Exploring the capacity of trigger factor to function as a shield for ribosome bound polypeptide chains

Tomic, Sladjana; Johnson, Arthur E.; Hartl, F. Ulrich; Etchells, Stephanie A.

doi:10.1016/j.febslet.2005.11.050

Cited by 35 publications

(42 citation statements)

References 19 publications

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“…6 A-D and Table 3). These results can explain previous observations of both increased protection from limited proteolysis and increased peak dispersion as the distance from the PTC increases (12,(25)(26)(27), suggesting that the increased protection is likely due to changes in global stability and not to interactions with the ribosome or changes in native state dynamics.…”

Section: Resultssupporting

confidence: 82%

Quantitative determination of ribosome nascent chain stability

Samelson

Jensen

Soto

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

133

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Accurate protein folding is essential for proper cellular and organismal function. In the cell, protein folding is carefully regulated; changes in folding homeostasis (proteostasis) can disrupt many cellular processes and have been implicated in various neurodegenerative diseases and other pathologies. For many proteins, the initial folding process begins during translation while the protein is still tethered to the ribosome; however, most biophysical studies of a protein's energy landscape are carried out in isolation under idealized, dilute conditions and may not accurately report on the energy landscape in vivo. Thus, the energy landscape of ribosome nascent chains and the effect of the tethered ribosome on nascent chain folding remain unclear. Here we have developed a general assay for quantitatively measuring the folding stability of ribosome nascent chains, and find that the ribosome exerts a destabilizing effect on the polypeptide chain. This destabilization decreases as a function of the distance away from the peptidyl transferase center. Thus, the ribosome may add an additional layer of robustness to the protein-folding process by avoiding the formation of stable partially folded states before the protein has completely emerged from the ribosome.protein folding | cotranslational folding | pulse proteolysis | protein stability P roper protein folding is necessary for the function of all cells, and changes in cellular protein folding capacity can lead to cell death and disease (1). For more than 50 years, experimental studies have probed the physical properties of proteins, paving the way for incredible advances in protein design and protein structure prediction, as well as for understanding the first principles of protein folding (2, 3). These in vitro studies, however, do not necessarily recapitulate the folding process in vivo, including the constraints that the ribosome imposes on the emerging nascent chain during translation (4).In the cell, proteins are synthesized by the ribosome one amino acid at a time on a time scale that is slower than most in vitro protein folding rates (5). Thus, the emerging chain has time to explore conformational space and adopt structured conformations before its entire sequence has been synthesized (6). This vectorial process, and the proximity of the ribosome itself, can modulate the emerging chain's energy landscape in ways that are just beginning to be appreciated. Translation can affect folding efficiency, enable the population of intermediates that are not revealed during offribosome studies, and even determine the final conformational fate of the nascent protein (6-10). To understand the folding process in vivo, general rules and biophysical mechanisms for these modulations of the emerging chain's energy landscape need to be elucidated.To unravel the in vivo folding landscape, we need to interrogate the energetics and dynamics of ribosome-bound nascent chains in a manner analogous to studies of the in vitro folding process. The challenge, however, is that standard ...

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Section: Resultssupporting

confidence: 82%

Quantitative determination of ribosome nascent chain stability

Samelson

Jensen

Soto

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

133

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“…TF(FRK/AAA), in which residues 44 -46 of the ribosome binding site of TF have been changed to alanines, has a strongly reduced ability to bind ribosomes (6). When TF326(FRK/AAA)-NBD was present during translation, no significant increase in NBD fluorescence was observed ( (34). Thus, our measurements with fluorescence labeled TF distinguish between ribosome-associated TF being merely in close proximity to the nascent chain and actual binding.…”

Section: Monitoring the Interaction Of Tf With Nascent Chains Duringmentioning

confidence: 81%

“…FtsQ was amplified from E. coli genomic DNA and cloned into pCDF1b vector under the T7 promoter resulting in pCDF1b-FtsQ. The nucleotide sequence of Luc encoding residues 87-100 was amplified from pET3a-Luc by PCR and ligated inframe at the region encoding residue 87 of ␣-synuclein in the plasmid pSTS500-␣-synuclein (34). The resulting plasmid was named pSTS500-␣-Syn (Luc).…”

Section: Methodsmentioning

confidence: 99%

“…3C). This could suggest that only a fraction of nascent chains are in close proximity to position 150, because the PPIase domain acts only as a secondary site for nascent chain binding (16,17,24,34). The dissociation of TF376-NBD from FL Luc was also biphasic, with t1 ⁄ 2 values of 8 Ϯ 2 and 88 Ϯ 23 s, respectively ( Fig.…”

Section: Monitoring the Interaction Of Tf With Nascent Chains Duringmentioning

confidence: 98%

“…We employed real-time fluorescence spectroscopy to monitor the interactions of TF with newly emerging nascent chains during translation. Firefly Luc was initially chosen as a model substrate for these experiments, based on previous observations indicating the high affinity of TF for Luc RNCs (16,17,34). TF was labeled at its substrate binding sites with the fluorophore NBD, which has previously been used to study the binding of signal recognition particle (SRP) to RNCs (37).…”

Section: Monitoring the Interaction Of Tf With Nascent Chains Duringmentioning

confidence: 99%

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Versatility of Trigger Factor Interactions with Ribosome-Nascent Chain Complexes

Lakshmipathy

Gupta²,

Pinkert

et al. 2010

Journal of Biological Chemistry

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Trigger factor (TF) is the first molecular chaperone that interacts with nascent chains emerging from bacterial ribosomes. TF is a modular protein, consisting of an N-terminal ribosome binding domain, a PPIase domain, and a C-terminal domain, all of which participate in polypeptide binding. To directly monitor the interactions of TF with nascent polypeptide chains, TF variants were site-specifically labeled with an environmentally sensitive NBD fluorophore. We found a marked increase in TF-NBD fluorescence during translation of firefly luciferase (Luc) chains, which expose substantial regions of hydrophobicity, but not with nascent chains lacking extensive hydrophobic segments. TF remained associated with Luc nascent chains for 111 ؎ 7 s, much longer than it remained bound to the ribosomes (t1 ⁄ 2 ϳ 10 -14 s). Thus, multiple TF molecules can bind per nascent chain during translation. The Escherichia coli cytosolic proteome was classified into predicted weak and strong interactors for TF, based on the occurrence of continuous hydrophobic segments in the primary sequence. The residence time of TF on the nascent chain generally correlated with the presence of hydrophobic regions and the capacity of nascent chains to bury hydrophobicity. Interestingly, TF bound the signal sequence of a secretory protein, pOmpA, but not the hydrophobic signal anchor sequence of the inner membrane protein FtsQ. On the other hand, proteins lacking linear hydrophobic segments also recruited TF, suggesting that TF can recognize hydrophobic surface features discontinuous in sequence. Moreover, TF retained significant affinity for the folded domain of the positively charged, ribosomal protein S7, indicative of an alternative mode of TF action. Thus, unlike other chaperones, TF appears to employ multiple mechanisms to interact with a wide range of substrate proteins.

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Protein Misfolding Diseases

Kolstoe

Wood

2010

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Exploring the capacity of trigger factor to function as a shield for ribosome bound polypeptide chains

Cited by 35 publications

References 19 publications

Quantitative determination of ribosome nascent chain stability

Quantitative determination of ribosome nascent chain stability

Versatility of Trigger Factor Interactions with Ribosome-Nascent Chain Complexes

Protein Misfolding Diseases

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