Nuclease Degradation and the Structure of Ribosomes

Abstract

Well-defined patterns of fragments are produced when Escherichia coli ribosomes, both native, dissociated, unfolded, and partially denuded of proteins by treatment with caesium chloride, are digested with pancreatic ribonuclease, and the extracted RNA is fractionated by polyacrylamide gel electrophoresis. Procedures are given for determining the degree of congruence of a pair of digestion patterns, and analysing the results in terms of probability of matching. By this means it has been established that all the digestion patterns which have been obtained are closely related. The similarity between native 70-8 ribosomes and completely unfolded nucleoprotein particles in EDTA indicates that the greater part of the RNA is accessible on the surface of the 70-S ribosome. It has not been possible to show a close relationship between the patterns generated by the nucleoprotein particles in various states and these from free ribosomal RNA. We infer that a number of the labile points in the RNA are protected by bound proteins. The digestion proceeds much more slowly with increasing stabilisation of structure by magnesium ions, both in free RNA and ribonucleoprotein. It is shown that the labile points of the chain are segments of some length, and not isolated phosphodiester links.The availability of RNA in the 70-S ribosome of Escherichia coli to pancreatic ribonuclease was first noted by Xanter [I], who deduced that the RNA is on the surface of the particle, but is in a more resistant state than free, extracted RNA. There have subsequently been many studies on the digestion of ribosomes with nucleases [2-61, and in one of the most recent [7], it has been reported that a considerable part of the RNA can be detached without elimination of biological activity. It has been found in this laboratory and elsewhere [8-121 that the controlled attack of nucleases on ribosomal RNA produces a series of sharply defined fragments which can readily be separated by polyacrylamide gel electrophoresis and can serve as a kind of fingerprint of an RNA species [12]. Although the lability of the RNA to nuclease is undoubteldy affected by basepairing, our evidence [Ill on digestion with T I ribonuclease points to a specificity of cleavage determined by primary structure, for essentially the same pattern of zones can be obtained from an RNA from which base-pairing has been eliminated by treatment with formaldehyde. The digestion pattern is thus a characteristic of the RNA chain, and one might hope to be able to use it in such a structure as the ribosome to establish to what extent the limited number of primary labile regions of the RNA are available to the enzyme in various conformational states, The results ofsuch a study are presented here. MATERIALS AND METHODS Preparation of RibosomesRibosomes from nuclease-deficient strain MRE 600 of E . coli were prepared by standard procedures [13], and RNA in general by extraction with phenol and detergent [14]. Run-off and "stuck" ribosomes, which differ in that the former contain no messenger, and are ...

Homology among trypsin/chymotrypsin inhibitors from red kidney bean, Brazilian pink bean, lima bean, and soybean

“…6 Total probability of finding the degree of congruence equal to or higher than the observed values on a random basis. The probabilities were calculated by using the equation [derived by Finder and Gratzer (1972)] chymotryptic digests than among tryptic digests according to their FPLC chromatograms. This may be due to the lower specificity of chymotrypsin than trypsin.…”

Effect of ribonuclease T1 on ribosomal subunits of rat liver

“…Limited digestion of subunits also led to a specific fragmentation of rRNA, according to the conformation of the particle (8). The RNA fragments were useful for sequence analysis (9 -11).…”

A comparative study of ribonuclease hydrolysis of rat brain-cortex and liver membrane-bound ribosomes

“…Heavy endoplasmic reticulum membrane fractions from brain cortex and liver were incubated with various RNAases in the sucrose-TKM buffer from brain cortex, which contains a high concentration of KCI (100mM). Brain polyribosomes are more susceptible to dissociation than those from liver (Zomzely et al, 1966) and, as the ribosomal subunit appears more susceptible to nuclease attack than the complete unit (Cox, 1969;Pinder & Gratzer, 1972), all the data were obtained with the higher-ionicstrength buffer. However, use of the more commonly used low K+ concentration buffer did not influence the amount of oligonucleotide material released from brain ribosomes.…”