Dataset Information

Atomic model of the Thermus thermophilus 70S ribosome developed in silico.

ABSTRACT: The ribosome is a large molecular complex that consists of at least three ribonucleic acid molecules and a large number of proteins. It translates genetic information from messenger ribonucleic acid and makes protein accordingly. To better understand ribosomal function and provide information for designing biochemical experiments require knowledge of the complete structure of the ribosome. For expanding the structural information of the ribosome, we took on the challenge of developing a detailed Thermus thermophilus ribosomal structure computationally. By combining information derived from the low-resolution x-ray structure of the 70S ribosome (providing the overall fold), high-resolution structures of the ribosomal subunits (providing the local structure), sequences, and secondary structures, we have developed an atomic model of the T. thermophilus ribosome using a homology modeling approach. Our model is stereochemically sound with a consistent single-species sequence. The overall folds of the three ribosomal ribonucleic acids in our model are consistent with those in the low-resolution crystal structure (root mean-square differences are all <1.9 angstroms). The large overall interface area (approximately 2500 angstroms2) of intersubunit bridges B2a, B3, and B5, and the inherent flexibility in regions connecting the contact residues are consistent with these bridges serving as anchoring patches for the ratcheting and rolling motions between the two subunits during translocation.

SUBMITTER: Tung CS

PROVIDER: S-EPMC1304690 | biostudies-literature | 2004 Oct

REPOSITORIES: biostudies-literature

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Atomic model of the Thermus thermophilus 70S ribosome developed in silico.

Tung Chang-Shung CS Sanbonmatsu Kevin Y KY

Biophysical journal 20041001 4

The ribosome is a large molecular complex that consists of at least three ribonucleic acid molecules and a large number of proteins. It translates genetic information from messenger ribonucleic acid and makes protein accordingly. To better understand ribosomal function and provide information for designing biochemical experiments require knowledge of the complete structure of the ribosome. For expanding the structural information of the ribosome, we took on the challenge of developing a detailed ...[more]

PMID: 15454463

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Project description:BackgroundThermus thermophilus, an extremely thermophilic bacterium, has been widely recognized as a model organism for studying how microbes can survive and adapt under high temperature environment. However, the thermotolerant mechanisms and cellular metabolism still remains mostly unravelled. Thus, it is highly required to consider systems biological approaches where T. thermophilus metabolic network model can be employed together with high throughput experimental data for elucidating its physiological characteristics under such harsh conditions.ResultsWe reconstructed a genome-scale metabolic model of T. thermophilus, iTT548, the first ever large-scale network of a thermophilic bacterium, accounting for 548 unique genes, 796 reactions and 635 unique metabolites. Our initial comparative analysis of the model with Escherichia coli has revealed several distinctive metabolic reactions, mainly in amino acid metabolism and carotenoid biosynthesis, producing relevant compounds to retain the cellular membrane for withstanding high temperature. Constraints-based flux analysis was, then, applied to simulate the metabolic state in glucose minimal and amino acid rich media. Remarkably, resulting growth predictions were highly consistent with the experimental observations. The subsequent comparative flux analysis under different environmental conditions highlighted that the cells consumed branched chain amino acids preferably and utilized them directly in the relevant anabolic pathways for the fatty acid synthesis. Finally, gene essentiality study was also conducted via single gene deletion analysis, to identify the conditional essential genes in glucose minimal and complex media.ConclusionsThe reconstructed genome-scale metabolic model elucidates the phenotypes of T. thermophilus, thus allowing us to gain valuable insights into its cellular metabolism through in silico simulations. The information obtained from such analysis would not only shed light on the understanding of physiology of thermophiles but also helps us to devise metabolic engineering strategies to develop T. thermophilus as a thermostable microbial cell factory.

Project description:Lysine acetylation in proteins has recently been globally identified in bacteria and eukaryotes. Even though acetylproteins are known to be involved in various cellular processes, its physiological significance has not yet been resolved. Using a proteomics approach in combination with immunoprecipitation, we identified 197 lysine acetylation sites and 4 N-terminal acetylation sites from 128 proteins in Thermus thermophilus HB8, an extremely thermophilic eubacterium. Our analyses revealed that identified acetylproteins are well conserved across all three domains of life and are mainly involved in central metabolism and translation. To further characterize functional significance, we successfully mapped 113 acetylation sites on their 54 authentic and 59 homologous protein structures. The acetylation in the majority of proteins occurs in ordered structures and the sites were situated near the negatively charged glutamic acid residues. In addition, 59 of 103 acetylations were located within considerable distance that can disrupt electrostatic interactions and hydrogen bonding networks on protein surface, demonstrating the physiological significances of the acetylations. Finally, we further summarized 22 critical acetylation sites related to Schiff-base formation, ligand binding, protein-RNA and protein-protein interaction. The structural information of 113 acetylation sites provides new molecular insight into the role of lysine acetylation in the proteins. Data processing, bioinformatics: MS and MS/MS spectral data were processed by DataAnalysis 4.0 software (Bruker Daltonics). The peak lists containing m/z of precursor ions with that of their product ions were generated by the Compound-Auto MS(n) option of the DataAnalysis 4.0 software. Fifty non-deconvoluted peaks over the intensity threshold 150 and charge deconvoluted peaks in each MS/MS spectrum were exported into peak list files. The spectra were searched against our in-house T. thermophilus HB8 database, containing 2,238 protein sequence entries from the complete genome sequence using Mascot search engine (version 2.3; Matrix Science, London, UK). The acetylated peptides were identified using a mass tolerance of ±0.05 Da for precursor and product ions and allowed a maximum of 6 mis-cleavage sites for trypsin. The carbamidomethylation of cysteine was selected as a fixed modification. The oxidation of methionine, deamidation of asparagine and glutamine, acetylation of lysine and acetylation of protein N-terminus were selected as variable modifications. Only peptides in the confidence range of 99% probability (P value < 0.01) in Mascot ion score were assumed to be identified.

Dataset Information

Atomic model of the Thermus thermophilus 70S ribosome developed in silico.

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Atomic model of the Thermus thermophilus 70S ribosome developed in silico.

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