Propriétés des ribosomes et du RNA synthétisés par Escherichia coli cultivé en présence d'éthionine
Authors: Beaud Georges , Hayes Donal H. 
Published: March 2005

Two isogenic derivatives of Esherichia coli K12: strains EA 1 (Met−, Biot−, RCstr) and EA 2 (Met−, Biot−, RCrel) when grown in the presence of ethionine in place of methionine, synthesize ribosomes whose RNA is submethylated and whose proteins contain ethionine. Net ribosome synthesis in the presence of ethionine is greater in the RCrel strain than in the RCstr strain. The preceding paper describes results obtained with the latter strain. Here we present further studies carried out with the isogenic RCrel strain EA 2. As in the case of the RCstr strain (previous paper) ribosomes synthesized by the RCrel strain grown in the presence of ethionine display defective association properties. A large fraction of these “ethionine” ribosomes is found as free subunits in crude extracts contianing 10 mM Mg++ prepared from strain EA 2 grown in an ethionine‐containing medium. These free “ethionine” subunits have sedimentation coefficients of 28 S and 45 S approximately while the “ethionine” subunits found associated as 70 S particles in crude bacterial extracts sediment at 30 S and 50 S respectively. Both classes of “ethionine” subunits (free, and associated) can be methylated in vitro in the presence of S‐adenosyl methionine with or without the addition of an external source of methylases (100 000 ×g supernatant of a homologous crude bacterial extract). The latter observation shows that the “ethionine” subunits contain bound methylases. These enzymes do not seem to be normal ribosomal proteins nor to be bound specifically to “ethionine” ribosomes since (a) they are removed by washing the “ethionine” ribosomes with 1 M NH4Cl, and (b) they are found bound to normal methionine ribosomes from which they are also removed by washing with 1 M NH4Cl.

In vitro methylation of free or associated 30 S “ethionine” subunits causes incorporation of methyl groups exclusively in the RNA of these particles while in the case of free or associated 50 S “ethionine” subunits a large fraction (approx. 60%) of the incorporated methyl groups is found to be present in their protein moiety. Free “ethionine” subunits both 30 S and 50 S, are found to have a higher specific accepting activity for methyl groups than the corresponding associated particles.

A study of the sites of in vitro methylation of the RNA of 30 S and 50 S “ethionine” subunits has shown that in all cases (free or associated particles) the only nucleic acid bases which are methylated are adenine (product N6‐dimethyl adenine) and guanine (product N2‐methyl guanine).

The number of adenine residues per particle which can be methylated in vitro is greater in the free than in the associated 30 S “ethionine” subunits (2.7 and 0.8 respectively), while the number of guanine residues which can be methylated in vitro is approximately the same in the two classes of 30 S ethionine particles (1.25 and 0.8 respectively).

Preliminary studies of the biological activities of ethionine ribosomes are described. Free and associated 30 S “ethionine” subunits are found to have the same capacity to bind N‐acetyl‐phenylalanyl tRNA in the presence of poly(U), and free 30 S “ethionine” subunits are found to be 2–3 times more active in polyphenylalanine synthesis than the associated 30 S subunits. Free 50 S “ethionine” subunits are inactive for polyphenylalanine synthesis. The possible significance of the observations is discussed.