Interplay between trigger factor and other protein biogenesis factors on the ribosome

Abstract

Nascent proteins emerging from translating ribosomes in bacteria are screened by a number of ribosome-associated protein biogenesis factors, among them the chaperone trigger factor (TF), the signal recognition particle (SRP) that targets ribosomes synthesizing membrane proteins to the membrane and the modifying enzymes, peptide deformylase (PDF) and methionine aminopeptidase (MAP). Here, we examine the interplay between these factors both kinetically and at equilibrium. TF rapidly scans the ribosomes until it is stabilized on ribosomes presenting TF-specific nascent chains. SRP binding to those complexes is strongly impaired. Thus, TF in effect prevents SRP binding to the majority of ribosomes, except those presenting SRP-specific signal sequences, explaining how the small amount of SRP in the cell can be effective in membrane targeting. PDF and MAP do not interfere with TF or SRP binding to translating ribosomes, indicating that nascent-chain processing can take place before or in parallel with TF or SRP binding.

Regulation by a chaperone improves substrate selectivity during cotranslational protein targeting

“…Collectively, these results provide more conclusive evidence that TF and SRP can bind to the same RNC, on which they alter the binding affinities and conformation of one another (17,19,20). Importantly, these modulations did not lead to significant consequences on the occupancy of SRP or TF on the RNCs.…”

“…S2A), others appeared consistent with a cobinding model (Fig. S2B) (20). The robust FRET between SRP (or TF) and signal sequence allowed us to more unambiguously test and distinguish between these models.…”

“…Nevertheless, a cobinding model was assumed, rather than tested, in this analysis (20). Indeed, the low signal-to-noise ratio of this assay (often 10-20%) led to ambiguous interpretations: Although some data appeared consistent with a competitive model (Fig.…”

“…How are nascent proteins sorted between them and committed to the correct pathway in a timely and accurate manner? Extensive past work to address these questions has led to different (and sometimes contradictory) models, including (i) TF and SRP compete for binding to the RNC (10,15,16,18); (ii) TF and SRP can bind to the same RNC simultaneously (17,(19)(20)(21); (iii) FtsY rejects TF from SRP-bound ribosomes (17); and (iv) TF preferentially occupies longer nascent chains (13,(45)(46)(47) and, by inference, SRP preferentially binds short nascent chains. A unifying model that reconciles all these observations and explains how nascent chains on the ribosome are selected by TF or SRP is still lacking.…”

“…Accumulating data indicate that this process begins at the ribosome exit site, where many protein biogenesis machineries can interact and gain access to the nascent polypeptide. This includes chaperones (1-5) such as trigger factor (TF) (1,4,6,7), Hsp70, and the nascent polypeptide-associated complex (8)(9)(10)(11)(12)(13); modification enzymes (10,(14)(15)(16) such as N-acetyl transferase, methionine aminopeptidase, and arginyl transferase; protein-targeting and translocation machineries such as signal recognition particle (SRP) (17)(18)(19)(20), SecA (21), the SecYEG (or Sec61p) (22,23) and YidC translocases (24,25), and the ribosome-bound quality control complex (26)(27)(28)(29)(30). Engagement of these factors with nascent polypeptides influences their folding, assembly, localization, processing, and quality control.…”