Structure of the ribosomal P stalk base in archaean Methanococcus jannaschii.

Complexes of archaeal ribosomal proteins uL11 and uL10/P0 (the two-domain N-terminal fragment of uL10, uL10NTF/P0NTF) with the adjacent 74 nucleotides of 23S rRNA fragment (23SrRNA(74)) from Methanococcus jannaschii (Mja) were obtained, crystallized and their structures were studied. The comparative structural analysis of the complexes of Mja uL10NTF•23SrRNA(74) and Mja uL10NTF•uL11•23SrRNA(74) shows that the insertion of uL11 in the binary complex does not change the conformation of the 23S rRNA fragment. On the other hand, the interaction with this specific RNA fragment leads to the restructuring of uL11 compared to the structure of this protein in the free state. Besides, although analysis confirmed the mobility of uL10/P0 domain II, disproved the assumption that it may be in contact with rRNA or uL11. In addition, the Mja uL10NTF•uL11•23SrRNA(74) complex was cocrystallized with the antibiotic thiostrepton, and the structure of this complex was solved. The thiostrepton binding site in this archaeal complex was found between the 23S rRNA and the N-terminal domain (NTD) of the Mja uL11 protein, similar to its binding site in the one of bacterial ribosome complex with thiostrepton. Upon binding of thiostrepton, the NTD of uL11 shifts toward rRNA by 7 Å. Such a shift may be the cause of the inhibitory effect of the antibiotic on the recruitment of translation factors to the GTPase-activating region in archaeal ribosomes, similar to its inhibitory effect on protein synthesis in bacterial ribosomes.

Crystallographic analysis of archaeal ribosomal protein L11.

Ribosomal protein L11 is an important part of the GTPase-associated centre in ribosomes of all organisms. L11 is a highly conserved two-domain ribosomal protein. The C-terminal domain of L11 is an RNA-binding domain that binds to a fragment of 23S rRNA and stabilizes its structure. The complex between L11 and 23S rRNA is involved in the GTPase activity of the translation elongation and release factors. Bacterial and archaeal L11-rRNA complexes are targets for peptide antibiotics of the thiazole class. To date, there is no complete structure of archaeal L11 owing to the mobility of the N-terminal domain of the protein. Here, the crystallization and X-ray analysis of the ribosomal protein L11 from Methanococcus jannaschii are reported. Crystals of the native protein and its selenomethionine derivative belonged to the orthorhombic space group I222 and were suitable for structural studies. Native and single-wavelength anomalous dispersion data sets have been collected and determination of the structure is in progress.

The base of the ribosomal P stalk from Methanococcus jannaschii: crystallization and preliminary X-ray studies.

The lateral P stalk in archaeal/eukaryotic ribosomes and the L12 stalk in bacterial ribosomes play a pivotal role in specific binding to the ribosome and recruiting translational factors during protein biosynthesis. The P stalk consists of the ribosomal proteins L11, P0 and P1. The proteins P0 and P1 form the complex that binds 23S rRNA through the N-terminal domain of the P0 protein. Ribosomal protein L11 binds to the same region of 23S rRNA and together with the protein P0 forms the base of the stalk. The structure of the ribosomal protein L11 from archaea has been solved, but with several missing segments. Here, the preparation and crystallization of a ternary complex consisting of the ribosomal protein L11, the two-domain N-terminal fragment of the ribosomal protein P0 and a specific fragment of 23S rRNA from the archaeon Methanococcus jannaschii are reported. The crystals belonged to the monoclinic space group P2(1), with unit-cell parameters a = 72.4, b = 88.5, c = 95.2 Å, ß = 102.2°. A complete diffraction data set has been collected to a resolution of 2.9 Šusing an in-house rotating-anode X-ray generator.

Structure of a two-domain N-terminal fragment of ribosomal protein L10 from Methanococcus jannaschii reveals a specific piece of the archaeal ribosomal stalk.

Ribosomal stalk is involved in the formation of the so-called "GTPase-associated site" and plays a key role in the interaction of ribosome with translation factors and in the control of translation accuracy. The stalk is formed by two or three copies of the L7/L12 dimer bound to the C-terminal tail of protein L10. The N-terminal domain of L10 binds to a segment of domain II of 23S rRNA near the binding site for ribosomal protein L11. The structure of bacterial L10 in complex with three L7/L12 N-terminal dimers has been determined in the isolated state, and the structure of the first third of archaeal L10 bound to domain II of 23S rRNA has been solved within the Haloarcula marismortui 50S ribosomal subunit. A close structural similarity between the RNA-binding domain of archaeal L10 and the RNA-binding domain of bacterial L10 has been demonstrated. In this work, a long RNA-binding N-terminal fragment of L10 from Methanococcus jannaschii has been isolated and crystallized. The crystal structure of this fragment (which encompasses two-thirds of the protein) has been solved at 1.6 A resolution. The model presented shows the structure of the RNA-binding domain and the structure of the adjacent domain that exist in archaeal L10 and eukaryotic P0 proteins only. Furthermore, our model incorporated into the structure of the H. marismortui 50S ribosomal subunit allows clarification of the structure of the archaeal ribosomal stalk base.