Nature uses 64 codons to encode the synthesis of proteins from the genome, and chooses 1 sense codon-out of up to 6 synonyms-to encode each amino acid. Synonymous codon choice has diverse and important roles, and many synonymous substitutions are detrimental. Here we demonstrate that the number of codons used to encode the canonical amino acids can be reduced, *

In addition to extruding DNA loops, cohesin entraps within its SMC-kleisin ring (S-K) individual DNAs during G1 and sister DNAs during S-phase. All three activities require related hook-shaped proteins called Scc2 and Scc3. Using thiol-specific crosslinking we provide rigorous proof of entrapment activity in vitro. Scc2 alone promotes entrapment of DNAs in the E-S and E-K compartments, between ATP-bound engaged heads and the SMC hinge and associated kleisin, respectively. This does not require ATP hydrolysis nor is it accompanied by entrapment within S-K rings, which is a slower process requiring Scc3. Cryo-EM reveals that DNAs transported into E-S/E-K compartments are ‘clamped’ in a sub-compartment created by Scc2’s association with engaged heads whose coiled coils are folded around their elbow. We suggest that clamping may be a recurrent feature of cohesin complexes active in loop extrusion and that this conformation precedes the S-K entrapment required for sister chromatid cohesion.

Cohesin’s association with and translocation along chromosomal DNAs depend on an ATP hydrolysis cycle driving the association and subsequent release of DNA. This involves DNA being ‘clamped’ by Scc2 and ATP-dependent engagement of cohesin’s Smc1 and Smc3 head domains. Scc2’s replacement by Pds5 abrogates cohesin’s ATPase and has an important role in halting DNA loop extrusion. The ATPase domains of all SMC proteins are separated from their hinge dimerisation domains by 50-nm-long coiled coils, which have been observed to zip up along their entire length and fold around an elbow, thereby greatly shortening the distance between hinges and ATPase heads. Whether folding exists in vivo or has any physiological importance is not known. We present here a cryo-EM structure of the apo form of cohesin that reveals the structure of folded and zipped-up coils in unprecedented detail and shows that Scc2 can associate with Smc1’s ATPase head even when it is fully disengaged from that of Smc3. Using cysteine-specific crosslinking, we show that cohesin’s coiled coils are frequently folded in vivo, including when cohesin holds sister chromatids together. Moreover, we describe a mutation (SMC1D588Y) within Smc1’s hinge that alters how Scc2 and Pds5 interact with Smc1’s hinge and that enables Scc2 to support loading in the absence of its normal partner Scc4. The mutant phenotype of loading without Scc4 is only explicable if loading depends on an association between Scc2/4 and cohesin’s hinge, which in turn requires coiled coil folding.

Bactofilins are small β-helical proteins that form cytoskeletal filaments in a range of bacteria. Bactofilins have diverse functions from cell stalk formation in C. crescentus to chromosome segregation and motility in M. xanthus. However, the precise molecular architecture of bactofilin filaments has remained unclear. Here we revealed by sequence analysis and electron microscopy that in addition to wide distribution across bacteria and archaea, bactofilins are also present in a few eukaryotic lineages such as the Oomycetes. Cryo-EM analysis demonstrated that the sole bactofilin from Thermus thermophilus (TtBac) forms constitutive filaments that polymerise through end-to-end association of the β-helical domains. With the aid of a nanobody, we determined the near-atomic filament structure, showing that the filaments are non-polar. A polymerisation-impairing mutation enabled crystallisation and structure determination, while reaffirming the lack of polarity and the strength of the beta-stacking interface. To confirm the generality of the lack of polarity, we performed co-evolutionary analysis of a large set of sequences. Finally, we determined that the widely conserved N-terminal disordered tail of TtBac is responsible for direct binding to lipid membranes both on liposomes and in E. coli cells. Membrane binding is likely a common feature of these widespread but only recently discovered filaments of the prokaryotic cytoskeleton.