Kaiser, C.M. et al. Real-time observation of Trigger factor function on translating ribosomes. Nature 444, 455-460
Department of Cellular Biochemistry, Max Planck Institute of Biochemistry, Am Klopferspitz 18, D-82152 Martinsried, Germany. Nature
(Impact Factor: 41.46).
12/2006; 444(7118):455-60. DOI: 10.1038/nature05225
The contribution of co-translational chaperone functions to protein folding is poorly understood. Ribosome-associated trigger factor (TF) is the first molecular chaperone encountered by nascent polypeptides in bacteria. Here we show, using fluorescence spectroscopy to monitor TF function and structural rearrangements in real time, that TF interacts with ribosomes and translating polypeptides in a dynamic reaction cycle. Ribosome binding stabilizes TF in an open, activated conformation. Activated TF departs from the ribosome after a mean residence time of approximately 10 s, but may remain associated with the elongating nascent chain for up to 35 s, allowing entry of a new TF molecule at the ribosome docking site. The duration of nascent-chain interaction correlates with the occurrence of hydrophobic motifs in translating polypeptides, reflecting a high aggregation propensity. These findings can explain how TF prevents misfolding events during translation and may provide a paradigm for the regulation of nucleotide-independent chaperones.
Available from: Bernd Bukau
- "Two algorithms predicting such motifs in the linear (unfolded) sequence were developed earlier: initially, TF-binding sites were predicted as stretches of 8 aa enriched in basic and aromatic residues with a positive net charge (Patzelt et al., 2001) (orange lines in Figure 1 and Figure S1). Later, TF was found to stay associated with nascent chains containing motifs of five or more consecutive amino acids of high mean hydrophobicity (Kaiser et al., 2006) (green lines in Figure 1 and Figure S1). b-lactamase (Figure 1C), ICDH (Figure S1A), MBP, and luciferase contain both types of linear binding motifs. "
[Show abstract] [Hide abstract]
ABSTRACT: How nascent polypeptides emerging from ribosomes fold into functional structures is poorly understood. Here, we monitor disulfide bond formation, protease resistance, and enzymatic activity in nascent polypeptides to show that in close proximity to the ribosome, conformational space and kinetics of folding are restricted. Folding constraints decrease incrementally with distance from the ribosome surface. Upon ribosome binding, the chaperone Trigger Factor counters folding also of longer nascent chains, to extents varying between different chain segments. Trigger Factor even binds and unfolds pre-existing folded structures, the unfolding activity being limited by the thermodynamic stability of nascent chains. Folding retardation and unfolding activities are not shared by the DnaK chaperone assisting later folding steps. These ribosome- and Trigger Factor-specific activities together constitute an efficient mechanism to prevent or even revert premature folding, effectively limiting misfolded intermediates during protein synthesis.
Available from: PubMed Central
- "The poor capacity of the bacterial cytosol to support efficient folding of certain model proteins has been exploited to investigate the mechanisms and molecules involved in these processes. It is possible that this inability may be due to the presence of incompatible bacterial chaperones [45, 46] or the absence of specialized eukaryotic chaperones [47, 48]. In addition to their distinct chaperone complements, a major difference between the protein biosynthetic machineries of bacteria and eukaryotes that has remained largely unexplored is the rate at which proteins are synthesized. "
[Show abstract] [Hide abstract]
ABSTRACT: The genetic code is said to be redundant in that the same amino acid residue can be encoded by multiple, so-called synonymous, codons. If all properties of synonymous codons were entirely equivalent, one would expect that they would be equally distributed along protein coding sequences. However, many studies over the last three decades have demonstrated that their distribution is not entirely random. It has been postulated that certain codons may be translated by the ribosome faster than others and thus their non-random distribution dictates how fast the ribosome moves along particular segments of the mRNA. The reasons behind such segmental variability in the rates of protein synthesis, and thus polypeptide emergence from the ribosome, have been explored by theoretical and experimental approaches. Predictions of the relative rates at which particular codons are translated and their impact on the nascent chain have not arrived at unequivocal conclusions. This is probably due, at least in part, to variation in the basis for classification of codons as "fast" or "slow", as well as variability in the number and types of genes and proteins analyzed. Recent methodological advances have allowed nucleotide-resolution studies of ribosome residency times in entire transcriptomes, which confirm the non-uniform movement of ribosomes along mRNAs and shed light on the actual determinants of rate control. Moreover, experiments have begun to emerge that systematically examine the influence of variations in ribosomal movement and the fate of the emerging polypeptide chain.
Available from: Taotao Chen
- "An effective functional cooperation apparently exists between TF and the DnaK system in the folding of a group of large multidomain proteins that aggregate substantially only in the absence of both chaperones. These proteins may normally interact sequentially with TF and DnaK during translation or with multiple TF molecules in the absence of DnaK, as shown with large model proteins in vitro (Agashe et al., 2004; Kaiser et al., 2006). Notably, most of these proteins would be unable to interact productively with GroEL, as the capacity of the GroEL/ES folding compartment is limited to proteins up to $60 kDa (Kerner et al., 2005). "
[Show abstract] [Hide abstract]
ABSTRACT: Cellular chaperone networks prevent potentially toxic protein aggregation and ensure proteome integrity. Here, we used Escherichia coli as a model to understand the organization of these networks, focusing on the cooperation of the DnaK system with the upstream chaperone Trigger factor (TF) and the downstream GroEL. Quantitative proteomics revealed that DnaK interacts with at least ~700 mostly cytosolic proteins, including ~180 relatively aggregation-prone proteins that utilize DnaK extensively during and after initial folding. Upon deletion of TF, DnaK interacts increasingly with ribosomal and other small, basic proteins, while its association with large multidomain proteins is reduced. DnaK also functions prominently in stabilizing proteins for subsequent folding by GroEL. These proteins accumulate on DnaK upon GroEL depletion and are then degraded, thus defining DnaK as a central organizer of the chaperone network. Combined loss of DnaK and TF causes proteostasis collapse with disruption of GroEL function, defective ribosomal biogenesis, and extensive aggregation of large proteins.
Data provided are for informational purposes only. Although carefully collected, accuracy cannot be guaranteed. The impact factor represents a rough estimation of the journal's impact factor and does not reflect the actual current impact factor. Publisher conditions are provided by RoMEO. Differing provisions from the publisher's actual policy or licence agreement may be applicable.