Dataset Information

Solution structure of ribosomal protein S28E from Methanobacterium thermoautotrophicum.

ABSTRACT: The ribosomal protein S28E from the archaeon Methanobacterium thermoautotrophicum is a component of the 30S ribosomal subunit. Sequence homologs of S28E are found only in archaea and eukaryotes. Here we report the three-dimensional solution structure of S28E by NMR spectroscopy. S28E contains a globular region and a long C-terminal tail protruding from the core. The globular region consists of four antiparallel beta-strands that are arranged in a Greek-key topology. Unique features of S28E include an extended loop L2-3 that folds back onto the protein and a 12-residue charged C-terminal tail with no regular secondary structure and greater flexibility relative to the rest of the protein. The structural and surface resemblance to OB-fold family of proteins and the presence of highly conserved basic residues suggest that S28E may bind to RNA. A broad positively charged surface extending over one side of the beta-barrel and into the flexible C terminus may present a putative binding site for RNA.

SUBMITTER: Wu B

PROVIDER: S-EPMC2366991 | biostudies-literature | 2003 Dec

REPOSITORIES: biostudies-literature

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Solution structure of ribosomal protein S28E from Methanobacterium thermoautotrophicum.

Wu Bin B Yee Adelinda A Pineda-Lucena Antonio A Semesi Anthony A Ramelot Theresa A TA Cort John R JR Jung Jin-Won JW Edwards Aled A Lee Weontae W Kennedy Michael M Arrowsmith Cheryl H CH

Protein science : a publication of the Protein Society 20031201 12

The ribosomal protein S28E from the archaeon Methanobacterium thermoautotrophicum is a component of the 30S ribosomal subunit. Sequence homologs of S28E are found only in archaea and eukaryotes. Here we report the three-dimensional solution structure of S28E by NMR spectroscopy. S28E contains a globular region and a long C-terminal tail protruding from the core. The globular region consists of four antiparallel beta-strands that are arranged in a Greek-key topology. Unique features of S28E inclu ...[more]

PMID: 14627743

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Project description:BACKGROUND: RNA ligases are essential reagents for many methods in molecular biology including NextGen RNA sequencing. To prevent ligation of RNA to itself, ATP independent mutant ligases, defective in self-adenylation, are often used in combination with activated pre-adenylated linkers. It is important that these ligases not have de-adenylation activity, which can result in activation of RNA and formation of background ligation products. An additional useful feature is for the ligase to be active at elevated temperatures. This has the advantage or reducing preferences caused by structures of single-stranded substrates and linkers. RESULTS: To create an RNA ligase with these desirable properties we performed mutational analysis of the archaeal thermophilic RNA ligase from Methanobacterium thermoautotrophicum. We identified amino acids essential for ATP binding and reactivity but dispensable for phosphodiester bond formation with 5' pre-adenylated donor substrate. The motif V lysine mutant (K246A) showed reduced activity in the first two steps of ligation reaction. The mutant has full ligation activity with pre-adenylated substrates but retained the undesirable activity of deadenylation, which is the reverse of step 2 adenylation. A second mutant, an alanine substitution for the catalytic lysine in motif I (K97A) abolished activity in the first two steps of the ligation reaction, but preserved wild type ligation activity in step 3. The activity of the K97A mutant is similar with either pre-adenylated RNA or single-stranded DNA (ssDNA) as donor substrates but we observed two-fold preference for RNA as an acceptor substrate compared to ssDNA with an identical sequence. In contrast, truncated T4 RNA ligase 2, the commercial enzyme used in these applications, is significantly more active using pre-adenylated RNA as a donor compared to pre-adenylated ssDNA. However, the T4 RNA ligases are ineffective in ligating ssDNA acceptors. CONCLUSIONS: Mutational analysis of the heat stable RNA ligase from Methanobacterium thermoautotrophicum resulted in the creation of an ATP independent ligase. The K97A mutant is defective in the first two steps of ligation but retains full activity in ligation of either RNA or ssDNA to a pre-adenylated linker. The ability of the ligase to function at 65°C should reduce the constraints of RNA secondary structure in RNA ligation experiments.

Dataset Information

Solution structure of ribosomal protein S28E from Methanobacterium thermoautotrophicum.

Publications

Solution structure of ribosomal protein S28E from Methanobacterium thermoautotrophicum.

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