Intrinsic structural differences between 'tight couples' and Kaltschmidt-Wittmann ribosomes evidenced by dielectric spectroscopy and scanning microcalorimetry

Measurements of dielectric spectroscopy (DS) and microcalorimetry (differen tial scanning calorimetry (DSC)) of Escherichia coli 70S, 50S and 30S were performed on particles prepared according either to the 'classical' twice N H4Cl-washed ribosomes, also known as loose couples (LC), or to the 'tight c ouples' preparative protocol (TC), Results show that 70S particles prepared according to the two different protocols exhibit different structural prop erties. Two subsequent relaxation processes occur in both samples as measur ed by DS. However, in LC ribosomes the first one is shifted towards a lower frequency with a higher dielectric increment, This is suggestive of a more sensitive exposure of RNA to the solvent and of an overall more relaxed st ructure. The smaller LC subunit exhibits only one relaxation while the TC 3 0S shows two dielectric dispersions as well as 70S. No substantial differen ces were evidenced in either 50S species. Tno typical melting peaks were ob served by DSC both in LC and TC 70S as well as in 50S. Thermograms obtained from the TC 30S show a single well structured peak while LC particles prod uce a large unstructured curie. On the basis of these results we conclude t hat TC 70S particles are more compact than LC ribosomes and that in the for mer ones the rRNA is less exposed to the solvent phase. Furthermore 30S par ticles obtained from TC show a more stable structure with respect to LC 30S , We conclude that the 30S subunit gi,es a major contribution to the compac t character of the whole TC 70S. These differences might be related to the intrinsic and well documented functional difference between the two ribosom e species. (C) 2001 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.