Non-bridging phosphate oxygen in 16S rRNA important for 30S subunit assembly and association with the 50S ribosomal subunit

Department of Chemistry and Biochemistry, University of California, San Diego, San Diego, California, United States
RNA (Impact Factor: 4.94). 06/2005; 11(5):657-67. DOI: 10.1261/rna.7224305
Source: PubMed


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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    • "sapiens: 876A; C v(G1835) = 1.861) has been shown to cause important growth defects under osmotic and oxidative stress, situations that might be relevant to mitochondrial translation (Osterman et al., 2011). Finally, deleterious mutations have also been found on the SSU side of the bridge in bacteria (Ghosh and Joseph, 2005; Rackham et al., 2006; Kim et al., 2007; Belanger et al., 2004) and we have previously identified two " likely disruptive " variants in the 12S mt-rRNA component of bridge mt-B2c (Smith et al., 2014). In summary, the data presented here is consistent with a " likely disruptive " potential for the two LSU mutations located on bridge mt-B2c, namely 875U N C "
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    ABSTRACT: Mitochondrial DNA mutations are well recognized as an important cause of disease, with over two hundred variants in the protein encoding and mt-tRNA genes associated with human disorders. In contrast, the two genes encoding the mitochondrial rRNAs (mt-rRNAs) have been studied in far less detail. This is because establishing the pathogenicity of mt-rRNA mutations is a major diagnostic challenge. Only two disease causing mutations have been identified at these loci, both mapping to the small subunit (SSU). On the large subunit (LSU), however, the evidence for the presence of pathogenic LSU mt-rRNA changes is particularly sparse. We have previously expanded the list of deleterious SSU mt-rRNA mutations by identifying highly disruptive base changes capable of blocking the activity of the mitoribosomal SSU. To do this, we used a new methodology named heterologous inferential analysis (HIA). The recent arrival of near-atomic-resolution structures of the human mitoribosomal LSU, has enhanced the power of our approach by permitting the analysis of the corresponding sites of mutation within their natural structural context. Here, we have used these tools to determine whether LSU mt-rRNA mutations found in the context of human disease and/or ageing could disrupt the function of the mitoribosomal LSU. Our results clearly show that, much like the for SSU mt-rRNA, LSU mt-rRNAs mutations capable of compromising the function of the mitoribosomal LSU are indeed present in clinical samples. Thus, our work constitutes an important contribution to an emerging view of the mitoribosome as an important element in human health.
    Full-text · Article · Sep 2015 · Mitochondrion
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    • "The evidence for the possible involvement of the 450G (m.1097G) in the incorrect functioning of bridge mt-B2c appears quite likely, since the disruption of the G809:C770 base pair by mutagenesis has been shown to be detrimental to ribosomal function (34,35). In addition, mutations disrupting Watson:Crick geometry at the adjacent G769:C810 base pair were highly deleterious (36,37). In the specific case of the 450G>A (m.1097G>A) mutation, the resulting C·A mismatch between 411C and 450G could adopt wobble geometry and thus, impose a rather small structural distortion at the base of m-h24. "
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    ABSTRACT: Mutations of mitochondrial DNA are linked to many human diseases. Despite the identification of a large number of variants in the mitochondrially-encoded rRNA (mt-rRNA) genes, the evidence supporting their pathogenicity is, at best, circumstantial. Establishing the pathogenicity of these variations is of major diagnostic importance. Here, we aim to estimate the disruptive effect of mt-rRNA variations on the function of the mitochondrial ribosome. In the absence of direct biochemical methods to study the effect of mt-rRNA variations, we relied on the universal conservation of the rRNA fold to infer their disruptive potential. Our method, named Heterologous Inferential Analysis or HIA, combines conservational information with functional and structural data obtained from heterologous ribosomal sources. Thus, HIA's predictive power is superior to the traditional reliance on simple conservation indexes. By using HIA we have been able to evaluate the disruptive potential for a subset of uncharacterized 12S mt-rRNA variations. Our analysis revealed the existence of variations in the rRNA component of the human mitoribosome with different degrees of disruptive power. In cases where sufficient information regarding the genetic and pathological manifestation of the mitochondrial phenotype is available, HIA data can be used to predict the pathogenicity of mt-rRNA mutations. In other cases, HIA analysis will allow the prioritisation of variants for additional investigation. Eventually, HIA-inspired analysis of potentially pathogenic mt-rRNA variations, in the context of a scoring system specifically designed for these variants, could lead to a powerful diagnostic tool.
    Full-text · Article · Oct 2013 · Human Molecular Genetics
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    • "Exact order of presenting proteins to the RNA may require quick recognition, and dipole-dipole and quadrupole interactions may facilitate this process by their long-range steering effect. Some high-resolution studies on specific electrostatic interactions such as the one formed by non-bridging phosphate oxygen have also been reported [24]. Thus, it is speculated that the requirement of orderly assembly and stabilizing electrostatic interactions, which are specific to rRNA-protein interactions, are reflected in higher electric moments in proteins interacting with them. "
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    ABSTRACT: Protein-RNA interactions play important role in many biological processes such as gene regulation, replication, protein synthesis and virus assembly. Although many structures of various types of protein-RNA complexes have been determined, the mechanism of protein-RNA recognition remains elusive. We have earlier shown that the simplest electrostatic properties viz. charge, dipole and quadrupole moments, calculated from backbone atomic coordinates of proteins are biased relative to other proteins, and these quantities can be used to identify DNA-binding proteins. Closely related, RNA-binding proteins are investigated in this study. In particular, discrimination between various types of RNA-binding proteins, evolutionary conservation of these bulk electrostatic features and effect of conformational changes by complex formation are investigated. Basic binding mechanism of a putative RNA-binding protein (HI1333 from Haemophilus influenza) is suggested as a potential application of this study. We found that similar to DNA-binding proteins (DBPs), RNA-binding proteins (RBPs) also show significantly higher values of electric moments. However, higher moments in RBPs are found to strongly depend on their functional class: proteins binding to ribosomal RNA (rRNA) constitute the only class with all three of the properties (charge, dipole and quadrupole moments) being higher than control proteins. Neural networks were trained using leave-one-out cross-validation to predict RBPs from control data as well as pair-wise classification capacity between proteins binding to various RNA types. RBPs and control proteins reached up to 78% accuracy measured by the area under the ROC curve. Proteins binding to rRNA are found to be best distinguished (AUC = 79%). Changes in dipole and quadrupole moments between unbound and bound structures were small and these properties are found to be robust under complex formation. Bulk electric moments of proteins considered here provide insights into target recognition by RNA-binding proteins, as well as ability to recognize one type of RBP from others. These results help in understanding the mechanism of protein-RNA recognition, and identifying RNA-binding proteins.
    Full-text · Article · Feb 2011 · BMC Structural Biology
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