Probing the alpha-sarcin region of Escherichia coli 23S rRNA with a cDNA oligomer

Abstract

The cause of 50S ribosomal subunit collapse reportedly triggered by hybridization of a 14-base cDNA probe to the alpha-sarcin region of 23S rRNA was investigated by physical measurement of probe-subunit complexes in varying buffer conditions. The results reported here show that this probe was unable to hybridize to its target site in the intact 50S subunit and the physical characteristics of 50S subunits remained unchanged in its presence. Subunit collapse was induced in buffer containing 20mM Tris-HCl (pH 7.5), 600 mM NH4Cl, 1 mM MgCl2, 1 mM DTT, and 0.1 mM EDTA in the absence of probe. The probe bound specifically to its target site in the collapsed particle, but did not promote further unfolding. The results demonstrate that a DNA probe bound to the alpha-sarcin region cannot cause the 50S subunit to unfold or cause 23S rRNA to degrade. We suggest that the previously reported collapse was most probably the result of the ionic conditions used.