Interaction of ribosomal proteins, S6, S8, S15 and S18 with the central domain of 16 S ribosomal RNA

Svensson, Patrik; Changchien, Li-Ming; Craven, Gary R.; Noller, Harry F.

doi:10.1016/0022-2836(88)90242-2

Cited by 90 publications

(81 citation statements)

References 26 publications

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“…The filled arrowheads indicate positions in 16S rRNA protected by the ribosomal proteins in native and reconstituted 30S subunits relative to 16S rRNA transcript. Shown are protection by primary binding proteins (S4, S8, S17, and S20), secondary binding proteins (S6, S11, S12, S16, and S18), and tertiary binding proteins (S2 and S21) (Powers et al 1988;Stern et al 1988a,c;Svensson et al 1988). Sites of enhanced reactivity observed in native and reconstituted 30S subunits relative to 16S rRNA transcripts are indicated by the unfilled arrowheads.…”

Section: Discussionmentioning

confidence: 99%

“…Chemical probing showed that the protection pattern of 16S rRNA in the reconstituted 30S subunit is similar to that in the native 30S subunit; however, the level of protection is incomplete. Especially, regions in the 16S rRNA that interact with the tertiary binding proteins show incomplete protection compared to regions that bind the primary and secondary binding proteins (Powers et al 1988;Stern et al 1988a,c;Svensson et al 1988). This further indicates that the reconstituted 30S subunits purified by a single sucrose gradient centrifugation are a mixture of fully and partially assembled 30S particles.…”

Section: Nonbridging Phosphate Oxygens In 16s Rrna That Are Importantmentioning

confidence: 99%

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Nonbridging phosphate oxygens in 16S rRNA important for 30S subunit assembly and association with the 50S ribosomal subunit

Ghosh

Joseph

2005

RNA

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Ribosomes are composed of RNA and protein molecules that associate together to form a supramolecular machine responsible for protein biosynthesis. Detailed information about the structure of the ribosome has come from the recent X-ray crystal structures of the ribosome and the ribosomal subunits. However, the molecular interactions between the rRNAs and the r-proteins that occur during the intermediate steps of ribosome assembly are poorly understood. Here we describe a modification-interference approach to identify nonbridging phosphate oxygens within 16S rRNA that are important for the in vitro assembly of the Escherichia coli 30S small ribosomal subunit and for its association with the 50S large ribosomal subunit. The 30S small subunit was reconstituted from phosphorothioate-substituted 16S rRNA and small subunit proteins. Active 30S subunits were selected by their ability to bind to the 50S large subunit and form 70S ribosomes. Analysis of the selected population shows that phosphate oxygens at specific positions in the 16S rRNA are important for either subunit assembly or for binding to the 50S subunit. The X-ray crystallographic structures of the 30S subunit suggest that some of these phosphate oxygens participate in r-protein binding, coordination of metal ions, or for the formation of intersubunit bridges in the mature 30S subunit. Interestingly, however, several of the phosphate oxygens identified in this study do not participate in any interaction in the mature 30S subunit, suggesting that they play a role in the early steps of the 30S subunit assembly.

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Section: Discussionmentioning

confidence: 99%

Section: Nonbridging Phosphate Oxygens In 16s Rrna That Are Importantmentioning

confidence: 99%

Nonbridging phosphate oxygens in 16S rRNA important for 30S subunit assembly and association with the 50S ribosomal subunit

Ghosh

Joseph

2005

RNA

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“…During 30S subunit assembly, S8 binds to the central domain of the 16S rRNA and interacts cooperatively with several other small-subunit proteins to form a well-defined ribonucleoprotein neighborhood (20,57). The binding site for this protein consists of an imperfect helix spanning nucleotides 583 to 653 within the 16S rRNA (20,37,67).…”

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confidence: 99%

Mutagenesis of ribosomal protein S8 from Escherichia coli: defects in regulation of the spc operon

et al. 1992

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The structural features of Escherichia coli ribosomal protein S8 that are involved in translational regulation of spc operon expression and, therefore, in its interaction with RNA have been investigated by use of a genetic approach. The rpsH gene, which encodes protein S8, was first inserted into an expression vector under the control of the lac promoter and subsequently mutagenized with methoxylamine or nitrous acid. A screening procedure based on the regulatory role of S8 was used to identify mutants that were potentially defective in their ability to associate with spc operon mRNA and, by inference, 16S rRNA. In this way, we isolated 39 variants of the S8 gene containing alterations at 34 different sites, including 37 that led to single amino acid substitutions and 2 that generated premature termination codons. As the mutations were distributed throughout the polypeptide chain, our results indicate that amino acid residues important for the structural integrity of the RNA-binding domain are not localized to a single segment. Nonetheless, the majority were located within three short sequences at the N terminus, middle, and C terminus that are phylogenetically conserved among all known eubacterial and chloroplast versions of this protein. We conclude that these sites encompass the main structural determinants required for the interaction of protein S8 with RNA.Protein S8, a small globular polypeptide component of the Escherichia coli ribosome, plays critical roles both in the assembly of the 30S ribosomal subunit (24) and in the translational regulation of ribosomal proteins encoded by the spc operon (12,65). During 30S subunit assembly, S8 binds to the central domain of the 16S rRNA and interacts cooperatively with several other small-subunit proteins to form a well-defined ribonucleoprotein neighborhood (20, 57). The binding site for this protein consists of an imperfect helix spanning nucleotides 583 to 653 within the 16S rRNA (20,37,67). A variety of methods, including comparative sequence analysis (59), chemical modification (36, 60), protein-RNA cross-linking (63), and site-directed mutagenesis (19,21), have been used to delineate the main structural determinants within the rRNA that mediate its association with protein S8. One of the most striking features of the S8 binding site is a phylogenetically conserved element that comprises either a small internal loop (19,20) or a slightly unwound helix containing three bulged adenine residues (36). Whatever the precise configuration of this region, alteration of the conserved features by mutagenesis reduces the affinity of S8 for the rRNA to below-detectable levels (19).Protein S8 participates in maintaining the balanced synthesis of ribosomal proteins through the mechanism of translational feedback control (7, 65). When expressed in excess, S8 is thought to bind to a specific site in the polycistronic spc mRNA, inhibiting the translation of at least eight ribosomal proteins and causing a significant reduction in the growth rate (12,32,39). A fragment of the m...

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“…Eubacterial S8 binds 16S rRNA in the 30S subunit and is considered crucial for ribosome assembly (Held et al 1974;Svensson et al 1988;Brodersen et al 2002). The Nterminal domain of S8 comprises a double-stranded DNAbinding motif present in several proteins including DNaseI, prokaryotic initiation factor 3 (IF3), and DNA methyltransferase (Davies et al 1996).…”

Section: Introductionmentioning

confidence: 99%

Analysis of RPS15aE, an Isoform of a Plant-Specific Evolutionarily Distinct Ribosomal Protein in Arabidopsis thaliana, Reveals its Potential Role as a Growth Regulator

Szick-Miranda

Zanial

et al. 2009

Plant Mol Biol Rep

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There is increasing evidence for ribosome heterogeneity in biological systems. In Arabidopsis thaliana, the ribosomal protein S15a is encoded by six separate genes, which fall into two evolutionarily distinct categories (Type I and Type II). Type I S15a is a universally conserved component of cytosolic ribosomes, whereas there is ambiguity as to the specific subcellular location of Type II S15a (cytosolic and/or mitochondrial ribosomes). In this study, we investigated the functional significance of the distinct form of ribosomal protein S15a (Type II) in Arabidopsis by examining: the evolutionary relationship of eukaryotic S15a proteins with respect to organellar homologs, the expression of individual Type II S15a genes during various developmental stages by RT-PCR, and the phenotypes of an insertional mutation into the RPS15aE gene. The Type II S15a proteins are plant specific, and the duplication event that gave rise to the Type II S15a genes appears to have occurred during the evolution of land plants. The genes encoding Type II S15a in Arabidopsis are differentially expressed, and mutant plants in which the gene encoding S15aE is knocked down produce larger leaves, longer roots, and possess larger cells than wild-type plants suggesting that the RPS15aE isoform of Type II S15a may act as a regulator of translational activity. Our results add significantly to the understanding of the protein constitution of plant ribosomes and the functional significance of ribosome heterogeneity.

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Interaction of ribosomal proteins, S6, S8, S15 and S18 with the central domain of 16 S ribosomal RNA

Cited by 90 publications

References 26 publications

Nonbridging phosphate oxygens in 16S rRNA important for 30S subunit assembly and association with the 50S ribosomal subunit

Nonbridging phosphate oxygens in 16S rRNA important for 30S subunit assembly and association with the 50S ribosomal subunit

Mutagenesis of ribosomal protein S8 from Escherichia coli: defects in regulation of the spc operon

Analysis of RPS15aE, an Isoform of a Plant-Specific Evolutionarily Distinct Ribosomal Protein in Arabidopsis thaliana, Reveals its Potential Role as a Growth Regulator

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