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Biomedical subjects

J Sykes

Publications and source records attributed to J Sykes.

52 records · Page 3Linked to original sources
PubMed1977

The nature of the proteins in 'chloramphenicol particles' from Escherichia coli A19 (Hfr rel met rns).

The unusual particles which accumulate in cell-free extracts from Escherichia coli A19 during chloramphenicol inhibition ('chloramphenicol particles') have been isolated by large-scale rate-zonal density gradient ultracentrifugation. The proteins and RNA species composing these particles have been examined. The rRNA species present are precursor and mature forms of 16S and 23S rRNA which accumulate during inhibition. The proteins prepared directly from the particles give strong multiple immunoprecipitates with antisera specific to 30S and 50S ribosomal proteins. The soluble proteins of the cell prepared in the same manner do not give this immunological reaction. Two-dimensional electrophoresis patterns of the proteins from the 'chloramphenicol particles' strongly resemble those for 30S and 50S ribosomal proteins, i.e. they are predominantly basic low molecular weight proteins, and are dissimilar to the patterns for the soluble proteins of the cell. It is concluded that the 'chloramphenicol particles' are a heterogeneous group of ribonucleoproteins comprising the bulk of the rRNA accumulating during inhibition in association with variable amounts of some of their corresponding ribosomal proteins. The particles are therefore not artefacts of preparation, as previously thought, but arrested ribosome precursors.

Bacterial Proteins↗
PubMed1977

The nature of the proteins present in the 'relaxed particles' from methionine-starved Escherichia coli A19 (Hfr rel met rns).

The 'relaxed particles' formed during methionine starvation of Escherichia coli A19 (Hfr rel met rns) have been isolated by large-scale rate-zonal density gradient ultracentrifugation. The proteins and rRNA species associated with these particles have been examined. The rRNA species present are precursor and mature forms of 16S and 23S rRNA. The bulk of the rRNA which accumulates during starvation is found within the particles. The proteins prepared directly from the particles give strong multiple immunoprecipitates with antisera specific to 30S and 50S ribosomal proteins. The soluble proteins, prepared and examined in the same manner, do not give this immunological reaction. Two-dimensional electrophoresis patterns of the proteins from the particles show that the proteins co-migrate with proteins from 30S and 50S ribosomes and are entirely dissimilar to the proteins prepared by the same methods from the soluble fraction of the cells. On the basis of these and other observations, it is concluded that the 'relaxed particles' are not artefacts but are arrested ribosome precursors containing both rRNA and certain ribosomal proteins. The free pool of ribosomal proteins is low in exponential-phase cells and is not significantly increased by a 2 h period of starvation for glucose. The implications of these observations concerning the proteins associated with 'relaxed' and 'chloramphenicol particles' are discussed in raltion to ribosome biogenesis and the stabilization of rRNA.

Bacterial Proteins↗
PubMed1974

The role of ribonuclease II in the maturation of precursor 16S ribosomal ribonucleic acid in Escherichia coli.

The suggested involvement of ribonuclease II in the maturation of rRNA has been examined directly by determining the activity of the enzyme and the amount of p16S rRNA in cell-free extracts from Escherichia coli A19 and its temperature-sensitive derivative N464 grown under experimental conditions designed to vary the amounts of enzyme and precursor independently. In strain A19 the enzyme showed maximum activity in circumstances where the amount of p16S rRNA was normal (e.g. exponential-phase cells) or raised eight times (e.g. during inhibition of growth by methionine starvation of the relaxed auxotroph or by chloramphenicol or puromycin treatment). In strain N464 at the non-permissive temperature the ribonuclease II activity may be decreased by 50% without effect upon the amount of p16S rRNA, whereas in methionine starvation of this strain the enzyme activity is at a maximum and the p16S rRNA is eight times that in exponential-phase cells. These observations are discussed in relation to the previously implied role of ribonuclease II in the maturation of rRNA within ribosome precursors.

Centrifugation↗
PubMed1973

A comparison of the unfolding and dissociation of the large ribosome subunits from Rhodopseudomonas spheroides N.C.I.B. 8253 and Escherichia coli M.R.E. 600.

1. The behaviour of the large ribosomal subunit from Rhodopseudomonas spheroides (45S) has been compared with the 50S ribosome from Escherichia coli M.R.E. 600 (and E. coli M.R.E. 162) during unfolding by removal of Mg(2+) and detachment of ribosomal proteins by high univalent cation concentrations. The extent to which these processes are reversible with these ribosomes has also been examined. 2. The R. spheroides 45S ribosome unfolds relatively slowly but then gives rise directly to two ribonucleoprotein particles (16.6S and 13.7S); the former contains the intact primary structure of the 16.25S rRNA species and the latter the 15.00S rRNA species of the original ribosome. No detectable protein loss occurs during unfolding. The E. coli ribosome unfolds via a series of discrete intermediates to a single, unfolded ribonucleoprotein unit (19.1S) containing the 23S rRNA and all the protein of the original ribosome. 3. The two unfolded R. spheroides ribonucleoproteins did not recombine when the original conditions were restored but each simply assumed a more compact configuration. Similar treatments reversed the unfolding of the E. coli 50S ribosomes; replacement of Mg(2+) caused the refolding of the initial products of unfolding and in the presence of Ni(2+) the completely unfolded species (19.1S) again sedimented at the same rate as the original ribosomes (44S). 4. Ribosomal proteins (25%) were dissociated from R. spheroides 45S ribosomes by dialysis against a solution with a Na(+)/Mg(2+) ratio of 250:1. During this process two core particles were formed (21.2S and 14.2S) and the primary structures of the two original rRNA species were conserved. This dissociation was not reversed. With E. coli 50S approximately 15% of the original ribosomal protein was dissociated, a single 37.6S core particle was formed, the 23S rRNA remained intact and the ribosomal proteins would reassociate with the core particle to give a 50S ribosome. 5. The ribonuclease activities in R. spheroides 45S and E. coli M.R.E. 600 and E. coli M.R.E. 162 50S ribosomes are compared. 6. The observations concerning unfolding and dissociation are consistent with previous reports showing the unusual rRNA complement of the mature R. spheroides 45S ribosome and show the dependence of these events upon the rRNA and the importance of protein-protein interactions in the structure of the R. spheroides ribosome.

Centrifugation, Density Gradient↗