Diauxic shift-dependent relocalization of decapping activators Dhh1 and Pat1 to polysomal complexes

Drummond, Sheona P.; Hildyard, John; Firczuk, Helena; Reamtong, Onrapak; Li, Ning; Kannambath, Shichina; Claydon, Amy J.; Beynon, Robert J.; Eyers, Claire E.; McCarthy, John E. G.

Strains and plasmids used in this study are listed in Tables 1 and 2 , respectively. TCM-tag strains were generated by chromosomal integration of the tetracysteine-motif and kanMX antibiotic selection cassette at the C-terminal of the genomic copy of the gene of interest. Briefly, the 'TCM-tag-kanMX cassette' for homologous recombination comprised two oligonucleotides complementary to the TCM tag-kanMXmarker cassette together with the appropriate region of homology with the yeast genome to facilitate in frame fusion of the TCM-tag downstream of the gene of interest. Strains were grown at 30 C unless otherwise stated in either yeast extract/peptone medium (YPD) or synthetic medium (SC) supplemented with appropriate amino acids and 2% glucose. Strain growth was initiated using a single colony from an agar plate for each 5-ml starter culture (containing the appropriate selective medium). Each starter culture was then used to inoculate a larger liquid culture (starting OD 600 = 0.2), which was shaken (220 rpm) at 30 C in a conical flask of volume 10 times the culture volume until the culture reached either OD 600 = 0.5 (exponential growth phase) or OD 600 = 2.0 (post-diauxic shift growth; this point was reached after at least 20 h of incubation). These two points are indicated on the growth curve shown in Supplementary Figure S1 .