Alexey Rak

NMR Structure of Bacterial Ribosomal Protein L20: Implications for Ribosome Assembly and Translational Control

L20 is a specific protein of the bacterial ribosome, which is involved in the early assembly steps of the 50S subunit and in the feedback control of the expression of its own gene. This dual function involves specific interactions with either the 23S rRNA or its messenger RNA. The solution structure of the free Aquifex aeolicus L20 has been solved. It is composed of an unstructured N-terminal domain comprising residues 1-58 and a C-terminal alpha-helical domain. This is in contrast with what is observed in the bacterial 50S subunit, where the N-terminal region folds as an elongated alpha-helical region. The solution structure of the C-terminal domain shows that several solvent-accessible, conserved residues are clustered on the surface of the molecule and are probably involved in RNA recognition. In vivo studies show that this domain is sufficient to repress the expression of the cistrons encoding L35 and L20 in the IF3 operon. The ability of L20 C-terminal domain to specifically recognise RNA suggests an assembly mechanism for L20 into the ribosome. The pre-folded C-terminal domain would make a primary interaction with a specific site on the 23S rRNA. The N-terminal domain would then fold within the ribosome, participating in its correct 3D assembly.

The solution structure of ribosomal protein L18 from Thermus thermophilus reveals a conserved RNA-binding fold.

We have determined the solution structure of ribosomal protein L18 from Thermus thermophilus. L18 is a 12.5 kDa protein of the large subunit of the ribosome and binds to both 5 S and 23 S rRNA. In the uncomplexed state L18 folds to a mixed alpha/beta globular structure with a long disordered N-terminal region. We compared our high-resolution structure with RNA-complexed L18 from Haloarcula marismortui and T. thermophilus to examine RNA-induced as well as species-dependent structural differences. We also identified T. thermophilus S11 as a structural homologue and found that the structures of the RNA-recognition sites are conserved. Important features, for instance a bulge in the RNA-contacting beta-sheet, are conserved in both proteins. We suggest that the L18 fold recognizes a specific RNA motif and that the resulting RNA-protein-recognition module is tolerant to variations in sequence.

Another piece of the ribosome: solution structure of S16 and its location in the 30S subunit

BACKGROUND X-ray crystallography has recently yielded much-improved electron-density maps of the bacterial ribosome and its two subunits and many structural details of bacterial ribosome subunits are now being resolved. One approach to complement the structures and elucidate the details of rRNA and protein packing is to determine structures of individual protein components and model these into existing intermediate resolution electron density. RESULTS We have determined the solution structure of the ribosomal protein S16 from Thermus thermophilus. S16 is a mixed alpha/beta protein with a novel folding scaffold based on a five-stranded antiparallel/parallel beta sheet. Three large loops, which are partially disordered, extend from the sheet and two alpha helices are packed against its concave surface. Calculations of surface electrostatic potentials show a large continuous area of positive electrostatic potential and smaller areas of negative potential. S16 was modeled into a 5.5 A electron-density map of the T. thermophilus 30S ribosomal subunit. CONCLUSIONS The location and orientation of S16 in a narrow crevice formed by helix 21 and several other unassigned rRNA helices is consistent with electron density corresponding to the shape of S16, hydroxyl radical protection data, and the electrostatic surface potential of S16. Two protein neighbors to S16 are S4 and S20, which facilitate binding of S16 to the 30S subunit. Overall, this work exemplifies the benefits of combining high-resolution nuclear magnetic resonance (NMR) structures of individual components with low-resolution X-ray maps to elucidate structures of large complexes.

NMR structure of the ribosomal protein L23 from Thermus thermophilus

The ribosomal protein L23 is a component of the large ribosomal subunit in which it is located close to the peptide exit tunnel. In this position L23 plays a central role both for protein secretion and folding. We have determined the solution structure of L23 from Thermus thermophilus. Uncomplexed L23 consists of a well-ordered part, with four anti-parallel β-strands and three α-helices connected as β-α-β-α-β-β-α, and a large and flexible loop inserted between the third and fourth β-strand. The observed topology is distantly related to previously known structures, primarily within the area of RNA biochemistry. A comparison with RNA-complexed crystal structures of L23 from T. thermophilus, Deinococcus radiodurans and Haloarcula marismourtui, shows that the conformation of the well-ordered part is very similar in the uncomplexed and complexed states. However, the flexible loop found in the uncomplexed solution structure forms a rigid extended structure in the complexed crystal structures as it interacts with rRNA and becomes part of the exit tunnel wall. Structural characteristics of importance for the interaction with rRNA and with the ribosomal protein L29, as well as the functional role of L23, are discussed.

The Thermus thermophilus5S rRNA–Protein Complex: Identification of Specific Binding Sites for Proteins L5 and L18 in the 5S rRNA

Three 5S rRNA-binding ribosomal proteins (L5, L18, TL5) of extremely thermophilic bacterium Thermus thermophilushave earlier been isolated. Structural analysis of their complexes with rRNA requires identification of their binding sites in the 5S rRNA. Previously, a TL5-binding site has been identified, a TL5–RNA complex crystallized, and its structure determined to 2.3 Å. The sites for L5 and L18 were characterized, and two corresponding 5S rRNA fragments constructed. Of these, a 34-nt fragment specifically interacted with L5, and a 55-nt fragment interacted with L5, L18, and with both proteins. The 34-nt fragment–L5 complex was crystallized; the crystals are suitable for high-resolution X-ray analysis.

Letter to the editor: assignment and secondary structure identification of the ribosomal protein L18 from Thermus thermophilus.

This thesis covers the process from expression of a heterologous gene in Escherichia coli to structure determination of a protein by nuclear magnetic resonance (NMR) spectroscopy. The first part concerns structural genomics-related parallel screening studies on the effect of fusion tags (in particular the His tag) on protein solubility and the use of fusion tags in fast, parallel purification protocols intended for initial biophysical characterization of human proteins produced in E. coli. It was found that for most proteins the His tag has a negative influence on protein solubility. This influence appears to be more pronounced for our C-terminal His tag than for the N-terminal His tags used in this study. Moreover, high ratios of soluble per total protein do not always guarantee a high yield of soluble protein after purification, as different vector - target protein combinations result in large differences in host cell growth rates. Protein purification protocols for different fusion tags were developed that make it possible to express, purify and study structural properties of low concentration samples of 15N-labeled proteins in one or two days. The second part of this thesis describes the assignment and solution structure determination of ribosomal protein L18 of Thermus thermophilus. The protein is a mixed α/β structure with two α-helices on one side of a four-stranded β-sheet. Comparison to RNA-bound L18 showed that the protein to a large extent adopts identical structures in free and bound states, with exception of the loop regions and the flexible N-terminus. Keywords: protein production, protein solubility, fusion tags, nuclear magnetic resonance, structure determination, ribosomal protein

Preliminary NMR studies of Thermus thermophilus ribosomal protein S19 overproduced in Escherichia coli

The gene for the ribosomal protein S19 from Thermus thermophilus was cloned, sequenced and overexpressed in Escherichia coli. A simple procedure for isolating the recombinant protein was developed. Preliminary NMR studies revealed a high content of α‐helical secondary structure in the protein.