit to the initiator codon.     

Erythromycin and 5S rRNA binding properties of the spinach chloroplast ribosomal protein CL22.

The spinach chloroplast ribosomal protein (r-protein) CL22 contains a central region homologous to the Escherichia coli r-protein L22 plus long N- and C-terminal extensions. We show in this study that the CL22 combines two properties which in E. coli ribosome are split between two separate proteins. The CL22 which binds to the 5S rRNA can also be linked to an erythromycin derivative added to the 50S ribosomal subunit. This latter property is similar to that of the E. coli L22 and suggests a similar localization in the 50S subunit. We have overproduced the r-protein CL22 and deleted forms of this protein in E. coli. We show that the overproduced CL22 binds to the chloroplast 5S rRNA and that the deleted protein containing the N- and C-terminal extensions only has lost the 5S rRNA binding property. We suggest that the central homologous regions of the CL22 contains the RNA binding domain.     

Activity of Euglena gracilis chloroplast ribosomes with procaryotic and eucaryotic initiation factors

A method that permits the preparation of Euglena gracilis chloroplast 30 S ribosomal subunits that are largely free of endogenous initiation factors and that are active in the binding of fMet-tRNA in response to poly(A, U, G), has been developed. These 30 S subunits have been tested for activity in initiation complex formation with initiation factors from both procaryotes and eucaryotes. We have observed that Escherichia coli IF-2 binds fMet-tRNA nearly as well to Euglena chloroplast ribosomal subunits as it does to its homologous subunits. Neither wheat germ eIF-2 nor Euglena eIF-2A can bind fMet-tRNA efficiently to Euglena chloroplast or E. coli 30 S subunits although both are active with wheat germ 40 S ribosomal subunits. Euglena chloroplast 68 S ribosomes will also bind the initiator tRNA. Both E. coli IF-2 and E. coli IF-3 stimulate this reaction on chloroplast ribosomes with approximately the same efficiency as they do on their homologous ribosomes. E. coli IF-1 enhances the binding of fMet-tRNA to the chloroplast 68 S ribosomes when either IF-2 or IF-3 is limiting. The chloroplast ribosomes unlike E. coli ribosomes show considerable activity over a broad range of Mg2+ ion concentrations.     

Structure and expression of the gene encoding ribosomal protein S1 from the cyanobacterium Synechococcus sp. strain PCC 6301: striking sequence similarity to the chloroplast ribosomal protein CS1

We isolated a 38 kDa ssDNA-binding protein from the unicellular cyanobacterium Synechococcus sp. strain PCC 6301 and determined its N-terminal amino acid sequence. A genomic clone encoding the 38 kDa protein was isolated by using a degenerate oligonucleotide probe based on the amino acid sequence. The nucleotide sequence and predicted amino acid sequence revealed that the 38 kDa protein is 306 amino acids long and homologous to the nuclear-encoded 370 amino acid chloroplast ribosomal protein CS1 of spinach (48% identity), therefore identifying it as ribosomal protein (r-protein) S1. Cyanobacterial and chloroplast S1 proteins differ in size from Escherichia coli r-protein S1 (557 amino acids). This provides an additional evidence that cyanobacteria are closely related to chloroplasts. The Synechococcus gene rps1 encoding S1 is located 1.1 kb downstream from psbB, which encodes the photosystem 11 P680 chlorophyll a apoprotein. An open reading frame encoding a potential protein of 168 amino acids is present between psbB and rps1 and its deduced amino acid sequence is similar to that of E. coli hypothetical 17.2 kDa protein. Northern blot analysis showed that rps1 is transcribed as a monocistronic mRNA.     

Structure and expression of the gene encoding ribosomal protein S1 from the cyanobacterium Synechococcus sp. strain PCC 6301: striking sequence similarity to the chloroplast ribosomal protein CS1

We isolated a 38 kDa ssDNA-binding protein from the unicellular cyanobacterium Synechococcus sp. strain PCC 6301 and determined its N-terminal amino acid sequence. A genomic clone encoding the 38 kDa protein was isolated by using a degenerate oligonucleotide probe based on the amino acid sequence. The nucleotide sequence and predicted amino acid sequence revealed that the 38 kDa protein is 306 amino acids long and homologous to the nuclear-encoded 370 amino acid chloroplast ribosomal protein CS1 of spinach (48% identity), therefore identifying it as ribosomal protein (r-protein) S1. Cyanobacterial and chloroplast S1 proteins differ in size from Escherichia coli r-protein S1 (557 amino acids). This provides an additional evidence that cyanobacteria are closely related to chloroplasts. The Synechococcus gene rps1 encoding S1 is located 1.1 kb downstream from psbB, which encodes the photosystem 11 P680 chlorophyll a apoprotein. An open reading frame encoding a potential protein of 168 amino acids is present between psbB and rps1 and its deduced amino acid sequence is similar to that of E. coli hypothetical 17.2 kDa protein. Northern blot analysis showed that rps1 is transcribed as a monocistronic mRNA.     

Proteomic characterization of the Chlamydomonas reinhardtii chloroplast ribosome. Identification of proteins unique to th e70 S ribosome

We have conducted a proteomic analysis of the 70 S ribosome from the Chlamydomonas reinhardtii chloroplast. Twenty-seven orthologs of Escherichia coli large subunit proteins were identified in the 50 S subunit, as well as an ortholog of the spinach plastid-specific ribosomal protein-6. Several of the large subunit proteins of C. reinhardtii have short extension or insertion sequences, but overall the large subunit proteins are very similar to those of spinach chloroplast and E. coli. Two proteins of 38 and 41 kDa, designated RAP38 and RAP41, were identified from the 70 S ribosome that were not found in either of the ribosomal subunits. Phylogenetic analysis identified RAP38 and RAP41 as paralogs of spinach CSP41, a chloroplast RNA-binding protein with endoribonuclease activity. Overall, the chloroplast ribosome of C. reinhardtii is similar to those of spinach chloroplast and E. coli, but the C. reinhardtii ribosome has proteins associated with the 70 S complex that are related to non-ribosomal proteins in other species. In addition, the 30 S subunit contains unusually large orthologs of E. coli S2, S3, and S5 and a novel S1-type protein (Yamaguchi, K. et al., (2002) Plant Cell 14, 2957-2974). These additional proteins and domains likely confer functions used to regulate chloroplast translation in C. reinhardtii.     

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.     

Escherichia coli poly(A)-binding proteins that interact with components of degradosomes or impede RNA decay mediated by polynucleotide phosphorylase and RNase E

The multifunctional ribonuclease RNase E and the 3'-exonuclease polynucleotide phosphorylase (PNPase) are major components of an Escherichia coli ribonucleolytic "machine" that has been termed the RNA degradosome. Previous work has shown that poly(A) additions to the 3' ends of RNA substrates affect RNA degradation by both of these enzymes. To better understand the mechanism(s) by which poly(A) tails can modulate ribonuclease action, we used selective binding in 1 m salt to identify E. coli proteins that interact at high affinity with poly(A) tracts. We report here that CspE, a member of a family of RNA-binding "cold shock" proteins, and S1, an essential component of the 30 S ribosomal subunit, are poly(A)-binding proteins that interact functionally and physically, respectively, with degradosome ribonucleases. We show that purified CspE impedes poly(A)-mediated 3' to 5' exonucleolytic decay by PNPase by interfering with its digestion through the poly(A) tail and also inhibits both internal cleavage and poly(A) tail removal by RNase E. The ribosomal protein S1, which is known to interact with sequences at the 5' ends of mRNA molecules during the initiation of translation, can bind to both RNase E and PNPase, but in contrast to CspE, did not affect the ribonucleolytic actions of these enzymes. Our findings raise the prospect that E. coli proteins that bind to poly(A) tails may link the functions of degradosomes and ribosomes.     

Characterization of protein-binding to the spinach chloroplast psbA mRNA 5' untranslated region.

RNA-binding proteins play a major role in regulating mRNA metabolism in chloroplasts. In this work we characterized two proteins, of 43 and 47 kDa, which bind to the spinach psbA mRNA 5' untranslated region (psbA encoding the D1 protein of photosystem II). The 43 kDa protein, which is present in the stroma and in membranes, co-sediments with a complex of 68S. It was purified, and the N-terminal sequence was determined. Upon homology search it was identified as the chloroplast homologue of the Escherichia coli ribosomal protein S1. The 47 kDa protein, which, in contrast with the 43 kDa protein, sediments with a small sedimentation coefficient, is only detected in the stromal fraction. It is soluble in an uncomplexed form. By deletion analysis, an element within the psbA mRNA 5' untranslated region was identified that is necessary but not sufficient for binding of stromal proteins. The 'central protein binding element' ranges from nucleotide -49 to -9 of the psbA mRNA 5' untranslated region. It comprises the Shine-Dalgarno-like GGAG motif and, 7 nucleotides upstream, an endonucleolytic cleavage site involved in psbA mRNA degradation in vitro . The mechanistic impacts of this region in relation to RNA-binding proteins are discussed.     

Proteolysis of ribosomal protein S1 from <Emphasis Type="Italic">Escherichia coli</Emphasis> and <Emphasis Type="Italic">Thermus thermophilus</Emphasis> leads to formation of two different fragments

As a result of limited tryptic proteolysis of S1 ribosomal protein (molecular mass 60 kD) from Thermus thermophilus, 25 N-terminal amino acid residues and 71 C-terminal amino acid residues are split off and a stable high-molecular-weight fragment with molecular mass of 49 kD is formed that retains RNA-binding properties and is capable of interacting with 30S ribosomal subunit. Earlier, application of a similar procedure for the formation of a fragment of S1 protein from Escherichia coli resulted in splitting of 171 N-terminal amino acid residues with the formation of a 41.3 kD fragment that possesses RNA-binding properties only. Thus, in spite of high homology between E. coli and T. thermophilus proteins, the proteolysis leads to the formation of two different fragments, which points, in our opinion, to the fact of significant differences between their structures.     

Differential phosphorylation controls Maskin association with eukaryotic translation initiation factor 4E and localization on the mitotic apparatus

Several cytoplasmic polyadenylation element (CPE)-containing mRNAs that are repressed in Xenopus oocytes become active during meiotic maturation. A group of factors that are anchored to the CPE are responsible for this repression and activation. Two of the most important are CPEB, which binds directly to the CPE, and Maskin, which associates with CPEB. In oocytes, Maskin also binds eukaryotic translation initiation factor 4E (eIF4E), an interaction that excludes eIF4G and prevents formation of the eIF4F initiation complex. When the oocytes are stimulated to reenter the meiotic divisions (maturation), CPEB promotes cytoplasmic polyadenylation. The newly elongated poly(A) tail becomes bound by poly(A) binding protein (PABP), which in turn binds eIF4G and helps it displace Maskin from eIF4E, thereby inducing translation. Here we show that Maskin undergoes several phosphorylation events during oocyte maturation, some of which are important for its dissociation from eIF4E and translational activation of CPE-containing mRNA. These sites are T58, S152, S311, S343, S453, and S638 and are phosphorylated by cdk1. Mutation of these sites to alanine alleviates the cdk1-induced dissociation of Maskin from eIF4E. Prior to maturation, Maskin is phosphorylated on S626 by protein kinase A. While this modification has no detectable effect on translation during oocyte maturation, it is critical for this protein to localize on the mitotic apparatus in somatic cells. These results show that Maskin activity and localization is controlled by differential phosphorylation.     

The solution structure of the loop E region of the 5S rRNA from spinach chloroplasts

A structure has been obtained for the loop E region of the 5S rRNA from Spinacia oleracia chloroplast ribosomes using residual dipolar coupling data as well as NOE, J coupling and chemical shift information. Even though the loop E sequence of this chloroplast 5S rRNA differs from that of Escherichia coli loop E at approximately 40% of its positions, its conformation is remarkably similar to that of E.coli loop E. Consistent with this conclusion, ribosomal protein L25 from E.coli, which binds to the loop E region of both intact E.coli 5S rRNA and to oligonucleotides containing that sequence, also binds to the chloroplast-derived oligonucleotide discussed here.     

Maize chloroplast DNA encodes a protein sequence homologous to the bacterial ribosome assembly protein S4.

A cloned restriction fragment of maize chloroplast DNA (Bam H1 fragment 5) is shown to contain an open reading frame which encodes a basic protein of 201 amino acid residues with 40-50% sequence homology to E. coli ribosomal protein S4. Based on the experimentally determined sequence homology between the highly conserved bacterial ribosomal protein L12 and its chloroplast homologue (Bartsch M., Kimura, M. and Subramanian, A.R. (1982) Proc. Natl. Acad. Sci. USA 79, 6871), we conclude that this reading frame represents the maize chloroplast S4 gene. The nucleotide sequence of a 1100 base pair DNA segment containing the putative gene is presented.     

Phylogenetic Analysis of L4-Mediated Autogenous Control of the S10 Ribosomal Protein Operon

We investigated the regulation of the S10 ribosomal protein (r-protein) operon among members of the gamma subdivision of the proteobacteria, which includes Escherichia coli. In E. coli, this 11-gene operon is autogenously controlled by r-protein L4. This regulation requires specific determinants within the untranslated leader of the mRNA. Secondary structure analysis of the S10 leaders of five enterobacteria (Salmonella typhimurium, Citrobacter freundii, Yersinia enterocolitica, Serratia marcescens, and Morganella morganii) and two nonenteric members of the gamma subdivision (Haemophilus influenzae and Vibrio cholerae) shows that these foreign leaders share significant structural homology with the E. coli leader, particularly in the region which is critical for L4-mediated autogenous control in E. coli. Moreover, these heterologous leaders produce a regulatory response to L4 oversynthesis in E. coli. Our results suggest that an E. coli-like L4-mediated regulatory mechanism may operate in all of these species. However, the mechanism is not universally conserved among the gamma subdivision members, since at least one, Pseudomonas aeruginosa, does not contain the required S10 leader features, and its leader cannot provide the signals for regulation by L4 in E. coli. We speculate that L4-mediated autogenous control developed during the evolution of the gamma branch of proteobacteria.