The binding of S-RNA by Escherichia coli ribosomes

Abstract

Washed ribosomes from E. coli will bind soluble RNA (S-RNA) in high concentrations of magnesium ions. The bound S-RNA will not wash off in high magnesium but can be displaced by additional S-RNA or can be freed by lowering the magnesium ion concentration. This binding takes place in the cold and does not require the supernatant enzymes or an energy source. It is not affected either by two inhibitors of protein synthesis, puromycin and chloramphenicol, or by the charging of the S-RNA with amino acids. The binding requires the integrity of the pCpCpA§ end of the S-RNA molecule. There is one site for this binding on the 70 s ribosome, on the 50 s subunit. After protein synthesis in a cell-free extract from E. coli , a small fraction of the S-RNA that is bound to the ribosomes in high magnesium becomes resistant to being washed off the ribosomes in low magnesium. This tightly bound fraction appears on the 50 s subunit of the “stuck” 70 s ribosome (those 70 s ribosomes that do not dissociate in low magnesium and that carry nascent polypeptide chains). It is resistant to ribonuclease under mild conditions. This bound S-RNA can be recovered as intact 4 s material if the ribosomes are broken up with sodium dodecyl sulfate.