For this week’s journal club, I presented the paper by Goldman et al, “Mechanical force releases nascent chain-mediated ribosome arrest in vitro and in vivo”. The reason for choosing this paper is that it discussed an influence on protein folding/creation/translation that is not considered in any of today’s modelling efforts and I think it is massively important that every so often we, as a community, step-back and appreciate the complexity of the system we attempt to understand. This work focuses on the the SecM protein, which is known to regulate SecA (which is part of the translocon) which in turn regulates SecM. The bio-mechanical manner in which this regulation takes place is not fully understood. However, SecM contains within its sequence a peptide motiff that binds so strongly to the ribosome tunnel wall that translation is stopped. It is hypothesised that SecA regulates SecM by applying a force to the nascent chain to pull it past this stalling point and, hence, allow translation to continue.
To begin their study, Goldman wanted to confirm that one could advance past the stall point merely by the application of force. By attaching the nascent chain and the ribosome to nano-tweezers and a micro-pipette respectively they could do this. However, to confirm that the system was stalled before applying a (larger) force, they created a sequence which included CaM, a protein which periodically hops between a folded and unfolded state when pulled at 7pN, followed by the section of SecM which causes the stalling. The nano-tweezers were able to sense the slight variations in length at 7pn from the unfolding and refolding of CaM, though no continuing extension, which would indicate translation, was found. This indicated the system had truly stalled due to the SecM sequence. Once at this point, Goldman increased the applied force, at which point distance between the pipette and the optical tweezers slowly increased until detachment when the stop codon was reached. As well as confirming that force on the nascent chain could make the SecM system proceed past the stalling point, they also noted a force dependence to the speed with which it would overcome this barrier.
With this force dependence established, they pondered whether a domain folding upchain of the stall point could generate enough force that it could cause translation to continue. To investigate, Goldman created a protein that contained Top7 followed by a linker of variable length, followed by the SeqM stalling motif, which was in turn followed by GFP. Shown in the figure above, altering the length of the linker region defined the location of Top7 while it attempts to fold. A long linker allows Top7 to fold completely clear from the ribosome tunnel. A short linker means that a it can’t fold due to many of its residues being inside the ribosome tunnel. Between these extremes, however, the protein may only have a few residues within the tunnel and by stretching the nascent chain it may access them so as to be able to fold. In addition, Top7 was chosen specifically as it was known to fold even under light pressure. Hence, by newtons third, Top7 would fold even while its C terminus would be under strain into the ribosome, it in turn generates an equal and opposite force on the stalling peptide sequence within the heart of the ribosome tunnel, which should allow translation to proceed past the stall. Crucially, if Top7 folded too far away from the ribosome, this interaction would not occur and translation would not continue.
Goldman’s experiments showed that this is in fact the case; they found that only linkers of 15 to 22 amino acid would successfully complete translation. This confirms that a protein folding at the mouth of the ribosome tunnel can generate sizeable force (they calculate roughly 12pN in this instance). Now I find this whole system especially interesting as the I wonder how this may generalise to all translation, both in terms of interactions of the nascent chain with the side wall and the domain folding at the ribosome tunnel mouth. Should I consider these when I calculate translation speeds for example? Oh well, we need a reasonable model for translation while ignoring these special cases first before I really need to worry!