The E-site story: the importance of maintaining two tRNAs on the ribosome during protein synthesis.

In the sixties James Watson suggested a twosite model for the ribosome comprising the P site for the peptidyl transfer RNA (tRNA) before peptide-bond formation and the A site, where decoding takes place according to the codon exposed there. In the eighties a third tRNA binding site was detected, the E site, which was specific for deacylated tRNA and turned out to be a universal feature of ribosomes. However, despite having three tRNA binding sites, only two tRNAs occupy the ribosome at a time during protein synthesis: at the A and P sites before translocation (PRE state) and at the P and E sites after translocation (POST state). The importance of having two tRNAs in the POST state has been revealed during the last 25 years, showing that the E site contributes two fundamental features: (i) the fact that incorporation of a wrong amino acid is not harmful for the cell (only 1 in about 400 misincorporations destroys the function of a protein) stems from the presence of an E-tRNA; (ii) maintenance of the reading frame is one of the most remarkable achievements of the ribosome, essential for faithful translation of the genetic information. The presence of the POST state E-tRNA prevents loss of the reading frame.

Functions and interplay of the tRNA-binding sites of the ribosome.

The ribosome translates the genetic information of an mRNA molecule into a sequence of amino acids. The ribosome utilizes tRNAs to connect elements of the RNA and protein worlds during protein synthesis, i.e. an anticodon as a unit of genetic information with the corresponding amino acid as a building unit of proteins. Three tRNA-binding sites are located on the ribosome, termed the A, P and E sites. In recent years the tRNA-binding sites have been localized on the ribosome by three different techniques, small-angle neutron scattering, cryo-electron microscopy and X-ray analyses of 70 S crystals. These high-resolution glimpses into various ribosomal states together with a large body of biochemical data reveal an intricate interplay between the tRNAs and the three ribosomal binding sites, providing an explanation for the remarkable features of the ribosome, such as the ability to select the correct ternary complex aminoacyl-tRNA.EF-Tu.GTP out of more than 40 extremely similar tRNA complexes, the precise movement of the tRNA(2).mRNA complex during translocation and the maintenance of the reading frame.

Complex of transfer-messenger RNA and elongation factor Tu. Unexpected modes of interaction.

Transfer-messenger RNA (tmRNA) is a stable RNA in bacteria of 360 +/- 40 nucleotides that can be charged with alanine and can function as both tRNA and mRNA. Ribosomes that are stalled either in a coding region of mRNA or at the 3' end of an mRNA fragment lacking a stop codon are rescued by replacing their mRNA for tmRNA. Here we demonstrate that the interaction of tmRNA with the elongation factor Tu shows unexpected features. Deacylated tmRNA can form a complex with either EF-Tu.GDP or EF-Tu.GTP, the association constants are about one order of magnitude smaller than that of an Ala-tRNA.EF-Tu.GTP complex. tmRNA as well as Ala-tmRNA can be efficiently cross-linked with EF-Tu.GDP using a zero-length cross-link. The efficiency of cross-linking in the case of deacylated tmRNA does not depend on an intact CCA-3' end and is about the same, regardless whether protein mixtures such as the post-ribosomal supernatant (S100 enzymes) or purified EF-Tu are present. Two cross-linking sites with EF-Tu.GDP have been identified that are located outside the tRNA part of tmRNA, indicating an unusual interaction of tmRNA with EF-Tu.GDP.

Localization of L11 protein on the ribosome and elucidation of its involvement in EF-G-dependent translocation.

L11 protein is located at the base of the L7/L12 stalk of the 50 S subunit of the Escherichia coli ribosome. Because of the flexible nature of the region, recent X-ray crystallographic studies of the 50 S subunit failed to locate the N-terminal domain of the protein. We have determined the position of the complete L11 protein by comparing a three-dimensional cryo-EM reconstruction of the 70 S ribosome, isolated from a mutant lacking ribosomal protein L11, with the three-dimensional map of the wild-type ribosome. Fitting of the X-ray coordinates of L11-23 S RNA complex and EF-G into the cryo-EM maps combined with molecular modeling, reveals that, following EF-G-dependent GTP hydrolysis, domain V of EF-G intrudes into the cleft between the 23 S ribosomal RNA and the N-terminal domain of L11 (where the antibiotic thiostrepton binds), causing the N-terminal domain to move and thereby inducing the formation of the arc-like connection with the G' domain of EF-G. The results provide a new insight into the mechanism of EF-G-dependent translocation.

Mapping proteins of the 50S subunit from Escherichia coli ribosomes.

Mapping of protein positions in the ribosomal subunits was first achieved for the 30S subunit by means of neutron scattering about 15 years ago. Since the 50S subunit is almost twice as large as the 30S subunit and consists of more proteins, it was difficult to apply classical contrast variation techniques for the localisation of the proteins. Polarisation dependent neutron scattering (spin-contrast variation) helped to overcome this restriction. Here a map of 14 proteins within the 50S subunit from Escherichia coli ribosomes is presented including the proteins L17 and L20 that are not present in archeal ribosomes. The results are compared with the recent crystallographic map of the 50S subunit from the archea Haloarcula marismortui.

Localization of the ribosomal protection protein Tet(O) on the ribosome and the mechanism of tetracycline resistance.

Tet(O) belongs to a class of ribosomal protection proteins that mediate tetracycline resistance. It is a G protein that shows significant sequence similarity to elongation factor EF-G. Here we present a cryo-electron microscopic reconstruction, at 16 A resolution, of its complex with the E. coli 70S ribosome. Tet(O) was bound in the presence of a noncleavable GTP analog to programmed ribosomal complexes carrying fMet-tRNA in the P site. Tet(O) is directly visible as a mass close to the A-site region, similar in shape and binding position to EF-G. However, there are important differences. One of them is the different location of the tip of domain IV, which in the Tet(O) case, does not overlap with the ribosomal A site but is directly adjacent to the primary tetracycline binding site. Our findings give insights into the mechanism of tetracycline resistance.

A short fragment of 23S rRNA containing the binding sites for two ribosomal proteins, L24 and L4, is a key element for rRNA folding during early assembly.

Previously we described an in vitro selection variant abbreviated SERF (in vitro selection from random rRNA fragments) that identifies protein binding sites within large RNAs. With this method, a small rRNA fragment derived from the 23S rRNA was isolated that binds simultaneously and independently the ribosomal proteins L4 and L24 from Escherichia coli. Until now the rRNA structure within the ternary complex L24-rRNA-L4 could not be studied due to the lack of an appropriate experimental strategy. Here we tackle the issue by separating the various complexes via native gel-electrophoresis and analyzing the rRNA structure by in-gel iodine cleavage of phosphorothioated RNA. The results demonstrate that during the transition from either the L4 or L24 binary complex to the ternary complex the structure of the rRNA fragment changes significantly. The identified protein binding sites are in excellent agreement with the recently reported crystal structure of the 50S subunit. Because both proteins play a prominent role in early assembly of the large subunit, the results suggest that the identified rRNA fragment is a key element for the folding of the 23S RNA during early assembly. The introduced in-gel cleavage method should be useful when an RNA structure within mixed populations of different but related complexes should be studied.

Intrinsic structural differences between "tight couples" and Kaltschmidt-Wittmann ribosomes evidenced by dielectric spectroscopy and scanning microcalorimetry.

Measurements of dielectric spectroscopy (DS) and microcalorimetry (differential scanning calorimetry (DSC)) of Escherichia coli 70S, 50S and 30S were performed on particles prepared according either to the "classical" twice NH(4)Cl-washed ribosomes, also known as loose couples (LC), or to the "tight couples" preparative protocol (TC). Results show that 70S particles prepared according to the two different protocols exhibit different structural properties. Two subsequent relaxation processes occur in both samples as measured by DS. However, in LC ribosomes the first one is shifted towards a lower frequency with a higher dielectric increment. This is suggestive of a more extensive exposure of RNA to the solvent and of an overall more relaxed structure. The smaller LC subunit exhibits only one relaxation while the TC 30S shows two dielectric dispersions as well as 70S. No substantial differences were evidenced in either 50S species. Two typical melting peaks were observed by DSC both in LC and TC 70S as well as in 50S. Thermograms obtained from the TC 30S show a single well structured peak while LC particles produce a large unstructured curve. On the basis of these results we conclude that TC 70S particles are more compact than LC ribosomes and that in the former ones the rRNA is less exposed to the solvent phase. Furthermore 30S particles obtained from TC show a more stable structure with respect to LC 30S. We conclude that the 30S subunit gives a major contribution to the compact character of the whole TC 70S. These differences might be related to the intrinsic and well documented functional difference between the two ribosome species.

Localization of the protein L2 in the 50 S subunit and the 70 S E. coli ribosome.

The protein L2 is found in all ribosomes and is one of the best conserved proteins of this mega-dalton complex. The protein was localized within both the isolated 50 S subunit and the 70 S ribosome of the Escherichia coli bacteria with the neutron-scattering technique of spin-contrast variation. L2 is elongated, exposing one end of the protein to the surface of the intersubunit interface of the 50 S subunit. The protein changes its conformation slightly when the 50 S subunit reassociates with the 30 S subunit to form a 70 S ribosome, becoming more elongated and moving approximately 30 A into the 50 S matrix. The results support a recent observation that L2 is essential for the association of the ribosomal subunits and might participate in the binding and translocation of the tRNAs.

SERF: in vitro election of random RNA fragments to identify protein binding sites within large RNAs.

In vitro selection experiments have various goals depending on the composition of the initial pool and the selection method applied. We developed an in vitro selection variant (SERF, selection of random RNA fragments) that is useful for the identification of short RNA fragments originating from large RNAs that bind specifically to a protein. A pool of randomly fragmented RNA is constructed from a large RNA, which is the natural binding partner for a protein. Such a pool contains all the potential binding sites and is therefore used as starting material for affinity selection with the purified protein to find its natural target. Here we provide a detailed experimental protocol of the method. SERF has been developed for ribosomal systems and is a general approach providing a basis for functional and structural characterization of RNA-protein interactions in large ribonucleoprotein particles.

Ribosomal protein L2 is involved in the association of the ribosomal subunits, tRNA binding to A and P sites and peptidyl transfer.

Ribosomal proteins L2, L3 and L4, together with the 23S RNA, are the main candidates for catalyzing peptide bond formation on the 50S subunit. That L2 is evolutionarily highly conserved led us to perform a thorough functional analysis with reconstituted 50S particles either lacking L2 or harboring a mutated L2. L2 does not play a dominant role in the assembly of the 50S subunit or in the fixation of the 3'-ends of the tRNAs at the peptidyl-transferase center. However, it is absolutely required for the association of 30S and 50S subunits and is strongly involved in tRNA binding to both A and P sites, possibly at the elbow region of the tRNAs. Furthermore, while the conserved histidyl residue 229 is extremely important for peptidyl-transferase activity, it is apparently not involved in other measured functions. None of the other mutagenized amino acids (H14, D83, S177, D228, H231) showed this strong and exclusive participation in peptide bond formation. These results are used to examine critically the proposed direct involvement of His229 in catalysis of peptide synthesis.

Periodic conformational changes in rRNA: monitoring the dynamics of translating ribosomes.

In protein synthesis, a tRNA transits the ribosome via consecutive binding to the A (acceptor), P (peptidyl), and E (exit) site; these tRNA movements are catalyzed by elongation factor G (EF-G) and GTP. Site-specific Pb2+ cleavage was applied to trace tertiary alterations in tRNA and all rRNAs on pre- and posttranslocational ribosomes. The cleavage pattern of deacylated tRNA and AcPhe-tRNA changed individually upon binding to the ribosome; however, these different conformations were unaffected by translocation. On the other hand, translocation affects 23S rRNA structure. Significantly, the Pb2+ cleavage pattern near the peptidyl transferase center was different before and after translocation. This structural rearrangement emerged periodically during elongation, thus providing evidence for a dynamic and mobile role of 23S rRNA in translocation.

Visualization of tRNA movements on the Escherichia coli 70S ribosome during the elongation cycle.

Three-dimensional cryomaps have been reconstructed for tRNA-ribosome complexes in pre- and posttranslocational states at 17-A resolution. The positions of tRNAs in the A and P sites in the pretranslocational complexes and in the P and E sites in the posttranslocational complexes have been determined. Of these, the P-site tRNA position is the same as seen earlier in the initiation-like fMet-tRNA(f)(Met)-ribosome complex, where it was visualized with high accuracy. Now, the positions of the A- and E-site tRNAs are determined with similar accuracy. The positions of the CCA end of the tRNAs at the A site are different before and after peptide bond formation. The relative positions of anticodons of P- and E-site tRNAs in the posttranslocational state are such that a codon-anticodon interaction at the E site appears feasible.

A map of protein-rRNA distribution in the 70 S Escherichia coli ribosome.

Neutron scattering exploits the enormous scattering difference between protons and deuterons. A set of 42 x-ray and neutron solution scattering curves from hybrid Escherichia coli ribosomes was obtained, where the proteins and rRNA moieties in the subunits were either protonated or deuterated in all possible combinations. This extensive data set is analyzed using a novel method. The volume defined by the cryoelectron microscopic model of Frank and co-workers (Frank, J., Zhu, J., Penczek, P., Li, Y. H., Srivastava, S., Verschoor, A., Radermacher, M., Grassucci, R., Lata, R. K., and Agrawal, R. K. (1995) Nature 376, 441-444) is divided into 7890 densely packed spheres of radius 0.5 nm. Simulated annealing is employed to assign each sphere to solvent, protein, or rRNA moieties to simultaneously fit all scattering curves. Twelve independent reconstructions starting from random approximations yielded reproducible results. The resulting model at a resolution of 3 nm represents the volumes occupied by rRNA and protein moieties at 95% probability threshold and displays 15 and 20 protein subvolumes in the 30 S and 50 S, respectively, connected by rRNA. 17 proteins with known atomic structure can be tentatively positioned into the protein subvolumes within the ribosome in agreement with the results from other methods. The protein-rRNA map enlarges the basis for the models of the rRNA folding and can further help to localize proteins in high-resolution crystallographic density maps.

Detection and selective dissociation of intact ribosomes in a mass spectrometer.

Intact Escherichia coli ribosomes have been projected into the gas phase of a mass spectrometer by means of nanoflow electrospray techniques. Species with mass/charge ratios in excess of 20,000 were detected at the level of individual ions by using time-of-flight analysis. Once in the gas phase the stability of intact ribosomes was investigated and found to increase as a result of cross-linking ribosomal proteins to the rRNA. By lowering the Mg(2+) concentration in solutions containing ribosomes the particles were found to dissociate into 30S and 50S subunits. The resolution of the charge states in the spectrum of the 30S subunit enabled its mass to be determined as 852,187 +/- 3,918 Da, a value within 0.6% of that calculated from the individual proteins and the 16S RNA. Further dissociation into smaller macromolecular complexes and then individual proteins could be induced by subjecting the particles to increasingly energetic gas phase collisions. The ease with which proteins dissociated from the intact species was found to be related to their known interactions in the ribosome particle. The results show that emerging mass spectrometric techniques can be used to characterize a fully functional biological assembly as well as its isolated components.

Selecting rRNA binding sites for the ribosomal proteins L4 and L6 from randomly fragmented rRNA: application of a method called SERF.

Two-thirds of the 54 proteins of the Escherichia coli ribosome interact directly with the rRNAs, but the rRNA binding sites of only a very few proteins are known. We present a method (selection of random RNA fragments; SERF) that can identify the minimal binding region for proteins within ribonucleo-protein complexes such as the ribosome. The power of the method is exemplified with the ribosomal proteins L4 and L6. Binding sequences are identified for both proteins and characterized by phosphorothioate footprinting. Surprisingly, the binding region of L4, a 53-nt rRNA fragment of domain I of 23S rRNA, can simultaneously and independently bind L24, one of the two assembly initiator proteins of the large subunit.