Site-Specific Fluorescent Labeling of Nascent Proteins on the Translating Ribosome

Saraogi, Ishu; Zhang, Dawei; Chandrasekaran, Sandhya; Shan, Shu‐ou

doi:10.1021/ja206626g

Cited by 39 publications

(53 citation statements)

References 28 publications

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“…To this end, we used amber suppression technology to incorporate a fluorescent nonnatural amino acid, 7-hydroxycoumaryl ethylglycine (Cm), into the nascent polypeptide downstream of the signal sequence ( Fig. 1 A and B, asterisks) (48). When paired with SRP labeled with BODIPY FL at residue 421 in the Ffh M domain, efficient Förster resonance energy transfer (FRET) was observed (48), providing a highly specific and sensitive assay to report on the interaction of SRP with nascent polypeptides on RNC.…”

Section: Resultsmentioning

confidence: 99%

“…Short-chain and long-chain RNCs contain 80-85 and 130-135 amino acids, respectively, between the N terminus of signal sequence and the peptidyl transferase center of ribosome. RNCs were affinity-purified via the strep 3 tag on the nascent polypeptide, as described (41,48,78). The quality of RNC was documented in ref.…”

Section: Methodsmentioning

confidence: 99%

“…Amber suppression technology to incorporate Cm into the nascent polypeptide is described in detail in ref. 48. Cotranslational Protein Targeting and Translocation Assay.…”

Section: Methodsmentioning

confidence: 99%

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Regulation by a chaperone improves substrate selectivity during cotranslational protein targeting

Ariosa

Lee

Wang

et al. 2015

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

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The ribosome exit site is a crowded environment where numerous factors contact nascent polypeptides to influence their folding, localization, and quality control. Timely and accurate selection of nascent polypeptides into the correct pathway is essential for proper protein biogenesis. To understand how this is accomplished, we probe the mechanism by which nascent polypeptides are accurately sorted between the major cotranslational chaperone trigger factor (TF) and the essential cotranslational targeting machinery, signal recognition particle (SRP). We show that TF regulates SRP function at three distinct stages, including binding of the translating ribosome, membrane targeting via recruitment of the SRP receptor, and rejection of ribosome-bound nascent polypeptides beyond a critical length. Together, these mechanisms enhance the specificity of substrate selection into both pathways. Our results reveal a multilayered mechanism of molecular interplay at the ribosome exit site, and provide a conceptual framework to understand how proteins are selected among distinct biogenesis machineries in this crowded environment.signal recognition particle | trigger factor | ribosome | protein biogenesis | GTPases P roper protein biogenesis is a prerequisite for the maintenance of a functional proteome. Accumulating data indicate that this process begins at the ribosome exit site, where many protein biogenesis machineries can interact and gain access to the nascent polypeptide. This includes chaperones (1-5) such as trigger factor (TF) (1, 4, 6, 7), Hsp70, and the nascent polypeptide-associated complex (8-13); modification enzymes (10, 14-16) such as N-acetyl transferase, methionine aminopeptidase, and arginyl transferase; protein-targeting and translocation machineries such as signal recognition particle (SRP) (17-20), SecA (21), the SecYEG (or Sec61p) (22, 23) and YidC translocases (24,25), and the ribosome-bound quality control complex (26)(27)(28)(29)(30). Engagement of these factors with nascent polypeptides influences their folding, assembly, localization, processing, and quality control. Within seconds after the nascent polypeptide emerges from the ribosomal exit tunnel, it must engage the correct set of factors and thus commit to the proper biogenesis pathway. How this is accomplished in the crowded environment at the ribosome exit site is an emerging question. In this work, we address this question by deciphering how nascent proteins are selected between two major protein biogenesis machineries in bacteria, SRP and TF.SRP is a universally conserved ribonucleoprotein complex responsible for the cotranslational targeting of proteins to the eukaryotic endoplasmic reticulum (ER), or the bacterial plasma membrane (31). SRP recognizes ribosome-nascent chain complexes (termed RNC or cargo) carrying strong signal sequences and delivers them to the SecYEG or YidC translocation machinery on the target membrane. SRP binds RNC via two interactions: a helical N domain in the SRP54 protein (called Ffh in bacteria) binds the ribosoma...

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Regulation by a chaperone improves substrate selectivity during cotranslational protein targeting

Ariosa

Lee

Wang

et al. 2015

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

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“…Although in principle, the kinetics of any of the steps in the targeting pathway, including cargo binding, delivery, and unloading (Fig. 7, k 1 , k 2 , and k 3 , respectively) could be slower with suboptimal signal sequences, SRP-cargo binding is a relatively fast process (k 1 ϭ ϳ4 ϫ 10 6 M Ϫ1 s Ϫ1 ) (49) and not strongly dependent on the signal sequence. 3 In contrast, stable assembly of the SRPFtsY complex, which mediates the delivery of cargo (k 2 ), differs up to 10 3 -fold between strong and weak signal sequences (15,17).…”

Section: Discussionmentioning

confidence: 99%

Translation Elongation Regulates Substrate Selection by the Signal Recognition Particle

Zhang

Shan

2012

Journal of Biological Chemistry

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Background: SRP serves as a paradigm for understanding the molecular basis of protein localization. Results: Varying translation elongation rates changes the stringency of substrate selection by the SRP. Conclusion: Kinetic competition with ongoing protein synthesis regulates the fidelity of SRP. Significance: Unraveling mechanisms that govern the fidelity of protein localization is essential for understanding this fundamental cellular process.

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“…Get3, Get4/5, Get1CD, and intein-cpSRP43 were expressed and purified as described (10,12,14,28). E. coli S30 lysate and T7 RNA polymerase were prepared as described (12,37). Microsomes were prepared from Δget3 yeast cells as described (14,38,39).…”

Section: Methodsmentioning

confidence: 99%

A Protean Clamp Guides Membrane Targeting of Tail-Anchored Proteins

Shan

Chio

2018

Biophysical Journal

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Site-Specific Fluorescent Labeling of Nascent Proteins on the Translating Ribosome

Cited by 39 publications

References 28 publications

Regulation by a chaperone improves substrate selectivity during cotranslational protein targeting

Regulation by a chaperone improves substrate selectivity during cotranslational protein targeting

Translation Elongation Regulates Substrate Selection by the Signal Recognition Particle

A Protean Clamp Guides Membrane Targeting of Tail-Anchored Proteins

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