Nils Burkhardt

Cryo-EM reveals an active role for aminoacyl-tRNA in the accommodation process.

During the elongation cycle of protein biosynthesis, the specific amino acid coded for by the mRNA is delivered by a complex that is comprised of the cognate aminoacyl‐tRNA, elongation factor Tu and GTP. As this ternary complex binds to the ribosome, the anticodon end of the tRNA reaches the decoding center in the 30S subunit. Here we present the cryo‐ electron microscopy (EM) study of an Escherichia coli 70S ribosome‐bound ternary complex stalled with an antibiotic, kirromycin. In the cryo‐EM map the anticodon arm of the tRNA presents a new conformation that appears to facilitate the initial codon–anticodon interaction. Furthermore, the elbow region of the tRNA is seen to contact the GTPase‐associated center on the 50S subunit of the ribosome, suggesting an active role of the tRNA in the transmission of the signal prompting the GTP hydrolysis upon codon recognition.

Effect of buffer conditions on the position of tRNA on the 70 S ribosome as visualized by cryoelectron microscopy.

The effect of buffer conditions on the binding position of tRNA on the Escherichia coli 70 S ribosome have been studied by means of three-dimensional (3D) cryoelectron microscopy. Either deacylated tRNAf Met or fMet-tRNAf Met were bound to the 70 S ribosomes, which were programmed with a 46-nucleotide mRNA having AUG codon in the middle, under two different buffer conditions (conventional buffer: containing Tris and higher Mg2+ concentration [10–15 mm]; and polyamine buffer: containing Hepes, lower Mg2+ concentration [6 mm], and polyamines). Difference maps, obtained by subtracting 3D maps of naked control ribosome in the corresponding buffer from the 3D maps of tRNA·ribosome complexes, reveal the distinct locations of tRNA on the ribosome. The position of deacylated tRNAf Metdepends on the buffer condition used, whereas that of fMet-tRNAf Met remains the same in both buffer conditions. The acylated tRNA binds in the classical P site, whereas deacylated tRNA binds mostly in an intermediate P/E position under the conventional buffer condition and mostly in the position corresponding to the classical P site, i.e. in the P/P state, under the polyamine buffer conditions.

Ribosomal Protection from Tetracycline Mediated by Tet(O): Tet(O) Interaction with Ribosomes Is GTP-Dependent

Tet(O) mediates tetracycline resistance by protecting the ribosome from inhibition. A recombinant Tet(O) protein with a histidine tag was purified and its activity in protein synthesis characterized. Tetracycline inhibited the rate of poly(Phe) synthesis, producing short peptide chains. Tet(O)-His was able to restore the elongation rate and processivity. 70S ribosomes bound tetracycline with high affinity. Tet(O)-His in the presence of GTP, but not GDP or GMP, reduced the affinity of the ribosomes for tetracycline. Non-hydrolyzable GTP analogs in the presence of the factor were also able to interfere with tetracycline binding. Ribosomes increased the affinity of Tet(O)-His for GTPgammaS. Tet(O), 70S ribosomes and GTPgammaS formed a complex that could be isolated by gel filtration. The GTP conformer is the active form of Tet(O) that interacts with the ribosome. GTP binding is necessary for Tet(O) activity.

Formation of 70S ribosomes: large activation energy is required for the adaptation of exclusively the small ribosomal subunit

Association of ribosomal subunits is an essential reaction during the initiation phase of protein synthesis. Optimal conditions for 70S formation in vitro were determined to 20 mM Mg2+ and 30 mM K+. Under these conditions, the association reaction proceeds with first order kinetics, suggesting a conformational change to be the rate-limiting step. 70S formation separates into two sub-reactions, the adaptation of the ribosomal subunits to the association conditions and the association step itself. The activation energy of the process was determined to 78 kJ/mol and revealed to be required exclusively for the adaptation of the small subunit, rather than the large subunit or the association step. The presence of mRNA [poly(U)] together with cognate AcPhe-tRNA, accelerates the association rate significantly, forming a well-defined 70S peak in sucrose gradient profiles. mRNA alone provokes an equivalent acceleration, however, the resulting 70S couple impresses as an ill-defined, broad peak, probably indicating the readiness of the ribosome for tRNA binding, upon which the ribosome flips into a defined state.

[17] Experimental prerequisites for determination of tRNA binding to ribosomes from Escherichia coli

Publisher Summary This chapter discusses the experimental prerequisites for the determination of transfer RNA (tRNA) binding to ribosomes from Escherichia coli. In studies, the tRNA binding features of 70S ribosomes from E. coli suggests that these ribosomes contain three tRNA binding sites: the A site, which accepts the aminoacyl-tRNA during the first step of the elongation phase in protein biosynthesis, can also accept peptidyl-tRNA; the P site, where the three possible forms of a tRNA, peptidyl-, aminoacyl-, or deacyl-tRNA can bind; and the E site that shows a strong specificity for deacylated tRNA. The functional links between the three sites have been also established for the elongation phase of protein biosynthesis. The growing peptide chain is prolonged by one amino acid via three basic reactions. The first reaction is the occupation of the A site by an aminoacyl-tRNA that separates into a selection step and a tight-binding step. The selection process analyzes mainly the correctness of codon–anticodon interaction. After occupation of the A site, peptide-bond formation occurs in the second reaction: the peptidyl moiety attached to the P-site-bound tRNA is transferred to the free amino group of the aminoacyl-tRNA at the A site via a peptide bond. This reaction is catalyzed by the peptidyltransferase center located on the large subunit of the ribosome. The third reaction is the elongation factor–G·guanosine triphosphate–dependent translocation of the new peptidyl-tRNA from the A to the P site.

Small angle scattering in ribosomal structure research: localization of the messenger RNA within ribosomal elongation states.

Besides EM and biochemical studies small angle scattering (SAS) examinations have contributed significantly to our current knowledge about the ribosomal structure. SAS does not only allow the validation of competing models but permits independent model building. However, the major contribution of SAS to ribosomal structure research derived from its ability to reveal the spatial distribution of the individual ribosomal components (57 in the E. coli ribosome) within the ribosomal structure. More recently, an improved scattering method (proton-spin contrast variation) made it possible also to address the question of mapping functional ligands in defined ribosomal elongation states. Here, we review the contributions of SAS to the current understanding of the ribosome. Furthermore we present the direct localization of a small mRNA fragment within 70S elongation complexes and describe its movement upon the translocation reaction. The successful mapping of this fragment comprising only about 0.6% of the total mass of the complex proves that proton-spin contrast-variation is a powerful tool in modern ribosome research.

Mapping the messenger RNA within the elongating ribosome

Abstract The method of proton-spin contrast-variation was applied for determining the position of the messenger RNA within the elongating ribosome. Using an artificial mRNA fragment the mass center of the mRNA sequence covered by the ribosome could be localized for the pre- and the post-translocational elongation states. The mass center moves about 12 ± 5 A upon translocation. The radius of gyration was 12 ± e A . The data give an independent contribution for refining a structural model including the RNA ligands of the elongating ribosome.

Architecture of the E.coli 70S ribosome

Abstract The 70S ribosome from E.coli was analysed by neutron scattering focusing on the shape and the internal protein-RNA-distribution of the complex. Measurements on selectively deuterated 70S particles and free 30S and 50S subunits applying conventional contrast variation and proton-spin contrast-variation resulted in a total of 42 scattering curves. Processing the data on the basis of the spherical harmonic technique, a four-phase model for the 70S ribosome could be generated, which describes the shape of the particle as well as the protein-and the RNA-moieties of each subunit at about 35 A resolution.