Fig 1: Chaperonin requirement for translation of the septin Cdc12 by prokaryotic ribosomes. (A) Coomassie-stained SDS–PAGE of proteins purified via Ni-NTA from wild-type or groEL-mutant E. coli cells carrying plasmids encoding the indicated septins, plus other plasmids as necessary for induction or viability. Strains were B002 (“WT”), B003 (“?clpB”), B004 (“?dnaJ”), B005 (“?dnaK”), B006 (“?htpG”), and 461 (“groEL(E461K)”). Septin-encoding plasmids were pMVB128, pMVB133, and pMAM54. Leftmost lane contains a molecular weight ladder (Li-Cor Biosciences #928-60000). (B) Immunoblot of bound proteins prepared as in A, plus a sample from wild-type cells (B002) carrying no septin-encoding plasmid (“No Septins”), using a cocktail of antibodies recognizing Cdc3, Cdc11, and the 6xHis tag. Magenta asterisk indicates an ~70 kDa protein weakly reactive with the Cdc3 and/or Cdc11 antibody. (C) As in B but for urea-solubilized pellets (cell debris and insoluble material) obtained following cell lysis. The blue asterisk indicates the band corresponding to Cdc12. (D) DNA agarose gel electrophoresis and ethidium bromide staining of products of RT-PCR for Cdc3 or Cdc12 mRNA from total RNA extracted from groEL-mutant strain 461 carrying plasmid pMAM54 and induced with IPTG at the indicated temperatures. The presence or absence of reverse transcriptase (“RT”) in the reactions is indicated. Leftmost lane contains molecular weight ladder (Thermo Fisher Scientific #SM0331). (E) Published Ribo-seq/ribosome profiling data (Sen et al., 2015) showing the positions of ribosomes along the Cdc12 mRNA as numbers of reads of ribosome-protected mRNA fragments. Below, the Cdc12 ORF is illustrated with Met codons in green, Pro codons in blue, and the stop codon in red. The first 14 amino acids of Cdc12 are indicated with the same color scheme. (F) As in B but including samples from groEL-mutant cells carrying the plasmid pMAM88, which encodes Cdc3 and 6xHis-tagged Cdc12 with the four Pro residues highlighted in E mutated to Ala (“(AVAAA)Cdc12”). Samples from cells with pMAM54, encoding wild-type Cdc12, are indicated by “(PVPPP)Cdc12.” The blue asterisk indicates the band corresponding to Cdc12. (G) Chaperone-free in vitro transcription/translation reactions lacking release factors (PURExpress ?RF123) and containing fluorescent puromycin to label translation products were resolved by SDS–PAGE. The template plasmid pMVB128 encodes Cdc10 and 6xHis-Cdc12. Purified GroEL/ES chaperonin was added to one reaction, as indicated. Leftmost lane contains a molecular weight ladder (Bio-Rad #1610395). (H) As in G but gel was run longer, the order of lanes is reversed, and brightness and contrast were adjusted in FIJI.