Site-specific labeling of the ribosome for single-molecule spectroscopy

Single-molecule fluorescence spectroscopy can reveal mechanistic and kinetic details that may not be observed in static structural and bulk biochemical studies of protein synthesis. One approach requires site-specific and stable attachment of fluorophores to the components of translation machinery. Fluorescent tagging of the ribosome is a prerequisite for the observation of dynamic changes in ribosomal conformation during translation using fluorescence methods. Modifications of the ribosomal particle are difficult due to its complexity and high degree of sequence and structural conservation. We have developed a general method to label specifically the prokaryotic ribosome by hybridization of fluorescent oligonucleotides to mutated ribosomal RNA. Functional, modified ribosomes can be purified as a homogenous population, and fluorescence can be monitored from labeled ribosomal complexes immobilized on a derivatized quartz surface.     

Accessibility of 18S rRNA in human 40S subunits and 80S ribosomes at physiological magnesium ion concentrations--implications for the study of ribosome dynamics

Protein biosynthesis requires numerous conformational rearrangements within the ribosome. The structural core of the ribosome is composed of RNA and is therefore dependent on counterions such as magnesium ions for function. Many steps of translation can be compromised or inhibited if the concentration of Mg(2+) is too low or too high. Conditions previously used to probe the conformation of the mammalian ribosome in vitro used high Mg(2+) concentrations that we find completely inhibit translation in vitro. We have therefore probed the conformation of the small ribosomal subunit in low concentrations of Mg(2+) that support translation in vitro and compared it with the conformation of the 40S subunit at high Mg(2+) concentrations. In low Mg(2+) concentrations, we find significantly more changes in chemical probe accessibility in the 40S subunit due to subunit association or binding of the hepatitis C internal ribosomal entry site (HCV IRES) than had been observed before. These results suggest that the ribosome is more dynamic in its functional state than previously appreciated.     

New insights into the interaction of ribosomal protein L1 with RNA

The RNA-binding ability of ribosomal protein L1 is of profound interest, since L1 has a dual function as a ribosomal structural protein that binds rRNA and as a translational repressor that binds its own mRNA. Here, we report the crystal structure at 2.6 A resolution of ribosomal protein L1 from the bacterium Thermus thermophilus in complex with a 38 nt fragment of L1 mRNA from Methanoccocus vannielii. The conformation of RNA-bound T.thermophilus L1 differs dramatically from that of the isolated protein. Analysis of four copies of the L1-mRNA complex in the crystal has shown that domain II of the protein does not contribute to mRNA-specific binding. A detailed comparison of the protein-RNA interactions in the L1-mRNA and L1-rRNA complexes identified amino acid residues of L1 crucial for recognition of its specific targets on the both RNAs. Incorporation of the structure of bacterial L1 into a model of the Escherichia coli ribosome revealed two additional contact regions for L1 on the 23S rRNA that were not identified in previous ribosome models.     

Nascent membrane and secretory proteins differ in FRET-detected folding far inside the ribosome and in their exposure to ribosomal proteins

Fluorescence resonance energy transfer measurements reveal that a transmembrane sequence within a nascent membrane protein folds into a compact conformation near the peptidyltransferase center and remains folded as the sequence moves through a membrane bound ribosome into the translocon. This compact conformation is compatible with an alpha helix because nearly the same energy transfer efficiency was observed when the transmembrane sequence was integrated into the lipid bilayer. Since the transmembrane sequence unfolds upon emerging from a free ribosome, this nascent chain folding is ribosome induced and stabilized. In contrast, a nascent secretory protein is in an extended conformation in the exit tunnel. Furthermore, two ribosomal proteins photo-crosslink to nascent membrane but not secretory proteins. These interactions coincide with the previously described sequential closing and opening of the two ends of the aqueous translocon pore, thereby suggesting that ribosomal recognition of nascent chain folding controls the operational mode of the translocon at the ER membrane.     

The size and conformation of Artemia (brine-shrimp) ribosomal RNA free in solution.

The RNA was isolated from the large ribosomal subunits of the brine shrimp Artemia, and its conformation free in solution was studied by determining its sedimentation and diffusion coefficients. A comparison was made of the hydrodynamic radius of the ribosomal subunit and its isolated RNA in various buffers. The conformation of the rRNA free in solution is more extended than when it is incorporated in the ribosome. This is not only the case when the rRNA solution lacks bivalent and polyvalent cations, but even in the presence of Mg2+ and spermidine, which cause a tightening of RNA. Thus the ribosomal proteins should induce a further tightening of the rRNA during the assembly of the ribosome. In the discussion, the reported data on Escherichia coli rRNA species are presented in such a way that large discrepancies between various studied are revealed, and that they can be compared with the data reported here on the larger rRNA of an eukaryote.     

Pokeweed antiviral protein depurinates the sarcin/ricin loop of the rRNA prior to binding of aminoacyl-tRNA to the ribosomal A-site

Ribosome-inactivating proteins, such as the pokeweed antiviral protein (PAP), inhibit translation by depurinating the conserved sarcin/ricin loop of the large ribosomal RNA. Depurinated ribosomes are unable to bind elongation factor 2, and, thus, the translocation step of the elongation cycle is inhibited. Though the consequences of depurination are well characterized, the ribosome conformation required for depurination to take place has not been described. In this report, we correlate biochemical and genetic data to conclude that pokeweed antiviral protein depurinates the sarcin/ricin loop when the A-site of the ribosomal peptidyl-transferase center is unoccupied. We show that prior incubation of ribosomes with puromycin, an analog of the 3'-terminus of aminoacyl-tRNA, inhibits both binding and depurination by PAP in a concentration-dependent manner. Expression of PAP in the yeast strain mak8-1 results in little depurination unless the cells are lysed, a process that would promote loss of aminoacyl-tRNA from the ribosome. The mak8-1 strain is known to exhibit a higher affinity for aminoacyl-tRNA compared with wild-type cells, and therefore, its ribosomes are more resistant to PAP in vivo. These data contribute to the mechanism of action of pokeweed antiviral protein; specifically, they have uncovered the ribosomal conformation required for depurination that leads to subsequent translation inhibition.     

Enhancement of peptidyl transferase activity by antibiotics acting on the 50 S ribosomal subunit

Interconversions of ribosomes, between forms that are active and inactive in peptidyl transfer, were studied and conditions favoring a state of equilibrium between the two forms were established. Under such conditions activity was enhanced two-to fivefold by the antibiotics erythromycin, vernamycin Bα, lincomycin, chloramphenicol and vernamycin A. The antibiotics puromycin, gougerotin, thiostrepton and siomycin, whose target site is also the 50 S ribosomal subunit, were ineffective.A common feature of the effective antibiotics is their ability to bind to ribosomes active in peptidyl transfer but not to enzymatically inactive ribosomes. The activity enhancing effect of antibiotics is therefore interpreted as being due to a shift in the equilibrium between the two ribosomal forms in favor of the active conformation, brought about by the preferential binding of the antibiotic to ribosomes in this form. The results stress the flexible nature of ribosome structure and suggest that antibiotics can function as allosteric effectors in modifying ribosome conformation.     

Streptomycin-induced conformational changes in the 70-S bacterial ribosome.

Conformational alterations induced by streptomycin in the bacterial ribosome have been investigated using as probes, ethidium bromide, N-[14C]ethylmaleimide and a spin label nitroxide analog of N-ethylmaleimide. 1. The binding of the antibiotic to the ribosome does not affect the reactivity of sulfhydryl groups towards N-ethylmaleimide. 2. The motional freedom of spin labels bound to ribosomal proteins S1 and S18 is increased but it is hardly affected at other labeled sites. This observation suggests that the binding of streptomycin causes a local loosening of the ribosomal structure. 3. Ribosomes are found to bind less ethidium bromide in the presence of streptomycin, which suggests that the binding of streptomycin decreases the degree of organization of ribosomal RNA.     

Ribosome association primes the stringent factor Rel for tRNA-dependent locking in the A-site and activation of (p)ppGpp synthesis

In the Gram-positive Firmicute bacterium Bacillus subtilis, amino acid starvation induces synthesis of the alarmone (p)ppGpp by the RelA/SpoT Homolog factor Rel. This bifunctional enzyme is capable of both synthesizing and hydrolysing (p)ppGpp. To detect amino acid deficiency, Rel monitors the aminoacylation status of the ribosomal A-site tRNA by directly inspecting the tRNA’s CCA end. Here we dissect the molecular mechanism of B. subtilis Rel. Off the ribosome, Rel predominantly assumes a ‘closed’ conformation with dominant (p)ppGpp hydrolysis activity. This state does not specifically select deacylated tRNA since the interaction is only moderately affected by tRNA aminoacylation. Once bound to the vacant ribosomal A-site, Rel assumes an ‘open’ conformation, which primes its TGS and Helical domains for specific recognition and stabilization of cognate deacylated tRNA on the ribosome. The tRNA locks Rel on the ribosome in a hyperactivated state that processively synthesises (p)ppGpp while the hydrolysis is suppressed. In stark contrast to non-specific tRNA interactions off the ribosome, tRNA-dependent Rel locking on the ribosome and activation of (p)ppGpp synthesis are highly specific and completely abrogated by tRNA aminoacylation. Binding pppGpp to a dedicated allosteric site located in the N-terminal catalytic domain region of the enzyme further enhances its synthetase activity.     

Production and characterization of a noncytotoxic deletion variant of the Aspergillus fumigatus allergen Aspf1 displaying reduced IgE binding

Aspergillus fumigatus is responsible for many allergic respiratory diseases, the most notable of which - due to its severity - is allergic bronchopulmonary aspergillosis. Aspf1 is a major allergen of this fungus: this 149-amino acid protein belongs to the ribotoxin family, whose best characterized member is alpha-sarcin (EC 3.1.27.10). The proteins of this group are cytotoxic ribonucleases that degrade a unique bond in ribosomal RNA impairing protein biosynthesis. Aspf1 and its deletion mutant Aspf1Delta(7-22) have been produced as recombinant proteins; the deleted region corresponds to an exposed beta-hairpin. The conformation of these two proteins has been studied by CD and fluorescence spectroscopy. Their enzymatic activity and cytotoxicity against human rhabdomyosarcoma cells was also measured and their allergenic properties have been studied by using 58 individual sera of patients sensitized to Aspergillus. Aspf1Delta(7-22) lacks cytotoxicity and shows a remarkably reduced IgE reactivity. From these studies it can be concluded that the deleted beta-hairpin is involved in ribosome recognition and is a significant allergenic region.     

Is the in situ accessibility of the 16S rRNA of Escherichia coli for Cy3-labeled oligonucleotide probes predicted by a three-dimensional structure model of the 30S ribosomal subunit?

Systematic studies on the hybridization of fluorescently labeled, rRNA-targeted oligonucleotides have shown strong variations in in situ accessibility. Reliable predictions of target site accessibility would contribute to more-rational design of probes for the identification of individual microbial cells in their natural environments. During the past 3 years, numerous studies of the higher-order structure of the ribosome have advanced our understanding of its spatial conformation. These studies range from the identification of rRNA-rRNA interactions based on covariation analyses to physical imaging of the ribosome for the identification of protein-rRNA interactions. Here we reevaluate our Escherichia coli 16S rRNA in situ accessibility data with regard to a tertiary-structure model of the small subunit of the ribosome. We localized target sequences of 176 oligonucleotides on a 3.0-A-resolution three-dimensional (3D) model of the 30S ribosomal subunit. Little correlation was found between probe hybridization efficiency and the proximity of the probe target region to the surface of the 30S ribosomal subunit model. We attribute this to the fact that fluorescence in situ hybridization is performed on fixed cells containing denatured ribosomes, whereas 3D models of the ribosome are based on its native conformation. The effects of different fixation and hybridization protocols on the fluorescence signals conferred by a set of 10 representative probes were tested. The presence or absence of the strongly denaturing detergent sodium dodecyl sulfate had a much more pronounced effect than a change of fixative from paraformaldehyde to ethanol.     

A chemical approach to the fine structure of biomolecular complexes: the amino terminal region of the 50S ribosomal "A" protein from Bacillus stearothermophilus.

An experimental approach and methodology are described for determining the reactive properties and ionization constants of individual functional groups of proteins within biomolecular complexes. The ionization constants and reactivities of the methionyl-l amino terminus and the lysyl-3 residue of the alanine rich 50S ribosomal "A" protein from Bacillus stearothermophilus have been determined by an extension of the competitive labeling technique used by H. Kaplan, K. J. Stevenson, and B. S. Hartley ((1971), Biochem. J. 124, 289-299). This approach employs (1-14C)- and (3H)acetic anhydride in a double-labeling procedure. In 0.1 M KCl-0.02 M Mg2+-0.05 M Veronal at 10 degrees the methionyl-l amino terminus has a pKa of 7.5 and is exposed on the surface of the ribosome. The lysyl-3 has a pKa of 10 and is also exposed to solvent at the surface of the 50S subunit. Based on a linear free energy relationship (Bronsted plot) obtained with a series of standard amines the methionyl amino terminus has a substantially higher reactivity than expected from its ionization constant. The lysyl epsilon-amino group has the expected reactivity. The abnormally high reactivity of the methionyl amino terminus can only be accounted for by a specific interaction with other functional groups in the ribosome. These data support the proposal that the charged state of this residue is important in the structure and function of the "A" protein at the surface of the ribosome.     

pKa of adenine 2451 in the ribosomal peptidyl transferase center remains elusive.

The universally conserved A2451 of 23S rRNA has been proposed to participate directly in the catalysis of peptide bond formation in the ribosomal peptidyl transferase center. An unusually high, near neutral, pKa of A2451 is a prerequisite for its action as a general acid-base catalyst. Increased reactivity of A2451 to dimethylsulfate (DMS) at pH 8.5 compared to pH 6.5 was taken as evidence that the pKa of this nucleotide falls within this pH range. Structural data suggested that the interaction between A2451 and G2447 in the ribosome is responsible for A2451 pKa perturbation. In contrast to expectation, our studies did not show pH dependence of A2451 dimethylsulfate modification in ribosomes of Thermus aquaticus and Mycobacterium smegmatis. Other rRNA regions, however, showed major alterations in DMS reactivity at pH 8.5 compared to pH 6.5, suggesting that conformational rearrangements in the structure of the large ribosomal subunit may occur upon the pH shift. The G2447U mutant of M. smegmatis was viable, indicating that the G2447-A2451 interaction is not critical for the ribosome function. We concluded that the proposed unusual pKa of A2451, if existing, may not be crucial for the ribosome activity and that the previously reported pH-dependent alterations in the DMS modification of A2451 do not necessarily reveal an unusual pKa of this nucleotide.     

Structural basis for interaction of the ribosome with the switch regions of GTP-bound elongation factors

Elongation factor G (EF-G) catalyzes tRNA translocation on the ribosome. Here a cryo-EM reconstruction of the 70S*EF-G ribosomal complex at 7.3 A resolution and the crystal structure of EF-G-2*GTP, an EF-G homolog, at 2.2 A resolution are presented. EF-G-2*GTP is structurally distinct from previous EF-G structures, and in the context of the cryo-EM structure, the conformational changes are associated with ribosome binding and activation of the GTP binding pocket. The P loop and switch II approach A2660-A2662 in helix 95 of the 23S rRNA, indicating an important role for these conserved bases. Furthermore, the ordering of the functionally important switch I and II regions, which interact with the bound GTP, is dependent on interactions with the ribosome in the ratcheted conformation. Therefore, a network of interaction with the ribosome establishes the active GTP conformation of EF-G and thus facilitates GTP hydrolysis and tRNA translocation.     

Following the dynamics of changes in solvent accessibility of 16 S and 23 S rRNA during ribosomal subunit association using synchrotron-generated hydroxyl radicals

We have probed the structure and dynamics of ribosomal RNA in the Escherichia coli ribosome using equilibrium and time-resolved hydroxyl radical (OH) RNA footprinting to explore changes in the solvent-accessible surface of the rRNA with single-nucleotide resolution. The goal of these studies is to better understand the structural transitions that accompany association of the 30 S and 50 S subunits and to build a foundation for the quantitative analysis of ribosome structural dynamics during translation. Clear portraits of the subunit interface surfaces for 16 S and 23 S rRNA were obtained by constructing difference maps between the OH protection maps of the free subunits and that of the associated ribosome. In addition to inter-subunit contacts consistent with the crystal structure, additional OH protections are evident in regions at or near the subunit interface that reflect association-induced conformational changes. Comparison of these data with the comparable difference maps of the solvent-accessible surface of the rRNA calculated for the Thermus thermophilus X-ray crystal structures shows extensive agreement but also distinct differences. As a prelude to time-resolved OH footprinting studies, the reactivity profiles obtained using Fe(II)EDTA and X-ray generated OH were comprehensively compared. The reactivity patterns are similar except for a small number of nucleotides that have decreased reactivity to OH generated from Fe(II)EDTA compared to X-rays. These nucleotides are generally close to ribosomal proteins, which can quench diffusing radicals by virtue of side-chain oxidation. Synchrotron X-ray OH footprinting was used to monitor the kinetics of association of the 30 S and 50 S subunits. The rates individually measured for the inter-subunit contacts are comparable within experimental error. The application of this approach to the study of ribosome dynamics during the translation cycle is discussed.     

Elongation factor Tu-induced conformational changes of ribosomes detected by iodination

The effect of elongation factor (EF) Tu, bound to the ribosome with the help of poly(uridylic) acid, Phe-tRNA and guanyl-5-yl methylene diphosphonate, on the conformation and/or chemical environment of ribosomal proteins has been examined using, as a probe, protein iodination. Ribosomes complexed only with poly(uridylic acid) and Phe-tRNA have been used as a control. EF-Tu on the ribosome significantly increases the iodination of proteins S7, S10 and L3 and decreases that of S21 and L18.     

Investigating molecular mechanisms of 2A-stimulated ribosomal pausing and frameshifting in Theilovirus

The 2A protein of Theiler''s murine encephalomyelitis virus (TMEV) acts as a switch to stimulate programmed –1 ribosomal frameshifting (PRF) during infection. Here, we present the X-ray crystal structure of TMEV 2A and define how it recognises the stimulatory RNA element. We demonstrate a critical role for bases upstream of the originally predicted stem–loop, providing evidence for a pseudoknot-like conformation and suggesting that the recognition of this pseudoknot by beta-shell proteins is a conserved feature in cardioviruses. Through examination of PRF in TMEV-infected cells by ribosome profiling, we identify a series of ribosomal pauses around the site of PRF induced by the 2A-pseudoknot complex. Careful normalisation of ribosomal profiling data with a 2A knockout virus facilitated the identification, through disome analysis, of ribosome stacking at the TMEV frameshifting signal. These experiments provide unparalleled detail of the molecular mechanisms underpinning Theilovirus protein-stimulated frameshifting.     

On the use of the antibiotic chloramphenicol to target polypeptide chain mimics to the ribosomal exit tunnel

The ribosomal exit tunnel had recently become the centre of many functional and structural studies. Accumulated evidence indicates that the tunnel is not simply a passive conduit for the nascent chain, but a rather functionally important compartment where nascent peptide sequences can interact with the ribosome to signal translation to slow down or even stop. To explore further this interaction, we have synthesized short peptides attached to the amino group of a chloramphenicol (CAM) base, such that when bound to the ribosome these compounds mimic a nascent peptidyl-tRNA chain bound to the A-site of the peptidyltransferase center (PTC). Here we show that these CAM-peptides interact with the PTC of the ribosome while their effectiveness can be modulated by the sequence of the peptide, suggesting a direct interaction of the peptide with the ribosomal tunnel. Indeed, chemical footprinting in the presence of CAM-P2, one of the tested CAM-peptides, reveals protection of 23S rRNA nucleotides located deep within the tunnel, indicating a potential interaction with specific components of the ribosomal tunnel. Collectively, our findings suggest that the CAM-based peptide derivatives will be useful tools for targeting polypeptide chain mimics to the ribosomal tunnel, allowing their conformation and interaction with the ribosomal tunnel to be explored using further biochemical and structural methods.     

GTPase activation of elongation factors Tu and G on the ribosome

The GTPase activity of elongation factors Tu and G is stimulated by the ribosome. The factor binding site is located on the 50S ribosomal subunit and comprises proteins L7/12, L10, L11, the L11-binding region of 23S rRNA, and the sarcin-ricin loop of 23S rRNA. The role of these ribosomal elements in factor binding, GTPase activation, or functions in tRNA binding and translocation, and their relative contributions, is not known. By comparing ribosomes depleted of L7/12 and reconstituted ribosomes, we show that, for both factors, interactions with L7/12 and with other ribosomal residues contribute about equally and additively to GTPase activation, resulting in an overall 10(7)-fold stimulation. Removal of L7/12 has little effect on factor binding to the ribosome. Effects on other factor-dependent functions, i.e., A-site binding of aminoacyl-tRNA and translocation, are fully explained by the inhibition of GTP hydrolysis. Based on these results, we propose that L7/12 stimulates the GTPase activity of both factors by inducing the catalytically active conformation of the G domain. This effect appears to be augmented by interactions of other structural elements of the large ribosomal subunit with the switch regions of the factors.     

Selection of tRNA by the ribosome requires a transition from an open to a closed form

A structural and mechanistic explanation for the selection of tRNAs by the ribosome has been elusive. Here, we report crystal structures of the 30S ribosomal subunit with codon and near-cognate tRNA anticodon stem loops bound at the decoding center and compare affinities of equivalent complexes in solution. In ribosomal interactions with near-cognate tRNA, deviation from Watson-Crick geometry results in uncompensated desolvation of hydrogen-bonding partners at the codon-anticodon minor groove. As a result, the transition to a closed form of the 30S induced by cognate tRNA is unfavorable for near-cognate tRNA unless paromomycin induces part of the rearrangement. We conclude that stabilization of a closed 30S conformation is required for tRNA selection, and thereby structurally rationalize much previous data on translational fidelity.