This blog post comments on the results published by Fujiwara and co-workers in the 2020 Cell Reports article “Proteome-wide capture of co-translational protein dynamics in Bacillus subtilis using TnDR, a transposable protein-dynamics reporter.”
The study of mechanical force generation and its influence on biological systems has expanded in recent years. In the realm of nascent protein folding, we now know that both unstructured and folded nascent proteins generate forces on the order of piconewtons that propagate down the nascent chain. These forces can distort the functional site of the ribosome and may influence the rate of translation (PMIDs: 30824598, 29577725). It has also been shown that translational arrest can be relieved by mechanical force (PMID: 25908824). Much study has focused on so-called arrest peptides, short peptide sequences that interact so strongly with the ribosome exit tunnel that they can completely stall translation (e.g., SecM, MifM).
Fujiwara and co-workers designed a clever reporter for mechanical force application to nascent proteins by combining the MifM arrest sequence, the Himar1 mariner transposon (ITR refers to its internal terminal repeat), the FLAG epitope tag sequence, and the LacZ gene (see figure, reproduced from Fujiwara et al. 2020). kanR is the kanamycin-resistance gene. Transposition into a random gene X generates an X’-FLAG-MifM-LacZ gene fusion. If the X’-FLAG junction is in frame and the nascent transposition product behaves dynamically to relieve MifM stalling, then the transposition colony will appear visually blue due to the expression of LacZ in the presence of X-Gal. Selecting these blue colonies amounts to selecting members of the proteome that engage in some dynamic maturation/assembly/localization procedure.
Analysing their results, Fujiwara and co-workers identified various processes that generate mechanical force and relieve stalling for diverse proteins.
Localization out of the cytosol: 62% of the proteins identified in blue colonies were membrane proteins. Removal of the signal sequence from a subset of 4 of these proteins led to large decreases in arrest cancellation, indicating that translocation into other cellular compartments generates force and causes peptide release. Further experiments confirmed that Sec-dependent translocation was the cause of force generation.
Molecular assembly: The remaining 38% of proteins captured in this study are cytosolic, so some mechanism other than localization must explain their arrest release. Possible sources of this force are co-translational folding and molecular assembly. For one protein, sigmaF, binding to its SpollAB partner seems to be the cause. For the ribosomal protein bL25_94, however, co-translational ribosome assembly appears to be lead to force generation.
This paper highlights the diverse ways in which protein biogenesis can create mechanical forces. The speed of translation influences co-translational folding, and co-translational folding itself, along with many other co-translational processes, can influence the speed of translation by exerting pulling forces. Decoupling and understanding this apparent feedback loop may prove an interesting puzzle in the coming years.
