A ribonucleoprotein fragment of the 30 S ribosome of E. coli: evidence for long range RNA-RNA interactions

Abstract

A stable homogeneous ribonucleoprotein fragment of the 30 S ribosomal subunit of E. coli has been prepared by mild nuclease digestion and heating in a constant ionic environment. The fragment contains about half of the 16 S ribosomal RNA and six proteins: S4, S7, S9, S13, S16 and S19. The RNA moiety contains the reported binding sites of all six proteins. After deproteinization, 80% of the RNA migrated as two major electrophoretic bands, which were isolated and sequenced. Each band contained sequences from the 5' and 3' thirds of the 16 S RNA but none from the central third. That these two noncontiguous RNA domains migrated together electrophoretically in Mg++-containing gels after deproteinization constitutes direct evidence that the 16 S RNA is folded in the intact ribosome so as to bring the two domains close together and that there are RNA-RNA interactions between them in the presence of Mg++.

Long range RNA-RNA interactions in the 30 S ribosomal subunit ofE. coli

“…All operations were carried out in buffer MTK 1-10-20 (1 mM magnesium acetate, 10 mM Tris acetate (pH 7.8), 20 mM potassium acetat4. The experimental details were as described previously (1) except that the time of heating at 570 was reduced to 10 min.…”

“…Processing of the fragments Sucrose gradient fractions and their RNA moieties were examined by analytical polyacrylamide gel electrophoresis (1) and were pooled according to the results. Three fragment preparations, denoted A, D and E, were concentrated and freed of sucrose by pressure filtration (Amicon, filter PM-10), dissolved in a small volume of buffer MTK 1-10-20, and kept at -70 .…”

“…Three fragment preparations, denoted A, D and E, were concentrated and freed of sucrose by pressure filtration (Amicon, filter PM-10), dissolved in a small volume of buffer MTK 1-10-20, and kept at -70 . Proteins were extracted and identified by two-dimensional gel electrophoresis (1), and the protein-free RNA moieties were taken for further processing. Fractionation and sequencing of the RNA moieties Nucleoprotein fragments were deproteinized in the presence of Mgt± (MTK 1-10-20) by three treatments with sodium dodecyl sulfate and phenol (12).…”

“…We have used this approach to investigate the 30 S subunit of E. coli ribosomes (1,2). Intact native subunits were subjected to mild ribonuclease digestion and warmed under controlled conditions, and came apart into fragments whose protein and RNA components were identified.…”

“…Intact native subunits were subjected to mild ribonuclease digestion and warmed under controlled conditions, and came apart into fragments whose protein and RNA components were identified. Each fragment proved to be not a random assortment of RNA and protein, but a well-defined region of the ribosome, showing that native ribosomal structure had survived the treatment (1,2).…”