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Previous characterizations of TF-ribosome and TF-substrate interactions were conducted with vacant ribosomes (Maier, R. et al. 2003) and free substrate, respectively (Huang et al. 2000; Liu et al. 2005; Maier, R. et al. 2001; Patzelt et al. 2001; Suno et al.

2004) and thus the mechanisms underlying TF function in its biologically-relevant context of active translation have remained elusive. Free dimeric TF may exhibit certain chaperone properties as measured by prevention of aggregation or refolding of certain denatured model proteins (Kramer et al. 2004; Liu et al. 2005; Liu & Zhou 2004; Maier, R. et al.

2001). However, its role in vivo, at least for multi-domain proteins, has been shown to involve a postponement in the acquisition of nascent chain structure relative to translation (Agashe et al. 2004). Therefore, its function in chaperoning virtually all nascent chains emerging from the ribosome probably requires the substrate to be presented in the context of the translating ribosome. Characterization of TF ribosome-binding and intra-molecular rearrangements during translation was attempted in an S30-based transcription/translation system with the fluorescence-based tools described above. However, due to the crude composition of the system, high background and poor reproducibility impaired the measurements.

Recently, a protein synthesis system has become available that is reconstituted from purified components (Shimizu et al. 2001). All proteins needed for translation, such as initiation, elongation and release factors, as well as the 20 different aminoacyl-tRNA synthetases, were purified individually as His6-tagged recombinant proteins. In addition,

the system contains purified tRNAs, amino acids, ribosomes and an energy regenerating system. In contrast to protein synthesis systems based on crude S30 extracts from E. coli, this system allows protein synthesis in a well defined environment. Originally developed for rapid and convenient protein production and isolation in vitro, this system was applied in the present study to accomplish a real-time analysis of processes occurring during translation.

When luciferase was translated in the reconstituted system, a pronounced increase of TF ribosome-recruitment was observed, as monitored by a decrease in TF-B fluorescence and an increase in steady state fluorescence anisotropy of TF-C. Thus, the affinity of TF is higher for ribosome-nascent chain complexes than for vacant ribosomes. A 30-fold decrease in the KD of the TF-ribosome complex occurred upon translation of FL.

This suggests a regulatory principle for TF function: When protein production in the cell is taking place at a high rate, e.g. in E. coli cells during mid-log phase, increased amounts of

monomeric TF become recruited to the ribosome-nascent chain complexes. Monomerization and conformational opening result in activation of TF. Activated TF is then able to efficiently bind to the nascent chain substrate, protecting it from the entry into non-productive folding pathways and aggregation. If less ribosomes are engaged in translation, e.g. during stationary phase, TF is shifted from a ribosome-bound to a free cytosolic form, which readily dimerizes. Dimerization leads to inactivation of TF in terms of substrate binding, providing a cytosolic pool that can be activated when protein synthesis is increased. The regulation of cytosolic TF levels according to the growth rate (Griffiths et al. 1995; Guthrie & Wickner 1990; Lill et al. 1988; Pedersen et al. 1978) is consistent with this model.

A decrease in KD is the result of changes in the rate constants: A decrease in the

off-rate, an increase in the on-rate, or both. The “cradle” model for TF function suggests that TF crouches over the ribosomal exit tunnel, providing a protected environment for domain-wise co-translational folding (Ferbitz et al. 2004; Maier, T. et al. 2005). This model, based solely on the modeling of full-length TF onto the H. marismortui 50S ribosomal subunit, would imply that TF stays associated with the translating ribosome long enough to allow synthesis and co-translational folding of at least an entire protein domain. Based on this model, a decreased off-rate would be expected to account for the increased affinity of TF for the ribosome that was observed upon translation. This aspect was investigated in competition experiments where ribosome-bound labeled TF was displaced from translating ribosomes by the addition of excess unlabeled TF. No difference in the displacement kinetics was observed in the different scenarios with and without translation. Instead, the dynamics of TF-ribosome interaction were found to be unaltered in the presence of translation, indicating that the time that TF resides on the ribosome is not affected by the presence of a nascent chain substrate.

An increased recruitment of TF to translating ribosomes was also reflected by a decrease in the efficiency of intra-molecular FRET. The FRET efficiency measured is an average of the FRET efficiencies for the bound and the free form of TF. Thus, an increase in the fraction of bound TF leads to decreased average FRET efficiency. Upon addition of excess unlabeled wild type TF, a return of the FRET efficiency to the value observed in the absence of ribosomes occurred. Strikingly, the kinetics of ribosome detachment and intra- molecular compaction no longer occurred concomitantly when ribosomes were actively engaged in translation. Instead, the change in intra-molecular FRET efficiency was delayed with respect to ribosome release, with a ~3-fold difference in the t1/2 values. This argues

that, even after TF has left the translating ribosome, it is kept in its open conformation, presumably by an interaction with the nascent chain. Thus, TF appears to leave the ribosome while still interacting with the elongating polypeptide chain.

This model was supported by data from steady state fluorescence anisotropy measurements, where the change in signal that was observed upon ribosome displacement happened with the similar kinetics as ribosome-release in the case of vacant ribosomes, but is markedly slower when ribosomes are translating. This also indicates that after ribosome dissociation, TF remains associated with a very large molecular weight component, presumably the ribosome-tethered nascent chain.

Importantly, in none of the competition experiments was the TF FRK/AAA mutant (deficient in ribosome binding) found to act as an efficient competitor. This demonstrates that binding to the ribosome is a pre-requisite for efficient substrate binding, consistent with the hypothesis that ribosome binding leads to TF activation. Only then does TF efficiently bind its substrate and remains associated even after departing from the ribosome. Thus, ribosome-binding serves several purposes: Besides creating a high local TF concentration at the ribosomal exit tunnel in close proximity to its substrate, it stabilizes the monomer and induces a conformational change, thereby activating TF.

The delay in intra-molecular compaction over ribosome-dissociation was also observed when the nascent chain was released from the ribosome by disruption of the ribosome-nascent chain complex with EDTA. This further strengthens the notion that beyond ribosome departure of both TF and the nascent chain, similar to a post-translational scenario, TF remains engaged with its substrate. After disruption of the complex, TF is unable to rebind to the substrate regardless of whether it has folded or not. Ribosome- binding is necessary to activate TF and restricts its chaperone activity to ribosome-tethered nascent chains. Other chaperone systems acting downstream of TF may then aid the further folding of the released polypeptide.