| 1. |
- Arenz, Stefan, et al.
(författare)
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The stringent factor RelA adopts an open conformation on the ribosome to stimulate ppGpp synthesis
- 2016
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 44:13, s. 6471-6481
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Tidskriftsartikel (refereegranskat)abstract
- Under stress conditions, such as nutrient starvation, deacylated tRNAs bound within the ribosomal A-site are recognized by the stringent factor RelA, which converts ATP and GTP/GDP to (p)ppGpp. The signaling molecules (p) ppGpp globally rewire the cellular transcriptional program and general metabolism, leading to stress adaptation. Despite the additional importance of the stringent response for regulation of bacterial virulence, antibiotic resistance and persistence, structural insight into how the ribosome and deacylated-tRNA stimulate RelA-mediated (p)ppGpp has been lacking. Here, we present a cryo-EM structure of RelA in complex with the Escherichia coli 70S ribosome with an average resolution of 3.7 angstrom and local resolution of 4 to > 10 angstrom for RelA. The structure reveals that RelA adopts a unique 'open' conformation, where the C-terminal domain (CTD) is intertwined around an A/T-like tRNA within the intersubunit cavity of the ribosome and the N-terminal domain (NTD) extends into the solvent. We propose that the open conformation of RelA on the ribosome relieves the autoinhibitory effect of the CTD on the NTD, thus leading to stimulation of (p)ppGpp synthesis by RelA.
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| 2. |
- Ban, Nenad, et al.
(författare)
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A new system for naming ribosomal proteins.
- 2014
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Ingår i: Current Opinion in Structural Biology. - : Elsevier BV. - 1879-033X .- 0959-440X. ; 24, s. 165-169
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Tidskriftsartikel (refereegranskat)abstract
- A system for naming ribosomal proteins is described that the authors intend to use in the future. They urge others to adopt it. The objective is to eliminate the confusion caused by the assignment of identical names to ribosomal proteins from different species that are unrelated in structure and function. In the system proposed here, homologous ribosomal proteins are assigned the same name, regardless of species. It is designed so that new names are similar enough to old names to be easily recognized, but are written in a format that unambiguously identifies them as 'new system' names.
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| 3. |
- Cheng, Kimberley
(författare)
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Single-particle cryo-electron microscopy of macromolecular assemblies
- 2009
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- In this thesis, single-particle cryo-electron microscopy (cryo-EM) was used to study the structure of three macromolecular assemblies: the two hemocyanin isoforms from Rapana thomasiana, the Pyrococcus furiosus chaperonin, and the ribosome from Escherichia coli. Hemocyanins are large respiratory proteins in arthropods and molluscs. Most molluscan hemocyanins exist as two distinct isoforms composed of related polypeptides. In most species the two isoforms differ in terms of their oligomeric stability, and thus we set out to investigate the two Rapana thomasiana hemocyanins (RtH) in order to explain this behaviour. Our findings showed that the two RtHio forms are identical at the experimental resolution. Furthermore, three previously unreported connections that most likely contribute to the oligomeric stability were identified. Chaperonins are double-ring protein complexes that assist the folding process of nascent, non-native polypeptide chains. The chaperonin from the hyperthermophilic archaea Pyrococcus furiosus belongs to Group II chaperonins, and unlike most othergroup II chaperonins it appears to be homo-oligomeric. The 3D reconstruction of the Pyrococcus furiousus chaperonin revealed a di-octameric structure in a partially closed/open state, something in between the closed folding-active state and the open substrate-accepting state. The ribosome is the molecular machine where protein synthesis takes place. In bacteria there is a unique RNA molecule called transfer-messenger RNA (tmRNA) that together with its helper protein SmpB rescues ribosomes trapped on defective messenger RNAs (mRNAs) through a process called trans-translation. tmRNA is about 4 times the size of a normal tRNA, and it is composed of a tRNA-like domain (TLD) that is connected to the mRNA-like domain (MLD) by several pseudoknots (PKs) and RNA helices. During trans-translation, tmRNA utilize its TLD to receive the incomplete polypeptide from the peptidyl-tRNA in the ribosomal P site of the stalledribosome. Subsequently, its MLD is used to tag the incomplete polypeptide with adegradation signal. When tmRNA enters a stalled ribosome the MLD and pseudoknots form a highly structured arc that encircles the beak of the small ribosomal subunit. Byutilizing maximum-likelihood based methods for heterogeneity analysis we could observe the Escherichia coli ribosome in a number of different tmRNA·SmpB-boundstates. The cryo-EM map of the post-accommodated state revealed that the TLD·SmpBpart of the tmRNA·SmpB complex mimics native tRNAs in the A site of stalled ribosomes. The density map also showed that the tmRNA arc remains well structuredand that it is still attached to the beak of the small ribosomal subunit. Thereconstructions of the double-translocation tmRNA-bound ribosome complex showed that the pseudoknots of tmRNA still form an arc, and that they are located at positions similar to the ones assigned for the pseudoknots in the post-accommodated state. In addition, the tmRNA arc exists in two states; one stable and highly structured and another more flexible and disorganized.
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| 4. |
- Marino, Jacopo, et al.
(författare)
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Small protein domains fold inside the ribosome exit tunnel
- 2016
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Ingår i: FEBS Letters. - : Wiley. - 0014-5793 .- 1873-3468. ; 590:5, s. 655-660
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Tidskriftsartikel (refereegranskat)abstract
- Cotranslational folding of small protein domains within the ribosome exit tunnel may be an important cellular strategy to avoid protein misfolding. However, the pathway of cotranslational folding has so far been described only for a few proteins, and therefore, it is unclear whether folding in the ribosome exit tunnel is a common feature for small protein domains. Here, we have analyzed nine small protein domains and determined at which point during translation their folding generates sufficient force on the nascent chain to release translational arrest by the SecM arrest peptide, both in vitro and in live E. coli cells. We find that all nine protein domains initiate folding while still located well within the ribosome exit tunnel.
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| 5. |
- Nilsson, Ola B., et al.
(författare)
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Cotranslational folding of spectrin domains via partially structured states
- 2017
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Ingår i: Nature Structural & Molecular Biology. - : Springer Science and Business Media LLC. - 1545-9993 .- 1545-9985. ; 24:3, s. 221-225
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Tidskriftsartikel (refereegranskat)abstract
- How do the key features of protein folding, elucidated from studies on native, isolated proteins, manifest in cotranslational folding on the ribosome? Using a well-characterized family of homologous α-helical proteins with a range of biophysical properties, we show that spectrin domains can fold vectorially on the ribosome and may do so via a pathway different from that of the isolated domain. We use cryo-EM to reveal a folded or partially folded structure, formed in the vestibule of the ribosome. Our results reveal that it is not possible to predict which domains will fold within the ribosome on the basis of the folding behavior of isolated domains; instead, we propose that a complex balance of the rate of folding, the rate of translation and the lifetime of folded or partly folded states will determine whether folding occurs cotranslationally on actively translating ribosomes.
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| 6. |
- Nilsson, Ola B., et al.
(författare)
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Cotranslational Protein Folding inside the Ribosome Exit Tunnel
- 2015
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Ingår i: Cell Reports. - : Elsevier BV. - 2211-1247. ; 12:10, s. 1533-1540
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Tidskriftsartikel (refereegranskat)abstract
- At what point during translation do proteins fold? It is well established that proteins can fold cotranslationally outside the ribosome exit tunnel, whereas studies of folding inside the exit tunnel have so far detected only the formation of helical secondary structure and collapsed or partially structured folding intermediates. Here, using a combination of co-translational nascent chain force measurements, inter-subunit fluorescence resonance energy transfer studies on single translating ribosomes, molecular dynamics simulations, and cryoelectron microscopy, we show that a small zinc-finger domain protein can fold deep inside the vestibule of the ribosome exit tunnel. Thus, for small protein domains, the ribosome itself can provide the kind of sheltered folding environment that chaperones provide for larger proteins.
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| 7. |
- Shanmuganathan, Vivekanandan, et al.
(författare)
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Structural and mutational analysis of the ribosome-arresting human XBP1u
- 2019
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Ingår i: eLIFE. - 2050-084X. ; 8
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Tidskriftsartikel (refereegranskat)abstract
- XBP1u, a central component of the unfolded protein response (UPR), is a mammalian protein containing a functionally critical translational arrest peptide (AP). Here, we present a 3 angstrom cryo-EM structure of the stalled human XBP1u AP. It forms a unique turn in the ribosomal exit tunnel proximal to the peptidyl transferase center where it causes a subtle distortion, thereby explaining the temporary translational arrest induced by XBP1u. During ribosomal pausing the hydrophobic region 2 (HR2) of XBP1u is recognized by SRP, but fails to efficiently gate the Sec61 translocon. An exhaustive mutagenesis scan of the XBP1u AP revealed that only 8 out of 20 mutagenized positions are optimal; in the remaining 12 positions, we identify 55 different mutations increase the level of translational arrest. Thus, the wildtype XBP1u AP induces only an intermediate level of translational arrest, allowing efficient targeting by SRP without activating the Sec61 channel.
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| 8. |
- Su, Ting, et al.
(författare)
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Structural basis of L-tryptophan-dependent inhibition of release factor 2 by the TnaC arrest peptide
- 2021
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 49:16, s. 9539-9547
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Tidskriftsartikel (refereegranskat)abstract
- In Escherichia coli, elevated levels of free l-tryptophan (l-Trp) promote translational arrest of the TnaC peptide by inhibiting its termination. However, the mechanism by which translation-termination by the UGA-specific decoding release factor 2 (RF2) is inhibited at the UGA stop codon of stalled TnaC-ribosome-nascent chain complexes has so far been ambiguous. This study presents cryo-EM structures for ribosomes stalled by TnaC in the absence and presence of RF2 at average resolutions of 2.9 and 3.5 Å, respectively. Stalled TnaC assumes a distinct conformation composed of two small α-helices that act together with residues in the peptide exit tunnel (PET) to coordinate a single L-Trp molecule. In addition, while the peptidyl-transferase center (PTC) is locked in a conformation that allows RF2 to adopt its canonical position in the ribosome, it prevents the conserved and catalytically essential GGQ motif of RF2 from adopting its active conformation in the PTC. This explains how translation of the TnaC peptide effectively allows the ribosome to function as a L-Trp-specific small-molecule sensor that regulates the tnaCAB operon.
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| 9. |
- Su, Ting, et al.
(författare)
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The force-sensing peptide VemP employs extreme compaction and secondary structure formation to induce ribosomal stalling
- 2017
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Ingår i: eLIFE. - 2050-084X. ; 6
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Tidskriftsartikel (refereegranskat)abstract
- Interaction between the nascent polypeptide chain and the ribosomal exit tunnel can modulate the rate of translation and induce translational arrest to regulate expression of downstream genes. The ribosomal tunnel also provides a protected environment for initial protein folding events. Here, we present a 2.9 angstrom cryo-electron microscopy structure of a ribosome stalled during translation of the extremely compacted VemP nascent chain. The nascent chain forms two a-helices connected by an a-turn and a loop, enabling a total of 37 amino acids to be observed within the first 50-55 angstrom of the exit tunnel. The structure reveals how a-helix formation directly within the peptidyltransferase center of the ribosome interferes with aminoacyl-tRNA accommodation, suggesting that during canonical translation, a major role of the exit tunnel is to prevent excessive secondary structure formation that can interfere with the peptidyltransferase activity of the ribosome.
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| 10. |
- Tian, Pengfei, et al.
(författare)
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Folding pathway of an Ig domain is conserved on and off the ribosome
- 2018
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 115:48, s. E11284-E11293
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Tidskriftsartikel (refereegranskat)abstract
- Proteins that fold cotranslationally may do so in a restricted configurational space, due to the volume occupied by the ribosome. How does this environment, coupled with the close proximity of the ribosome, affect the folding pathway of a protein? Previous studies have shown that the cotranslational folding process for many proteins, including small, single domains, is directly affected by the ribosome. Here, we investigate the cotranslational folding of an all-beta Ig domain, titin I27. Using an arrest peptide-based assay and structural studies by cryo-EM, we show that I27 folds in the mouth of the ribosome exit tunnel. Simulations that use a kinetic model for the force dependence of escape from arrest accurately predict the fraction of folded protein as a function of length. We used these simulations to probe the folding pathway on and off the ribosome. Our simulations-which also reproduce experiments on mutant forms of I27-show that I27 folds, while still sequestered in the mouth of the ribosome exit tunnel, by essentially the same pathway as free I27, with only subtle shifts of critical contacts from the C to the N terminus.
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