Abstract
In all organisms ribosome-associated chaperones assist early steps of protein folding. To elucidate the mechanism of their action, we determined the kinetics of individual steps of the ribosome binding/release cycle of bacterial trigger factor (TF), using fluorescently labeled chaperone and ribosome-nascent chain complexes. Both the association and dissociation rates of TF-ribosome complexes are modulated by nascent chains, whereby their length, sequence, and folding status are influencing parameters. However, the effect of the folding status is modest, indicating that TF can bind small globular domains and accommodate them within its substrate binding cavity. In general, the presence of a nascent chain causes an up to 9-fold increase in the rate of TF association, which provides a kinetic explanation for the observed ability of TF to efficiently compete with other cytosolic chaperones for binding to nascent chains. Furthermore, a subset of longer nascent polypeptides promotes the stabilization of TF-ribosome complexes, which increases the half-life of these complexes from 15 to 50 s. Nascent chains thus regulate their folding environment generated by ribosome-associated chaperones.
Highlights
To promote the folding of cytosolic proteins to the native state, cells employ a large arsenal of chaperones, some of which transiently associate with ribosomes
To elucidate the mechanism of these early steps of chaperone-assisted folding of newly synthesized proteins, we investigated whether the properties of the nascent polypeptide chains, such as length, amino acid composition, and folding status affect the dynamics of trigger factor (TF) binding to the ribosome
This is evident by the severe protein aggregation and lethality phenotype of E. coli mutants lacking both TF as well as DnaK system, which presumably backs up TF to rescue misfolded proteins [32, 33]
Summary
Design of Arrested Nascent Chain Constructs—See Supplemental Material for details. Purification of Ribosomes and RNCs—Ribosomes were purified from strain MC4100 ⌬tig as described [18]. RNCs were eluted with Buffer I containing 2.5 mM desthiobiotin and 1 mM phenylmethylsulfonyl fluoride (6 times, 380-l fractions), and the concentration of RNCs was determined by measuring the absorption at 260 nm. The second-order association rate constant was obtained by performing a global fit of the general solution (Equation 1) of the differential equation of the association reaction to each set of traces with constant ribosome concentration and different TF concentrations using nonlinear regression analysis and the GraphPad Prism 4.0 program (GraphPad Software, San Diego). Where k1 indicates second-order association rate constant; k2 indicates dissociation rate constant; [A] and [B] indicate concentrations of BADAN-R14C-TF and vacant ribosomes, respectively, with [A] Ն [B]; ⌬ ϭ (k1[A] ϩ k1[B] ϩ k2) Ϫ 4k12[A][B]. The BADAN-R14C-TF-AAA mutant deficient in ribosome binding was used instead of BADAN-R14C-TF
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