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Interplay of signal recognition particle and trigger factor at L23 near the nascent chain exit site on the Escherichia coli ribosome.

Ullers RS, Houben EN, Raine A, ten Hagen-Jongman CM, Ehrenberg M, Brunner J, Oudega B, Harms N, Luirink J - J. Cell Biol. (2003)

Bottom Line: Both TF and SRP were found to interact with the SA with partially overlapping binding specificity.In addition, extensive contacts with L23 and L29 were detected.Both purified TF and SRP could be cross-linked to L23 on nontranslating ribosomes with a competitive advantage for SRP.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Microbiology, Vrije Universiteit, 1081 HV Amsterdam, The Netherlands.

ABSTRACT
As newly synthesized polypeptides emerge from the ribosome, they interact with chaperones and targeting factors that assist in folding and targeting to the proper location in the cell. In Escherichia coli, the chaperone trigger factor (TF) binds to nascent polypeptides early in biosynthesis facilitated by its affinity for the ribosomal proteins L23 and L29 that are situated around the nascent chain exit site on the ribosome. The targeting factor signal recognition particle (SRP) interacts specifically with the signal anchor (SA) sequence in nascent inner membrane proteins (IMPs). Here, we have used photocross-linking to map interactions of the SA sequence in a short, in vitro-synthesized, nascent IMP. Both TF and SRP were found to interact with the SA with partially overlapping binding specificity. In addition, extensive contacts with L23 and L29 were detected. Both purified TF and SRP could be cross-linked to L23 on nontranslating ribosomes with a competitive advantage for SRP. The results suggest a role for L23 in the targeting of IMPs as an attachment site for TF and SRP that is close to the emerging nascent chain.

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Scanning photocross-linking of nascent 77FtsQ. (A) Schematic representation of the position of (Tmd)Phe in nascent 77FtsQ. (B) In vitro translation of nascent 77FtsQTAG mutants was performed in the presence of (Tmd)Phe-tRNASup. After translation, samples were irradiated with UV light to induce cross-linking, and the ribosome–nascent chain complexes were purified and analyzed by SDS-PAGE. UV-irradiated ribosome–nascent chain complexes of 77FtsQTAG27 and 77FtsQTAG40 were immunoprecipitated as indicated. (C) Quantification of Ffh, TF, and L23 cross-linking adducts. The highest value for cross-linking efficiency was taken as 100%.
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fig1: Scanning photocross-linking of nascent 77FtsQ. (A) Schematic representation of the position of (Tmd)Phe in nascent 77FtsQ. (B) In vitro translation of nascent 77FtsQTAG mutants was performed in the presence of (Tmd)Phe-tRNASup. After translation, samples were irradiated with UV light to induce cross-linking, and the ribosome–nascent chain complexes were purified and analyzed by SDS-PAGE. UV-irradiated ribosome–nascent chain complexes of 77FtsQTAG27 and 77FtsQTAG40 were immunoprecipitated as indicated. (C) Quantification of Ffh, TF, and L23 cross-linking adducts. The highest value for cross-linking efficiency was taken as 100%.

Mentions: We have analyzed the molecular environment of a short nascent IMP in the E. coli cytosol using a scanning in vitro photocross-linking approach. FtsQ, a bitopic type II IMP, was synthesized from truncated mRNA to a length of 77 amino acids in a cell- and membrane-free E. coli extract. At this nascent chain length, the majority of the SA sequence is expected to be exposed outside the ribosome (Fig. 1 A). Previous studies have indicated that 77FtsQ represents a short targeting intermediate of FtsQ (Urbanus et al., 2001). It is efficiently recognized by the Sec-translocon in the inner membrane, whereas a truncate that is seven residues shorter is defective in targeting. Consequently, it is expected that 77FtsQ interacts in the cytosol with factors that force the decision for cotranslational targeting to the membrane. A single stop codon (TAG) was introduced at positions 25–43 and 49 in the SA sequence and at positions 10 and 24 in the flanking hydrophilic region of 77FtsQ (Fig. 1 A). The TAG codons were suppressed during in vitro synthesis by the addition of (Tmd)Phe-tRNASup, a suppressor tRNA that carries a photoreactive probe (Brunner, 1996). The translation mixture also contained [35S]methionine to label the nascent chains. After translation, the samples were irradiated with UV light to induce cross-linking.


Interplay of signal recognition particle and trigger factor at L23 near the nascent chain exit site on the Escherichia coli ribosome.

Ullers RS, Houben EN, Raine A, ten Hagen-Jongman CM, Ehrenberg M, Brunner J, Oudega B, Harms N, Luirink J - J. Cell Biol. (2003)

Scanning photocross-linking of nascent 77FtsQ. (A) Schematic representation of the position of (Tmd)Phe in nascent 77FtsQ. (B) In vitro translation of nascent 77FtsQTAG mutants was performed in the presence of (Tmd)Phe-tRNASup. After translation, samples were irradiated with UV light to induce cross-linking, and the ribosome–nascent chain complexes were purified and analyzed by SDS-PAGE. UV-irradiated ribosome–nascent chain complexes of 77FtsQTAG27 and 77FtsQTAG40 were immunoprecipitated as indicated. (C) Quantification of Ffh, TF, and L23 cross-linking adducts. The highest value for cross-linking efficiency was taken as 100%.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199365&req=5

fig1: Scanning photocross-linking of nascent 77FtsQ. (A) Schematic representation of the position of (Tmd)Phe in nascent 77FtsQ. (B) In vitro translation of nascent 77FtsQTAG mutants was performed in the presence of (Tmd)Phe-tRNASup. After translation, samples were irradiated with UV light to induce cross-linking, and the ribosome–nascent chain complexes were purified and analyzed by SDS-PAGE. UV-irradiated ribosome–nascent chain complexes of 77FtsQTAG27 and 77FtsQTAG40 were immunoprecipitated as indicated. (C) Quantification of Ffh, TF, and L23 cross-linking adducts. The highest value for cross-linking efficiency was taken as 100%.
Mentions: We have analyzed the molecular environment of a short nascent IMP in the E. coli cytosol using a scanning in vitro photocross-linking approach. FtsQ, a bitopic type II IMP, was synthesized from truncated mRNA to a length of 77 amino acids in a cell- and membrane-free E. coli extract. At this nascent chain length, the majority of the SA sequence is expected to be exposed outside the ribosome (Fig. 1 A). Previous studies have indicated that 77FtsQ represents a short targeting intermediate of FtsQ (Urbanus et al., 2001). It is efficiently recognized by the Sec-translocon in the inner membrane, whereas a truncate that is seven residues shorter is defective in targeting. Consequently, it is expected that 77FtsQ interacts in the cytosol with factors that force the decision for cotranslational targeting to the membrane. A single stop codon (TAG) was introduced at positions 25–43 and 49 in the SA sequence and at positions 10 and 24 in the flanking hydrophilic region of 77FtsQ (Fig. 1 A). The TAG codons were suppressed during in vitro synthesis by the addition of (Tmd)Phe-tRNASup, a suppressor tRNA that carries a photoreactive probe (Brunner, 1996). The translation mixture also contained [35S]methionine to label the nascent chains. After translation, the samples were irradiated with UV light to induce cross-linking.

Bottom Line: Both TF and SRP were found to interact with the SA with partially overlapping binding specificity.In addition, extensive contacts with L23 and L29 were detected.Both purified TF and SRP could be cross-linked to L23 on nontranslating ribosomes with a competitive advantage for SRP.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Microbiology, Vrije Universiteit, 1081 HV Amsterdam, The Netherlands.

ABSTRACT
As newly synthesized polypeptides emerge from the ribosome, they interact with chaperones and targeting factors that assist in folding and targeting to the proper location in the cell. In Escherichia coli, the chaperone trigger factor (TF) binds to nascent polypeptides early in biosynthesis facilitated by its affinity for the ribosomal proteins L23 and L29 that are situated around the nascent chain exit site on the ribosome. The targeting factor signal recognition particle (SRP) interacts specifically with the signal anchor (SA) sequence in nascent inner membrane proteins (IMPs). Here, we have used photocross-linking to map interactions of the SA sequence in a short, in vitro-synthesized, nascent IMP. Both TF and SRP were found to interact with the SA with partially overlapping binding specificity. In addition, extensive contacts with L23 and L29 were detected. Both purified TF and SRP could be cross-linked to L23 on nontranslating ribosomes with a competitive advantage for SRP. The results suggest a role for L23 in the targeting of IMPs as an attachment site for TF and SRP that is close to the emerging nascent chain.

Show MeSH
Related in: MedlinePlus