Decoding in the absence of a codon by tmRNA and SmpB in the ribosome

In bacteria, ribosomes stalled at the end of truncated messages are rescued by transfer-messenger RNA (tmRNA), a bifunctional molecule that acts as both a transfer RNA (tRNA) and a messenger RNA (mRNA), and SmpB, a small protein that works in concert with tmRNA. Here, we present the crystal structure of a tmRNA fragment, SmpB and elongation factor Tu bound to the ribosome at 3.2 angstroms resolution. The structure shows how SmpB plays the role of both the anticodon loop of tRNA and portions of mRNA to facilitate decoding in the absence of an mRNA codon in the A site of the ribosome and explains why the tmRNA-SmpB system does not interfere with normal translation.     

Visualizing tmRNA entry into a stalled ribosome

Bacterial ribosomes stalled on defective messenger RNAs (mRNAs) are rescued by tmRNA, an approximately 300-nucleotide-long molecule that functions as both transfer RNA (tRNA) and mRNA. Translation then switches from the defective message to a short open reading frame on tmRNA that tags the defective nascent peptide chain for degradation. However, the mechanism by which tmRNA can enter and move through the ribosome is unknown. We present a cryo-electron microscopy study at approximately 13 to 15 angstroms of the entry of tmRNA into the ribosome. The structure reveals how tmRNA could move through the ribosome despite its complicated topology and also suggests roles for proteins S1 and SmpB in the function of tmRNA.     

Crystal structure of the eukaryotic ribosome

Crystal structures of prokaryotic ribosomes have described in detail the universally conserved core of the translation mechanism. However, many facets of the translation process in eukaryotes are not shared with prokaryotes. The crystal structure of the yeast 80S ribosome determined at 4.15 angstrom resolution reveals the higher complexity of eukaryotic ribosomes, which are 40% larger than their bacterial counterparts. Our model shows how eukaryote-specific elements considerably expand the network of interactions within the ribosome and provides insights into eukaryote-specific features of protein synthesis. Our crystals capture the ribosome in the ratcheted state, which is essential for translocation of mRNA and transfer RNA (tRNA), and in which the small ribosomal subunit has rotated with respect to the large subunit. We describe the conformational changes in both ribosomal subunits that are involved in ratcheting and their implications in coordination between the two associated subunits and in mRNA and tRNA translocation.     

How hibernation factors RMF, HPF, and YfiA turn off protein synthesis

Eubacteria inactivate their ribosomes as 100S dimers or 70S monomers upon entry into stationary phase. In Escherichia coli, 100S dimer formation is mediated by ribosome modulation factor (RMF) and hibernation promoting factor (HPF), or alternatively, the YfiA protein inactivates ribosomes as 70S monomers. Here, we present high-resolution crystal structures of the Thermus thermophilus 70S ribosome in complex with each of these stationary-phase factors. The binding site of RMF overlaps with that of the messenger RNA (mRNA) Shine-Dalgarno sequence, which prevents the interaction between the mRNA and the 16S ribosomal RNA. The nearly identical binding sites of HPF and YfiA overlap with those of the mRNA, transfer RNA, and initiation factors, which prevents translation initiation. The binding of RMF and HPF, but not YfiA, to the ribosome induces a conformational change of the 30S head domain that promotes 100S dimer formation.     

Structure of the 70S ribosome complexed with mRNA and tRNA

The crystal structure of the bacterial 70S ribosome refined to 2.8 angstrom resolution reveals atomic details of its interactions with messenger RNA (mRNA) and transfer RNA (tRNA). A metal ion stabilizes a kink in the mRNA that demarcates the boundary between A and P sites, which is potentially important to prevent slippage of mRNA. Metal ions also stabilize the intersubunit interface. The interactions of E-site tRNA with the 50S subunit have both similarities and differences compared to those in the archaeal ribosome. The structure also rationalizes much biochemical and genetic data on translation.     

RIBOSOMAL RNA ON THE SURFACE OF RIBOSOMES

Ribonuclease from pancreas releases nucleotide from Escherichiacoli ribosomes while not altering significantly the base composition of the total ribosomal RNA. Ribonuclease hydrolyzes ribsomal RNA without destroying the structure of 70S or 30S and 50S ribosomes. The RNA in 30S and 50S ribosomes appears more sensitive to the action of ribonuclease. The data suggest that RNA may be a surface component of the ribosome.     

核糖体的结构与功能研究

从核糖体功能中长期存在之谜出发,阐述了关于核糖体亚基高分辨率晶体结构的研究,分析了核糖体结构与mRNA转录精确性的关系,及肽键形成机制与50S亚基结构的关系,论述了核糖体亚基结构在抗生素研制中的应用,并对核糖体功能复合物晶体结构研究的新方向进行了展望。     

Structures of the bacterial ribosome at 3.5 A resolution

We describe two structures of the intact bacterial ribosome from Escherichia coli determined to a resolution of 3.5 angstroms by x-ray crystallography. These structures provide a detailed view of the interface between the small and large ribosomal subunits and the conformation of the peptidyl transferase center in the context of the intact ribosome. Differences between the two ribosomes reveal a high degree of flexibility between the head and the rest of the small subunit. Swiveling of the head of the small subunit observed in the present structures, coupled to the ratchet-like motion of the two subunits observed previously, suggests a mechanism for the final movements of messenger RNA (mRNA) and transfer RNAs (tRNAs) during translocation.     

Translational operator of mRNA on the ribosome: how repressor proteins exclude ribosome binding

The ribosome of Thermus thermophilus was cocrystallized with initiator transfer RNA (tRNA) and a structured messenger RNA (mRNA) carrying a translational operator. The path of the mRNA was defined at 5.5 angstroms resolution by comparing it with either the crystal structure of the same ribosomal complex lacking mRNA or with an unstructured mRNA. A precise ribosomal environment positions the operator stem-loop structure perpendicular to the surface of the ribosome on the platform of the 30S subunit. The binding of the operator and of the initiator tRNA occurs on the ribosome with an unoccupied tRNA exit site, which is expected for an initiation complex. The positioning of the regulatory domain of the operator relative to the ribosome elucidates the molecular mechanism by which the bound repressor switches off translation. Our data suggest a general way in which mRNA control elements must be placed on the ribosome to perform their regulatory task.     

Structure of RNA in ribosomes

The 50S and 30S ribosomes and 23S and 16S RNA were hydrolyzed with ribonuclease A. The rate constants and number of fragments produced were determined for each reaction. The conformation of 23S RNA changes when the RNA is extracted from the ribosome. Specific regions of the RNA in 50S and 30S ribosomes are protected from hydrolysis by the ribosomal proteins.     

The structure of the eukaryotic ribosome at 3.0 Å resolution

Ribosomes translate genetic information encoded by messenger RNA into proteins. Many aspects of translation and its regulation are specific to eukaryotes, whose ribosomes are much larger and intricate than their bacterial counterparts. We report the crystal structure of the 80S ribosome from the yeast Saccharomyces cerevisiae--including nearly all ribosomal RNA bases and protein side chains as well as an additional protein, Stm1--at a resolution of 3.0 angstroms. This atomic model reveals the architecture of eukaryote-specific elements and their interaction with the universally conserved core, and describes all eukaryote-specific bridges between the two ribosomal subunits. It forms the structural framework for the design and analysis of experiments that explore the eukaryotic translation apparatus and the evolutionary forces that shaped it.     

Ribosome profiling shows that miR-430 reduces translation before causing mRNA decay in zebrafish

MicroRNAs regulate gene expression through deadenylation, repression, and messenger RNA (mRNA) decay. However, the contribution of each mechanism in non-steady-state situations remains unclear. We monitored the impact of miR-430 on ribosome occupancy of endogenous mRNAs in wild-type and dicer mutant zebrafish embryos and found that miR-430 reduces the number of ribosomes on target mRNAs before causing mRNA decay. Translational repression occurs before complete deadenylation, and disrupting deadenylation with use of an internal polyadenylate tail did not block target repression. Lastly, we observed that ribosome density along the length of the message remains constant, suggesting that translational repression occurs by reducing the rate of initiation rather than affecting elongation or causing ribosomal drop-off. These results show that miR-430 regulates translation initiation before inducing mRNA decay during zebrafish development.     

Proliferation, but not growth, blocked by conditional deletion of 40S ribosomal protein S6

Because ribosome biogenesis plays an essential role in cell proliferation, control mechanisms may have evolved to recognize lesions in this critical anabolic process. To test this possibility, we conditionally deleted the gene encoding 40S ribosomal protein S6 in the liver of adult mice. Unexpectedly, livers from fasted animals deficient in S6 grew in response to nutrients even though biogenesis of 40S ribosomes was abolished. However, liver cells failed to proliferate or induce cyclin E expression after partial hepatectomy, despite formation of active cyclin D-CDK4 complexes. These results imply that abrogation of 40S ribosome biogenesis may induce a checkpoint control that prevents cell cycle progression.     

Signal recognition particle receptor exposes the ribosomal translocon binding site

Signal sequences of secretory and membrane proteins are recognized by the signal recognition particle (SRP) as they emerge from the ribosome. This results in their targeting to the membrane by docking with the SRP receptor, which facilitates transfer of the ribosome to the translocon. Here, we present the 8 angstrom cryo-electron microscopy structure of a "docking complex" consisting of a SRP-bound 80S ribosome and the SRP receptor. Interaction of the SRP receptor with both SRP and the ribosome rearranged the S domain of SRP such that a ribosomal binding site for the translocon, the L23e/L35 site, became exposed, whereas Alu domain-mediated elongation arrest persisted.     

Crystal structure of the ribosome at 5.5 A resolution

We describe the crystal structure of the complete Thermus thermophilus 70S ribosome containing bound messenger RNA and transfer RNAs (tRNAs) at 5.5 angstrom resolution. All of the 16S, 23S, and 5S ribosomal RNA (rRNA) chains, the A-, P-, and E-site tRNAs, and most of the ribosomal proteins can be fitted to the electron density map. The core of the interface between the 30S small subunit and the 50S large subunit, where the tRNA substrates are bound, is dominated by RNA, with proteins located mainly at the periphery, consistent with ribosomal function being based on rRNA. In each of the three tRNA binding sites, the ribosome contacts all of the major elements of tRNA, providing an explanation for the conservation of tRNA structure. The tRNAs are closely juxtaposed with the intersubunit bridges, in a way that suggests coupling of the 20 to 50 angstrom movements associated with tRNA translocation with intersubunit movement.     

Mendelian and uniparental alterations in erythromycin binding by plastid ribosomes

Erythromycin binds specifically to the 52S subunit of the chloroplast ribosome of Chlamydomonas reinhardi. A number of erythromycin-resistant mutants whose ribosomes have lost their affinity for the antibiotic have been isolated, but the sedimentation properties of their ribosomes are indistinguishable from those of the wild-type strain. These mutants represent at least three genetic loci. Two of them show Mendelian inheritance, and one of them is inherited in a uniparental manner.     

Catalysis of ribosomal translocation by sparsomycin

During protein synthesis, transfer RNAs (tRNAs) are translocated from the aminoacyl to peptidyl to exit sites of the ribosome, coupled to the movement of messenger RNA (mRNA), in a reaction catalyzed by elongation factor G (EF-G) and guanosine triphosphate (GTP). Here, we show that the peptidyl transferase inhibitor sparsomycin triggers accurate translocation in vitro in the absence of EF-G and GTP. Our results provide evidence that translocation is a function inherent to the ribosome and that the energy to drive this process is stored in the tRNA-mRNA-ribosome complex after peptide-bond formation. These findings directly implicate the peptidyl transferase center of the 50S subunit in the mechanism of translocation, a process involving large-scale movement of tRNA and mRNA in the 30S subunit, some 70 angstroms away.     

The mechanism for activation of GTP hydrolysis on the ribosome

Protein synthesis requires several guanosine triphosphatase (GTPase) factors, including elongation factor Tu (EF-Tu), which delivers aminoacyl-transfer RNAs (tRNAs) to the ribosome. To understand how the ribosome triggers GTP hydrolysis in translational GTPases, we have determined the crystal structure of EF-Tu and aminoacyl-tRNA bound to the ribosome with a GTP analog, to 3.2 angstrom resolution. EF-Tu is in its active conformation, the switch I loop is ordered, and the catalytic histidine is coordinating the nucleophilic water in position for inline attack on the γ-phosphate of GTP. This activated conformation is due to a critical and conserved interaction of the histidine with A2662 of the sarcin-ricin loop of the 23S ribosomal RNA. The structure suggests a universal mechanism for GTPase activation and hydrolysis in translational GTPases on the ribosome.