Interplay between the Ribosomal Tunnel, Nascent Chain, and Macrolides Influences Drug Inhibition

An unbiased photo–cross-linking approach was used to probe the “molecular path” of a growing nascent Escherichia coli inner membrane protein (IMP) from the peptidyl transferase center to the surface of the ribosome. The nascent chain was initially in proximity to the ribosomal proteins L4 and L22 and subsequently contacted L23, which is indicative of progression through the ribosome via the main ribosomal tunnel. The signal recognition particle (SRP) started to interact with the nascent IMP and to target the ribosome–nascent chain complex to the Sec–YidC complex in the inner membrane when maximally half of the transmembrane domain (TM) was exposed from the ribosomal exit. The combined data suggest a flexible tunnel that may accommodate partially folded nascent proteins and parts of the SRP and SecY. Intraribosomal contacts of the nascent chain were not influenced by the presence of a functional TM in the ribosome.

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Ribosome binding induces repositioning of the signal recognition particle receptor on the translocon

The Journal of Cell Biology ◽

10.1083/jcb.201502103 ◽

2015 ◽

Vol 211 (1) ◽

pp. 91-104 ◽

Cited By ~ 24

Author(s):

Patrick Kuhn ◽

Albena Draycheva ◽

Andreas Vogt ◽

Narcis-Adrian Petriman ◽

Lukas Sturm ◽

...

Keyword(s):

Protein Targeting ◽

Signal Recognition Particle ◽

Resonance Energy Transfer ◽

Chain Complex ◽

Resonance Energy ◽

Endoplasmic Reticulum Membrane ◽

Signal Recognition ◽

Ribosomal Tunnel ◽

Nascent Chain

Cotranslational protein targeting delivers proteins to the bacterial cytoplasmic membrane or to the eukaryotic endoplasmic reticulum membrane. The signal recognition particle (SRP) binds to signal sequences emerging from the ribosomal tunnel and targets the ribosome-nascent-chain complex (RNC) to the SRP receptor, termed FtsY in bacteria. FtsY interacts with the fifth cytosolic loop of SecY in the SecYEG translocon, but the functional role of the interaction is unclear. By using photo-cross-linking and fluorescence resonance energy transfer measurements, we show that FtsY–SecY complex formation is guanosine triphosphate independent but requires a phospholipid environment. Binding of an SRP–RNC complex exposing a hydrophobic transmembrane segment induces a rearrangement of the SecY–FtsY complex, which allows the subsequent contact between SecY and ribosomal protein uL23. These results suggest that direct RNC transfer to the translocon is guided by the interaction between SRP and translocon-bound FtsY in a quaternary targeting complex.

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Functional ramifications of FRET-detected nascent chain folding far inside the membrane-bound ribosome

Biochemical Society Transactions ◽

10.1042/bst0320668 ◽

2004 ◽

Vol 32 (5) ◽

pp. 668-672 ◽

Cited By ~ 14

Author(s):

A.E. Johnson

Keyword(s):

Protein Trafficking ◽

Protein Biosynthesis ◽

Ribosomal Subunit ◽

Regulatory Control ◽

Secretory Proteins ◽

Induced Folding ◽

Ribosomal Tunnel ◽

Nascent Chain ◽

Membrane Bound ◽

Transmembrane Sequence

During protein biosynthesis, nascent protein chains are directed along a long narrow tunnel that spans the large ribosomal subunit. It has recently become clear that this structural feature has evolved to effect regulatory control over aspects of protein synthesis and protein trafficking. Since this control is nascent chain-specific, ribosomal components that form the tunnel must be involved in recognizing selected nascent proteins as they pass by. The present study focuses on one such situation in which nascent secretory proteins and membrane proteins are distinguished by the ribosome-induced folding of the latter's hydrophobic transmembrane sequence far inside the ribosomal tunnel and close to the peptidyltransferase centre.

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NAC functions as a modulator of SRP during the early steps of protein targeting to the endoplasmic reticulum

Molecular Biology of the Cell ◽

10.1091/mbc.e12-02-0112 ◽

2012 ◽

Vol 23 (16) ◽

pp. 3027-3040 ◽

Cited By ~ 41

Author(s):

Ying Zhang ◽

Uta Berndt ◽

Hanna Gölz ◽

Arlette Tais ◽

Stefan Oellerer ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Protein Targeting ◽

Signal Sequence ◽

Signal Recognition Particle ◽

Signal Recognition ◽

Signal Sequences ◽

Chain Complexes ◽

Ribosomal Tunnel ◽

Nascent Chain ◽

Combined Data

Nascent polypeptide-associated complex (NAC) was initially found to bind to any segment of the nascent chain except signal sequences. In this way, NAC is believed to prevent mistargeting due to binding of signal recognition particle (SRP) to signalless ribosome nascent chain complexes (RNCs). Here we revisit the interplay between NAC and SRP. NAC does not affect SRP function with respect to signalless RNCs; however, NAC does affect SRP function with respect to RNCs targeted to the endoplasmic reticulum (ER). First, early recruitment of SRP to RNCs containing a signal sequence within the ribosomal tunnel is NAC dependent. Second, NAC is able to directly and tightly bind to nascent signal sequences. Third, SRP initially displaces NAC from RNCs; however, when the signal sequence emerges further, trimeric NAC·RNC·SRP complexes form. Fourth, upon docking to the ER membrane NAC remains bound to RNCs, allowing NAC to shield cytosolically exposed nascent chain domains not only before but also during cotranslational translocation. The combined data indicate a functional interplay between NAC and SRP on ER-targeted RNCs, which is based on the ability of the two complexes to bind simultaneously to distinct segments of a single nascent chain.

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Protein quality control at the ribosome: focus on RAC, NAC and RQC

Essays in Biochemistry ◽

10.1042/ebc20160011 ◽

2016 ◽

Vol 60 (2) ◽

pp. 203-212 ◽

Cited By ~ 12

Author(s):

Martin Gamerdinger

Keyword(s):

Quality Control ◽

Protein Production ◽

Protein Quality ◽

Nascent Polypeptide ◽

Life And Death ◽

Ribosomal Tunnel ◽

Protein Biogenesis ◽

Nascent Chain ◽

Cellular Compartments ◽

Polypeptide Chains

The biogenesis of new polypeptides by ribosomes and their subsequent correct folding and localization to the appropriate cellular compartments are essential key processes to maintain protein homoeostasis. These complex mechanisms are governed by a repertoire of protein biogenesis factors that directly bind to the ribosome and chaperone nascent polypeptide chains as soon as they emerge from the ribosomal tunnel exit. This nascent chain ‘welcoming committee’ regulates multiple co-translational processes including protein modifications, folding, targeting and degradation. Acting at the front of the protein production line, these ribosome-associated protein biogenesis factors lead the way in the cellular proteostasis network to ensure proteome integrity. In this article, I focus on three different systems in eukaryotes that are critical for the maintenance of protein homoeostasis by controlling the birth, life and death of nascent polypeptide chains.

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A pair of non-optimal codons are necessary for the correct biosynthesis of the Aspergillus nidulans urea transporter, UreA

Royal Society Open Science ◽

10.1098/rsos.190773 ◽

2019 ◽

Vol 6 (11) ◽

pp. 190773 ◽

Cited By ~ 1

Author(s):

Manuel Sanguinetti ◽

Andrés Iriarte ◽

Sotiris Amillis ◽

Mónica Marín ◽

Héctor Musto ◽

...

Keyword(s):

Aspergillus Nidulans ◽

Signal Recognition Particle ◽

Signal Recognition ◽

Urea Transporter ◽

Translation Rate ◽

Synonymous Codons ◽

Optimal Codons ◽

Chain Complexes ◽

Ribosomal Tunnel ◽

Nascent Chain

In both prokaryotic and eukaryotic genomes, synonymous codons are unevenly used. Such differential usage of optimal or non-optimal codons has been suggested to play a role in the control of translation initiation and elongation, as well as at the level of transcription and mRNA stability. In the case of membrane proteins, codon usage has been proposed to assist in the establishment of a pause necessary for the correct targeting of the nascent chains to the translocon. By using as a model UreA, the Aspergillus nidulans urea transporter, we revealed that a pair of non-optimal codons encoding amino acids situated at the boundary between the N -terminus and the first transmembrane segment are necessary for proper biogenesis of the protein at 37°C. These codons presumably regulate the translation rate in a previously undescribed fashion, possibly contributing to the correct interaction of ureA -translating ribosome-nascent chain complexes with the signal recognition particle and/or other factors, while the polypeptide has not yet emerged from the ribosomal tunnel. Our results suggest that the presence of the pair of non-optimal codons would not be functionally important in all cellular conditions. Whether this mechanism would affect other proteins remains to be determined.

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Polytopic membrane protein folding at L17 in the ribosome tunnel initiates cyclical changes at the translocon

The Journal of Cell Biology ◽

10.1083/jcb.201103118 ◽

2011 ◽

Vol 195 (1) ◽

pp. 55-70 ◽

Cited By ~ 31

Author(s):

Pen-Jen Lin ◽

Candice G. Jongsma ◽

Martin R. Pool ◽

Arthur E. Johnson

Keyword(s):

Membrane Protein ◽

Conformational Changes ◽

Membrane Protein Folding ◽

Multiple Components ◽

Induced Folding ◽

Ribosomal Tunnel ◽

Nascent Chain ◽

Polytopic Membrane Protein ◽

Peptidyltransferase Center ◽

Compact Conformation

Multi-spanning membrane protein loops are directed alternately into the cytosol or ER lumen during cotranslational integration. Nascent chain exposure is switched after a newly synthesized transmembrane segment (TMS) enters the ribosomal tunnel. FRET measurements revealed that each TMS is initially extended, but folds into a compact conformation after moving 6–7 residues from the peptidyltransferase center, irrespective of loop size. The ribosome-induced folding of each TMS coincided with its photocrosslinking to ribosomal protein L17 and an inversion of compartmental exposure. This correlation indicates that successive TMSs fold and bind at a specific ribosomal tunnel site that includes L17, thereby triggering structural rearrangements of multiple components in and on both sides of the ER membrane, most likely via TMS-dependent L17 and/or rRNA conformational changes transmitted to the surface. Thus, cyclical changes at the membrane during integration are initiated by TMS folding, even though nascent chain conformation and location vary dynamically in the ribosome tunnel. Nascent chains therefore control their own trafficking.

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Molecular Mimicry of SecA and Signal Recognition Particle Binding to the Bacterial Ribosome

mBio ◽

10.1128/mbio.01317-19 ◽

2019 ◽

Vol 10 (4) ◽

Cited By ~ 5

Author(s):

Lara Knüpffer ◽

Clara Fehrenbach ◽

Kärt Denks ◽

Veronika Erichsen ◽

Narcis-Adrian Petriman ◽

...

Keyword(s):

Protein Transport ◽

Molecular Mimicry ◽

Signal Recognition Particle ◽

Transport Systems ◽

Secretory Proteins ◽

Signal Recognition ◽

Bacterial Protein ◽

Ribosomal Tunnel ◽

N Terminus ◽

Nascent Chain

ABSTRACT Bacteria execute a variety of protein transport systems for maintaining the proper composition of their different cellular compartments. The SecYEG translocon serves as primary transport channel and is engaged in transporting two different substrate types. Inner membrane proteins are cotranslationally inserted into the membrane after their targeting by the signal recognition particle (SRP). In contrast, secretory proteins are posttranslationally translocated by the ATPase SecA. Recent data indicate that SecA can also bind to ribosomes close to the tunnel exit. We have mapped the interaction of SecA with translating and nontranslating ribosomes and demonstrate that the N terminus and the helical linker domain of SecA bind to an acidic patch on the surface of the ribosomal protein uL23. Intriguingly, both also insert deeply into the ribosomal tunnel to contact the intratunnel loop of uL23, which serves as a nascent chain sensor. This binding pattern is remarkably similar to that of SRP and indicates an identical interaction mode of the two targeting factors with ribosomes. In the presence of a nascent chain, SecA retracts from the tunnel but maintains contact with the surface of uL23. Our data further demonstrate that ribosome and membrane binding of SecA are mutually exclusive, as both events depend on the N terminus of SecA. Our study highlights the enormous plasticity of bacterial protein transport systems and reveals that the discrimination between SRP and SecA substrates is already initiated at the ribosome. IMPORTANCE Bacterial protein transport via the conserved SecYEG translocon is generally classified as either cotranslational, i.e., when transport is coupled to translation, or posttranslational, when translation and transport are separated. We show here that the ATPase SecA, which is considered to bind its substrates posttranslationally, already scans the ribosomal tunnel for potential substrates. In the presence of a nascent chain, SecA retracts from the tunnel but maintains contact with the ribosomal surface. This is remarkably similar to the ribosome-binding mode of the signal recognition particle, which mediates cotranslational transport. Our data reveal a striking plasticity of protein transport pathways, which likely enable bacteria to efficiently recognize and transport a large number of highly different substrates within their short generation time.

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The ribosome modulates folding inside the ribosomal exit tunnel

10.1101/2020.06.30.180224 ◽

2020 ◽

Cited By ~ 1

Author(s):

Florian Wruck ◽

Pengfei Tian ◽

Renuka Kudva ◽

Robert B. Best ◽

Gunnar von Heijne ◽

...

Keyword(s):

Optical Tweezers ◽

Single Molecule ◽

Electrostatic Interactions ◽

Zinc Finger Domain ◽

Single Molecule Fret ◽

Ribosomal Tunnel ◽

Nascent Chain ◽

Folding Dynamics ◽

Exit Tunnel ◽

Dynamics Simulations

Proteins commonly fold cotranslationally on the ribosome, while the nascent chain emerges from the ribosomal tunnel. Protein domains that are sufficiently small can even fold while still located inside the tunnel. However, the effect of the tunnel on the folding dynamics of these domains is still not well understood. Here, we combine optical tweezers with single-molecule FRET and molecular dynamics simulations to investigate folding of the small zinc-finger domain ADR1a inside and at the vestibule of the ribosomal tunnel. The tunnel is found to accelerate folding and stabilize the folded state, reminiscent of the effects of chaperonins. However, a simple mechanism involving stabilization by confinement does not reproduce the results. Instead, it appears that electrostatic interactions between the protein and ribosome contribute to the observed folding acceleration and stabilization of ADR1a.

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Translation and folding of single proteins in real time

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1617873114 ◽

2017 ◽

Vol 114 (22) ◽

pp. E4399-E4407 ◽

Cited By ~ 39

Author(s):

Florian Wruck ◽

Alexandros Katranidis ◽

Knud H. Nierhaus ◽

Georg Büldt ◽

Martin Hegner

Keyword(s):

Protein Folding ◽

Amino Acid ◽

Amino Acid Sequence ◽

Real Time ◽

Electrostatic Interactions ◽

Tertiary Structure ◽

Chain Elongation ◽

Ribosomal Tunnel ◽

Cotranslational Folding ◽

Nascent Chain

Protein biosynthesis is inherently coupled to cotranslational protein folding. Folding of the nascent chain already occurs during synthesis and is mediated by spatial constraints imposed by the ribosomal exit tunnel as well as self-interactions. The polypeptide’s vectorial emergence from the ribosomal tunnel establishes the possible folding pathways leading to its native tertiary structure. How cotranslational protein folding and the rate of synthesis are linked to a protein’s amino acid sequence is still not well defined. Here, we follow synthesis by individual ribosomes using dual-trap optical tweezers and observe simultaneous folding of the nascent polypeptide chain in real time. We show that observed stalling during translation correlates with slowed peptide bond formation at successive proline sequence positions and electrostatic interactions between positively charged amino acids and the ribosomal tunnel. We also determine possible cotranslational folding sites initiated by hydrophobic collapse for an unstructured and two globular proteins while directly measuring initial cotranslational folding forces. Our study elucidates the intricate relationship among a protein’s amino acid sequence, its cotranslational nascent-chain elongation rate, and folding.

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