ABSTRACT
The core ribosomal protein S8 binds to the central domain of 16S rRNA independently of other ribosomal proteins and is required for assembling the 30S subunit. It has been shown with E. coli ribosomes that a short rRNA fragment restricted by nucleotides 588-602 and 636-651 is sufficient for strong and specific protein S8 binding. In this work, we studied the complexes formed by ribosomal protein S8 from Thermus thermophilus and Methanococcus jannaschii with short rRNA fragments isolated from the same organisms. The dissociation constants of the complexes of protein S8 with rRNA fragments were determined. Based on the results of binding experiments, rRNA fragments of different length were designed and synthesized in preparative amounts in vitro using T7 RNA-polymerase. Stable S8-RNA complexes were crystallized. Crystals were obtained both for homologous bacterial and archaeal complexes and for hybrid complexes of archaeal protein with bacterial rRNA. Crystals of the complex of protein S8 from M. jannaschii with the 37-nucleotide rRNA fragment from the same organism suitable for X-ray analysis were obtained.
Subject(s)
RNA, Ribosomal/chemistry , RNA, Ribosomal/metabolism , Ribosomal Proteins/chemistry , Ribosomal Proteins/metabolism , Binding Sites , Crystallization , Magnesium/chemistry , Magnesium/metabolism , Methanococcus/chemistry , Methanococcus/genetics , Nucleic Acid Conformation , RNA, Archaeal/chemistry , RNA, Archaeal/metabolism , RNA, Bacterial/chemistry , RNA, Bacterial/metabolism , Ribosomal Proteins/isolation & purification , Thermus thermophilus/chemistry , Thermus thermophilus/geneticsABSTRACT
In this work we show for the first time that the overproduced N-terminal fragment (residues 1-91) of ribosomal protein TL5 binds specifically to 5S rRNA and that the region of this fragment containing residues 80-91 is a necessity for its RNA-binding activity. The fragment of Escherichia coli 5S rRNA protected by TL5 against RNase A hydrolysis was isolated and sequenced. This 39 nucleotides fragment contains loop E and helices IV and V of 5S rRNA. The isolated RNA fragment forms stable complexes with TL5 and its N-terminal domain. Crystals of TL5 in complex with the RNA fragment diffracting to 2.75 A resolution were obtained.
Subject(s)
Bacterial Proteins/metabolism , RNA, Ribosomal, 5S/metabolism , RNA-Binding Proteins/metabolism , Ribosomal Proteins/metabolism , Thermus thermophilus/metabolism , Amino Acid Sequence , Binding Sites , Molecular Sequence Data , Nucleic Acid Conformation , RNA, Ribosomal, 5S/chemistry , Sequence Homology, Amino AcidABSTRACT
The gene encoding the 5S rRNA-binding ribosomal protein TL5 from Thermus thermophilus, an extremely thermophilic species, was expressed in E. coli. A method for isolation of TL5 from the overproducing strain was developed. Samples of TL5 protein isolated from ribosomes and the overproducing strain displayed identical RNA-binding properties. Circular dichroic spectroscopy was used to calculate the secondary structure of the protein. TL5 was shown to form a stable complex with the 3'-terminal fragment of 5S rRNA, which is similar to the fragment of E. coli RNA that binds to L25 protein. The data suggest that TL5 from T. thermophilus and L25 from E. coli bind to similar sites on the 5S rRNA molecule.
