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. 2003 May 26;161(4):679-84.
doi: 10.1083/jcb.200302130. Epub 2003 May 19.

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

Ronald S Ullers  1 , Edith N G Houben, Amanda Raine, Corinne M ten Hagen-Jongman, Måns Ehrenberg, Joseph Brunner, Bauke Oudega, Nellie Harms, Joen Luirink
Affiliations

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

Ronald S Ullers et al. J Cell Biol. .

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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Figures

Figure 1.
Figure 1.
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%.
Figure 2.
Figure 2.
Cross-linking of 77FtsQ upon translation in a reconstituted translation system. (A–C) 77FtsQTAG27 (A) and 77FtsQTAG40 (B and C) were synthesized in a reconstituted translation system in the presence of (Tmd)Phe-tRNASup and various concentrations of purified SRP (A), purified TF (B), or combinations of SRP and TF (C), as indicated. After translation, the ribosome–nascent chain complexes were purified and analyzed by SDS-PAGE.
Figure 3.
Figure 3.
L23 is a ribosome attachment site for Ffh. Purified ribosomes (1 μM) were incubated with SRP (300 nM) for 5 min at 26°C. Cross-linking was induced by the addition of 10 mM EDC, and samples were incubated for another 10 min at 26°C. Cross-linking was quenched, and the samples were TCA precipitated and analyzed by SDS-PAGE and immunoblotting using antisera against Ffh and L23, as indicated.
Figure 4.
Figure 4.
TF and SRP compete for cross-linking to L23. Purified ribosomes (1 μM) were incubated with various concentrations of SRP and TF for 5 min at 26°C as indicated. The samples were cross-linked and analyzed as described under Fig. 3.
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