Nascent Polypeptide Exit Tunnel Research Articles

Tetracenomycin X sequesters peptidyl-tRNA during translation of QK motifs.

As antimicrobial resistance threatens our ability to treat common bacterial infections, new antibiotics with limited cross-resistance are urgently needed. In this regard, natural products that target the bacterial ribosome have the potential to be developed into potent drugs through structure-guided design, provided their mechanisms of action are well understood. Here we use inverse toeprinting coupled to next-generation sequencing to show that the aromatic polyketide tetracenomycin X primarily inhibits peptide bond formation between an incoming aminoacyl-tRNA and a terminal Gln-Lys (QK) motif in the nascent polypeptide. Using cryogenic electron microscopy, we reveal that translation inhibition at QK motifs occurs via an unusual mechanism involving sequestration of the 3' adenosine of peptidyl-tRNALys in the drug-occupied nascent polypeptide exit tunnel of the ribosome. Our study provides mechanistic insights into the mode of action of tetracenomycin X on the bacterial ribosome and suggests a path forward for the development of novel aromatic polyketide antibiotics.

A male germ-cell-specific ribosome controls male fertility.

Ribosomes are highly sophisticated translation machines that have been demonstrated to be heterogeneous in the regulation of protein synthesis1,2. Male germ cell development involves complex translational regulation during sperm formation3. However, it remains unclear whether translation during sperm formation is performed by a specific ribosome. Here we report a ribosome with a specialized nascent polypeptide exit tunnel, RibosomeST, that is assembled with the male germ-cell-specific protein RPL39L, the paralogue of core ribosome (RibosomeCore) protein RPL39. Deletion of RibosomeST in mice causes defective sperm formation, resulting in substantially reduced fertility. Our comparison of single-particle cryo-electron microscopy structures of ribosomes from mouse kidneys and testes indicates that RibosomeST features a ribosomal polypeptide exit tunnel of distinct size and charge states compared with RibosomeCore. RibosomeST predominantly cotranslationally regulates the folding of a subset of male germ-cell-specific proteins that are essential for the formation of sperm. Moreover, we found that specialized functions of RibosomeST were not replaceable by RibosomeCore. Taken together, identification of this sperm-specific ribosome should greatly expand our understanding of ribosome function and tissue-specific regulation of protein expression pattern in mammals.

Ribosomal protein eL39 is important for maturation of the nascent polypeptide exit tunnel and proper protein folding during translation.

During translation, nascent polypeptide chains travel from the peptidyl transferase center through the nascent polypeptide exit tunnel (NPET) to emerge from 60S subunits. The NPET includes portions of five of the six 25S/5.8S rRNA domains and ribosomal proteins uL4, uL22, and eL39. Internal loops of uL4 and uL22 form the constriction sites of the NPET and are important for both assembly and function of ribosomes. Here, we investigated the roles of eL39 in tunnel construction, 60S biogenesis, and protein synthesis. We show that eL39 is important for proper protein folding during translation. Consistent with a delay in processing of 27S and 7S pre-rRNAs, eL39 functions in pre-60S assembly during middle nucleolar stages. Our biochemical assays suggest the presence of eL39 in particles at these stages, although it is not visualized in them by cryo-electron microscopy. This indicates that eL39 takes part in assembly even when it is not fully accommodated into the body of pre-60S particles. eL39 is also important for later steps of assembly, rotation of the 5S ribonucleoprotein complex, likely through long range rRNA interactions. Finally, our data strongly suggest the presence of alternative pathways of ribosome assembly, previously observed in the biogenesis of bacterial ribosomal subunits.

Cryo-EM structure of Mycobacterium tuberculosis 50S ribosomal subunit bound with clarithromycin reveals dynamic and specific interactions with macrolides

ABSTRACT Tuberculosis (TB) is the leading infectious disease caused by Mycobacterium tuberculosis (Mtb). Clarithromycin (CTY), an analog of erythromycin (ERY), is more potent against multidrug-resistance (MDR) TB. ERY and CTY were previously reported to bind to the nascent polypeptide exit tunnel (NPET) near peptidyl transferase center (PTC), but the only available CTY structure in complex with D. radiodurans (Dra) ribosome could be misinterpreted due to resolution limitation. To date, the mechanism of specificity and efficacy of CTY for Mtb remains elusive since the Mtb ribosome-CTY complex structure is still unknown. Here, we employed new sample preparation methods and solved the Mtb ribosome-CTY complex structure at 3.3Å with cryo-EM technique, where the crucial gate site A2062 (E. coli numbering) is located at the CTY binding site within NPET. Two alternative conformations of A2062, a novel syn-conformation as well as a swayed conformation bound with water molecule at interface, may play a role in coordinating the binding of specific drug molecules. The previously overlooked C–H hydrogen bond (H-bond) and π interaction may collectively contribute to the enhanced binding affinity. Together, our structure data provide a structural basis for the dynamic binding as well as the specificity of CTY and explain of how a single methyl group in CTY improves its potency, which provides new evidence to reveal previously unclear mechanism of translational modulation for future drug design and anti-TB therapy. Furthermore, our sample preparation method may facilitate drug discovery based on the complexes with low water solubility drugs by cryo-EM technique.

Structural insights into assembly of the ribosomal nascent polypeptide exit tunnel

The nascent polypeptide exit tunnel (NPET) is a major functional center of 60S ribosomal subunits. However, little is known about how the NPET is constructed during ribosome assembly. We utilized molecular genetics, biochemistry, and cryo-electron microscopy (cryo-EM) to investigate the functions of two NPET-associated proteins, ribosomal protein uL4 and assembly factor Nog1, in NPET assembly. Structures of mutant pre-ribosomes lacking the tunnel domain of uL4 reveal a misassembled NPET, including an aberrantly flexible ribosomal RNA helix 74, resulting in at least three different blocks in 60S assembly. Structures of pre-ribosomes lacking the C-terminal extension of Nog1 demonstrate that this extension scaffolds the tunnel domain of uL4 in the NPET to help maintain stability in the core of pre-60S subunits. Our data reveal that uL4 and Nog1 work together in the maturation of ribosomal RNA helix 74, which is required to ensure proper construction of the NPET and 60S ribosomal subunits.

Coupling of 5S RNP rotation with maturation of functional centers during large ribosomal subunit assembly

The protein composition and structure of assembling 60S ribosomal subunits undergo numerous changes as pre-ribosomes transition from the nucleolus to the nucleoplasm. This includes stable anchoring of the Rpf2 subcomplex containing 5S rRNA, rpL5, rpL11, Rpf2 and Rrs1, which initially docks onto the flexible domain V of rRNA at earlier stages of assembly. In this work, we tested the function of the C-terminal domain (CTD) of Rpf2 during these anchoring steps, by truncating this extension and assaying effects on middle stages of subunit maturation. The rpf2Δ255-344 mutation affects proper folding of rRNA helices H68-70 during anchoring of the Rpf2 subcomplex. In addition, several assembly factors (AFs) are absent from pre-ribosomes or in altered conformations. Consequently, major remodeling events fail to occur: rotation of the 5S RNP, maturation of the peptidyl transferase center (PTC) and the nascent polypeptide exit tunnel (NPET), and export of assembling subunits to the cytoplasm.

High-resolution crystal structures of ribosome-bound chloramphenicol and erythromycin provide the ultimate basis for their competition.

The 70S ribosome is a major target for antibacterial drugs. Two of the classical antibiotics, chloramphenicol (CHL) and erythromycin (ERY), competitively bind to adjacent but separate sites on the bacterial ribosome: the catalytic peptidyl transferase center (PTC) and the nascent polypeptide exit tunnel (NPET), respectively. The previously reported competitive binding of CHL and ERY might be due either to a direct collision of the two drugs on the ribosome or due to a drug-induced allosteric effect. Because of the resolution limitations, the available structures of these antibiotics in complex with bacterial ribosomes do not allow us to discriminate between these two possible mechanisms. In this work, we have obtained two crystal structures of CHL and ERY in complex with the Thermus thermophilus 70S ribosome at a higher resolution (2.65 and 2.89 Å, respectively) allowing unambiguous placement of the drugs in the electron density maps. Our structures provide evidence of the direct collision of CHL and ERY on the ribosome, which rationalizes the observed competition between the two drugs.

Intracellular Antimicrobial Peptides Targeting the Protein Synthesis Machinery.

While antimicrobial peptides (AMPs) are well-known for their disruptive effects on bacterial membranes, the mechanism of many intracellular AMPs is still being elucidated. In the recent years, it has been demonstrated that the subclass of proline-rich AMPs (PrAMPs) can pass through the bacterial membrane and kill bacteria by inhibiting protein synthesis. PrAMPs are a product of the innate immune system and are secreted in response to bacterial infection. So farPrAMPs have been identified in many arthropods, such as beetles, wasps, and flies, as well as some mammals, such as sheep, cows, and goats. PrAMPs show high potency against Gram-negative bacteria, while exhibiting low toxicity in eukaryotes, suggesting that they may represent a promising avenue for the development of future antimicrobial agents to combat the increase of multidrug-resistant bacterial pathogens. Structural and biochemical data have revealed the PrAMP binding sites on the ribosome as well as insight into their mechanisms of action. While the binding site of all so far investigated PrAMPs is situated within nascent polypeptide exit tunnel, the mechanism of action is distinct between class I and II PrAMPs. Specifically, class I PrAMPs, such as Bac7, Onc112, pyrrhocoricin, and metalnikowin, block the delivery of aa-tRNA by EF-Tu to the ribosomal A-site, whereas the class II PrAMPs, such as apidaecin 1b and Api137, act during translation termination and inhibit protein synthesis by trapping of release factors on the 70S ribosome following hydrolysis of the nascent polypeptide chain.

SecA mediates cotranslational targeting and translocation of an inner membrane protein.

Protein targeting to the bacterial plasma membrane was generally thought to occur via two major pathways: cotranslational targeting by signal recognition particle (SRP) and posttranslational targeting by SecA and SecB. Recently, SecA was found to also bind ribosomes near the nascent polypeptide exit tunnel, but the function of this SecA-ribosome contact remains unclear. In this study, we show that SecA cotranslationally recognizes the nascent chain of an inner membrane protein, RodZ, with high affinity and specificity. In vitro reconstitution and in vivo targeting assays show that SecA is necessary and sufficient to direct the targeting and translocation of RodZ to the bacterial plasma membrane in an obligatorily cotranslational mechanism. Sequence elements upstream and downstream of the RodZ transmembrane domain dictate nascent polypeptide selection by SecA instead of the SRP machinery. These findings identify a new route for the targeting of inner membrane proteins in bacteria and highlight the diversity of targeting pathways that enables an organism to accommodate diverse nascent proteins.

The 55S mammalian mitochondrial ribosome and its tRNA-exit region.

Mitochondria carry their own genetic material and gene-expression machinery, including ribosomes, which are responsible for synthesizing polypeptides that form essential components of the complexes involved in oxidative phosphorylation (or ATP generation) for the eukaryotic cell. Mitochondrial ribosomes (mitoribosomes) are quite divergent from cytoplasmic ribosomes in both composition and structure even as their main functional cores, such as the mRNA decoding and peptidyl transferase sites, are highly conserved. Remarkable progress has been made recently towards understanding the structure of mitoribosomes, by obtaining high-resolution cryo-electron microscopic (cryo-EM) maps. These studies confirm previous structural findings that had revealed that a significant reduction in size of ribosomal RNAs has caused topological changes in some of the functionally relevant regions, including the transfer RNA (tRNA)-binding sites and the nascent polypeptide-exit tunnel, within the structure of the mammalian mitoribosome. In addition, these studies provide unprecedented detailed views of the molecular architecture of those regions. In this review, we summarize the current state of knowledge of the structure of the mammalian mitoribosome and describe the molecular environment of its tRNA-exit region.

Structure and Assembly Pathway of the Ribosome Quality Control Complex

SummaryDuring ribosome-associated quality control, stalled ribosomes are split into subunits and the 60S-housed nascent polypeptides are poly-ubiquitinated by Listerin. How this low-abundance ubiquitin ligase targets rare stall-generated 60S among numerous empty 60S is unknown. Here, we show that Listerin specificity for nascent chain-60S complexes depends on nuclear export mediator factor (NEMF). The 3.6 Å cryo-EM structure of a nascent chain-containing 60S-Listerin-NEMF complex revealed that NEMF makes multiple simultaneous contacts with 60S and peptidyl-tRNA to sense nascent chain occupancy. Structural and mutational analyses showed that ribosome-bound NEMF recruits and stabilizes Listerin’s N-terminal domain, while Listerin’s C-terminal RWD domain directly contacts the ribosome to position the adjacent ligase domain near the nascent polypeptide exit tunnel. Thus, highly specific nascent chain targeting by Listerin is imparted by the avidity gained from a multivalent network of context-specific individually weak interactions, highlighting a new principle of client recognition during protein quality control.

Tagging truncated proteins with CAT tails

Protein Synthesis During the translation of a messenger RNA (mRNA) into protein, ribosomes can sometimes stall. Truncated proteins thus formed can be toxic to the cell and must be destroyed. Shen et al. show that the proteins Ltn1p and Rqc2p, subunits of the ribosome quality control complex, bind to the stalled and partially disassembled ribosome. Ltn1p, a ubiquitin ligase, binds near the nascent polypeptide exit tunnel on the ribosome, well placed to tag the truncated protein for destruction. The Rqc2p protein interacts with the transfer RNA binding sites on the partial ribosome and recruits alanine- and threonine-bearing tRNAs. Rqc2p then catalyzes the addition of these amino acids onto the unfinished protein, in the absence of both the fully assembled ribosome and mRNA. These so-called CAT tails may promote the heat shock response, which helps buffer against malformed proteins. Science , this issue p. [75][1] [1]: /lookup/doi/10.1126/science.1259724

Protein synthesis. Rqc2p and 60S ribosomal subunits mediate mRNA-independent elongation of nascent chains.

In Eukarya, stalled translation induces 40S dissociation and recruitment of the ribosome quality control complex (RQC) to the 60S subunit, which mediates nascent chain degradation. Here we report cryo-electron microscopy structures revealing that the RQC components Rqc2p (YPL009C/Tae2) and Ltn1p (YMR247C/Rkr1) bind to the 60S subunit at sites exposed after 40S dissociation, placing the Ltn1p RING (Really Interesting New Gene) domain near the exit channel and Rqc2p over the P-site transfer RNA (tRNA). We further demonstrate that Rqc2p recruits alanine- and threonine-charged tRNA to the A site and directs the elongation of nascent chains independently of mRNA or 40S subunits. Our work uncovers an unexpected mechanism of protein synthesis, in which a protein--not an mRNA--determines tRNA recruitment and the tagging of nascent chains with carboxy-terminal Ala and Thr extensions ("CAT tails").

Structure of a mitochondrial ribosome with minimal RNA

The Leishmania tarentolae mitochondrial ribosome (Lmr) is a minimal ribosomal RNA (rRNA)-containing ribosome. We have obtained a cryo-EM map of the Lmr. The map reveals several features that have not been seen in previously-determined structures of eubacterial or eukaryotic (cytoplasmic or organellar) ribosomes to our knowledge. Comparisons of the Lmr map with X-ray crystallographic and cryo-EM maps of the eubacterial ribosomes and a cryo-EM map of the mammalian mitochondrial ribosome show that (i) the overall structure of the Lmr is considerably more porous, (ii) the topology of the intersubunit space is significantly different, with fewer intersubunit bridges, but more tunnels, and (iii) several of the functionally-important rRNA regions, including the alpha-sarcin-ricin loop, have different relative positions within the structure. Furthermore, the major portions of the mRNA channel, the tRNA passage, and the nascent polypeptide exit tunnel contain Lmr-specific proteins, suggesting that the mechanisms for mRNA recruitment, tRNA interaction, and exiting of the nascent polypeptide in Lmr must differ markedly from the mechanisms deduced for ribosomes in other organisms. Our study identifies certain structural features that are characteristic solely of mitochondrial ribosomes and other features that are characteristic of both mitochondrial and chloroplast ribosomes (i.e., organellar ribosomes).

Distinct Mode of Interaction of a Novel Ketolide Antibiotic That Displays Enhanced Antimicrobial Activity

Ketolides represent the latest generation of macrolide antibiotics, displaying improved activities against some erythromycin-resistant strains, while maintaining their activity against erythromycin-susceptible ones. In this study, we present a new ketolide, K-1325, that carries an alkyl-aryl side chain at C-13 of the lactone ring. According to our genetic and biochemical studies, K-1325 binds within the nascent polypeptide exit tunnel, at a site previously described as the primary attachment site of all macrolide antibiotics. Compared with telithromycin, K-1325 displays enhanced antimicrobial activity against wild-type Escherichia coli strains, as well as against strains bearing the U2609C mutation in 23S rRNA. Chemical protection experiments showed that the alkyl-aryl side chain of K-1325 interacts specifically with helix 35 of 23S rRNA, a fact leading to an increased affinity of U2609C mutant ribosomes for the drug and rationalizing the enhanced effectiveness of this new ketolide.

Collective dynamics of the ribosomal tunnel revealed by elastic network modeling

The collective dynamics of the nascent polypeptide exit tunnel are investigated with the computationally efficient elastic network model using normal mode analysis. The calculated normal modes are considered individually and in linear combinations with different coefficients mimicking the phase angles between modes, in order to follow the mechanistic motions of tunnel wall residues. The low frequency fluctuations indicate three distinct regions along the tunnel-the entrance, the neck, and the exit-each having distinctly different domain motions. Generally, the lining of the entrance region moves in the exit direction, with the exit region having significantly larger motions, but in a perpendicular direction, whereas the confined neck region has rotational motions. Especially the universally conserved extensions of ribosomal proteins L4 and L22 located at the narrowest and mechanistically strategic region of tunnel undergo generally anti- or non-correlated motions, which may have an important role in nascent polypeptide gating mechanism. These motions appear to be sufficiently robust so as to be unaffected by the presence of a peptide chain in the tunnel.