Run off ribosomes of Escherichia coli: their accumulation in response to cold treatment and their dissociation during centrifugation

An Investigation of the Conformational Properties of Ribosomes Using N‐Ethylmalemide as a Probe

“…Cells were grown in a rich medium [7] to midlog phase and either rapidly chilled by the addition of crushed ice or slowly cooled from 37 "C to 10 "C by placing the culture in an ice bath, holding at 10 "C for 10 min, and finally bringing it to 0 "C by the addition of ice. A slow cooling process of this kind produces run-off, vacant couples from polysomes which are free from mRNA and peptidyl tRNA [8,9]. Cells were washed with buffer A and stored at -70 "C. Ribosomes with tritium-labelled protein were obtained from cells grown on 3H-labelled synthetic algal hydrolysate amino acid mixtures (New England Nuclear).…”

“…Complexed ribosomes, that is to say ribosomes having mRNA and tRNA bound [lo], and run-off ribosomes, free of such ligands [8,9] were obtained by alumina grinding of rapidly cooled and slowly cooled cells respectively and extraction in buffer A [3]. High-salt-washed ribosomes were derived from complexed ribosomes by two cycles of washing in ammonium chloride.…”

Kinectics of beta-galactosidase synthesis in Escherichia coli at 5 C

“…As t increases and messenger decay becomes impor- 10.0o tant, Yave will no longer increase linearily with t and EFC5 will no longer increase linearily with t2. It is important to note that a similar dependence of EFC5 on t2 would follow from any 4…”

“…The minimum temperature that permits sustained growth of Escherichia coli is in the vicinity of 7.8 C (22). Das and Goldstein (6) studied macromolecular syntheses after a transfer of exponentially growing cells of E. coli from 37 to 0 C. They observed that the incorporation of radioactive leucine into protein proceeded at a slowly decreasing rate over a period of 4 h, but that incorporation of radioactive uridine into ribonucleic acid (RNA) continued in a linear fashion during this same period. Based on this and other indirect evidence, they inferred that initiation of translation at the 5' end of messengers is blocked at 0 C, but that polypeptide chain elongation can continue.…”

“…Based on this and other indirect evidence, they inferred that initiation of translation at the 5' end of messengers is blocked at 0 C, but that polypeptide chain elongation can continue. In addition, it has been shown (1,4,6) that low-temperature incubation of E. coli leads to polysomal run-off, with the consequent accumulation of free 70S ribosomes (1,4). Friedman et al (9) concluded that f2 viral coat protein is completed but not initiated after transfer from 37 to 6 C. They (9) also observed that initiation of polypeptide chain synthesis from E. coli messengers is severely restricted at 4 C; however, it was not clear from their experiments whether this restriction was due to a block at the level of initiation of transcription or of translation.…”