scholarly journals The control of ribonucleic acid synthesis in bacteria. The synthesis and stability of ribonucleic acid in rifampicin-inhibited cultures of Escherichia coli

1971 ◽  
Vol 122 (2) ◽  
pp. 161-169 ◽  
Author(s):  
W. J. H. Gray ◽  
J. E. M. Midgley
Keyword(s):  
Escherichia Coli ◽  
Ribonucleic Acid ◽  
Acid Synthesis ◽  
Polymerization Rate ◽  
23S Rrna ◽  
Considerable Proportion ◽  
Bacterial Growth Rate ◽  
Ribonucleic Acids ◽  
E Coli ◽  
Kinetics Of

A study was made of the kinetics of labelling of the stable ribonucleic acids (rRNA+tRNA) and the unstable mRNA fraction in cultures of Escherichia coli M.R.E.600, inhibited by the addition of 0.1g of rifampicin/l. Labelling was carried out by adding either [2-14C]- or [5-3H]-uracil as an exogenous precursor of the cellular nucleic acids. From studies using DNA RNA hybridization, the kinetics of the synthesis and degradation of mRNA was followed in the inhibited cultures. Although a considerable proportion of the mRNA labelled in the presence of rifampicin decayed to non-hybridizable products, about 25% was stabilized beyond the point where protein synthesis had finally ceased. It therefore seems unwise to extrapolate the results of studies on mRNA stability in rifampicin-inhibited cultures to the situation existing in the rate of steady growth, where there appears to be little, if any, stable messenger. The kinetics of labelling of RNA in inhibited cultures indicated that the clapsed time from the addition of rifampicin to the point at which radioactivity no longer enters the total cellular ribonucleic acids is a measure of the time required to polymerize a molecule of rRNA. At 37°C, in culture grown in broth, glucose–salts or lactate salts media, exogenous [2-14C]uracil entered rifampicin-inhibited cells and was incorporated into RNA for 2 3min after the antibiotic was added. Taking this time as that required to polymerize a complete chain of 23S rRNA, the polymerization rate of this fraction in the three media was 25, 22 and 19 nucleotides added/s to the growing chains. Similar experiments in cultures previously inhibited by 0.2g of chloramphenicol/l showed virtually identical behaviour. This confirmed the work of Midgley & Gray (1971), who, by a different approach, showed that the polymerization rate of rRNA in steadily growing and chloramphenicol-inhibited cultures of E. coli at 37°C was essentially constant at about 22 nucleotides added/s. It was thus confirmed that the rate of polymerization of at least the rRNA fraction in E. coli is virtually unaffected by the nature of the growth medium and therefore by bacterial growth rate.

scholarly journals The control of ribonucleic acid synthesis in bacteria. The synthesis and stability of ribonucleic acid in chloramphenicol-inhibited cultures of Escherichia coli

1971 ◽  
Vol 122 (2) ◽  
pp. 149-159 ◽  
Author(s):  
J. E. M. Midgley ◽  
W. J. H. Gray
Keyword(s):  
Escherichia Coli ◽  
Ribonucleic Acid ◽  
Acid Synthesis ◽  
Chain Elongation ◽  
23S Rrna ◽  
Initiation Rate ◽  
Rate Of Polymerization ◽  
Antibiotic Inhibition ◽  
Kinetics Of ◽  
Small Acceleration

The rate of polymerization of ribosomal ribonucleic acid chains was estimated for steadily growing cultures of Escherichia coli M.R.E.600, from the kinetics of incorporation of exogenous [5-3H]uracil into completed 23S rRNA molecules. The analytical method of Avery & Midgley (1971) was used. Measurements were made at 37°C, in the presence or the absence of chloramphenicol, in each of three media; enriched broth, glucose–salts or sodium lactate–salts. The rate of chain elongation of 23S rRNA was virtually constant in all media at 37°C, as 24±4 nucleotides added/s. Accelerations in the rate of biosynthesis of rRNA by chloramphenicol in growth-limiting media are due primarily to an increase in the rate of initiation of new RNA chains, up to the rates existing in cultures growing rapidly in broth. Thus, in poorer media, only a small fraction of the available DNA-dependent RNA polymerase molecules are active at any given instant, since the chain-initiation rate is limiting in these conditions. In cultures growing rapidly in enriched broth, antibiotic inhibition caused a rise of some 12% in the rate of incorporation of exogenous uracil into total RNA. This small acceleration was due entirely to the partial stabilization of the mRNA fraction, which accumulated as 14′ of the RNA formed after the addition of chloramphenicol. In cultures growing more slowly in glucose–salts or lactate–salts media, chloramphenicol caused an immediate acceleration of two- to three-fold in the overall rate of RNA synthesis. Studies by DNA–RNA hybridization showed that the synthesis of mRNA was accelerated in harmony with the other affected species. However, just over half the mRNA formed after the addition of chloramphenicol quickly decayed to acid-soluble products, whereas the remainder was more stable and accumulated in the cells. The mRNA fraction constituted about 6% of the total cellular RNA after 3h inhibition. A model was suggested to explain the partial stabilization and accumulation of the mRNA fraction and the acceleration in the rate of synthesis of mRNA when chloramphenicol was added to cultures in growth-limiting media.

scholarly journals The control of ribonucleic acid synthesis in bacteria. The synthesis and stability of ribonucleic acids in relaxed and stringent amino acid auxotrophs of Escherichia coli

1972 ◽  
Vol 128 (5) ◽  
pp. 1007-1020 ◽  
Author(s):  
W. J. H. Gray ◽  
J. E. M. Midgley
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Heterogeneous Material ◽  
Acid Synthesis ◽  
Sucrose Density Gradient ◽  
23S Rrna ◽  
Ribonucleic Acids ◽  
Mrna Content ◽  
High Concentrations ◽  
The Stability

The biosynthesis and stability of various RNA fractions was studied in RCstr and RCrel multiple amino acid auxotrophs of Escherichia coli. In conditions of amino acid deprivation, RCstr mutants were labelled with exogenous nucleotide bases at less than 1% of the rate found in cultures growing normally in supplemented media. Studies by DNA–RNA hybridization and by other methods showed that, during a period of amino acid withdrawal, not more than 60–70% of the labelled RNA formed in RCstr mutants had the characteristics of mRNA. Evidence was obtained for some degradation of newly formed 16S and 23S rRNA species to heterogeneous material of lower molecular weight. This led to overestimations of the mRNA content of rapidly labelled RNA from such methods as simple examination of sucrose-density-gradient profiles. In RCrel strains the absolute and relative rates of synthesis of the various RNA fractions were not greatly affected. However, the stability of about half of the mRNA fraction was increased in RCrel strains during amino acid starvation, giving kinetics of mRNA labelling and turnover that were identical with those found in either RCstr or RCrel strains inhibited by high concentrations of chloramphenicol. Coincidence hybridization techniques showed that the mRNA content of amino acid-starved RCstr auxotrophs was unchanged from that found in normally growing cells. In contrast, RCrel strains deprived of amino acids increased their mRNA content about threefold. In such cultures the mRNA content of accumulating newly formed RNA was a constant 16% by wt.

scholarly journals Patterns of Ribonucleic Acid Synthesis in T5-infected Escherichia coli

1970 ◽  
Vol 245 (10) ◽  
pp. 2679-2692
Author(s):  
David A. Sirbasku ◽  
John M. Buchanan
Keyword(s):  
Escherichia Coli ◽  
Ribonucleic Acid ◽  
Acid Synthesis

scholarly journals The Control of Ribonucleic Acid Synthesis in Escherichia coli

1970 ◽  
Vol 245 (9) ◽  
pp. 2309-2318 ◽  
Author(s):  
Michael Cashel ◽  
Barbara Kalbacher
Keyword(s):  
Escherichia Coli ◽  
Ribonucleic Acid ◽  
Acid Synthesis

scholarly journals Patterns of Ribonucleic Acid Synthesis in T5-infected Escherichia coli

1971 ◽  
Vol 246 (6) ◽  
pp. 1665-1676
Author(s):  
David A. Sirbasku ◽  
John M. Buchanan
Keyword(s):  
Escherichia Coli ◽  
Ribonucleic Acid ◽  
Acid Synthesis

scholarly journals Binding Site of the Bridged Macrolides in the Escherichia coli Ribosome

2005 ◽  
Vol 49 (1) ◽  
pp. 281-288 ◽  
Author(s):  
Liqun Xiong ◽  
Yakov Korkhin ◽  
Alexander S. Mankin
Keyword(s):  
Escherichia Coli ◽  
Tight Binding ◽  
Dimethyl Sulfate ◽  
23S Rrna ◽  
Side Chain ◽  
Macrolide Antibiotics ◽  
Side Chains ◽  
Alkyl Aryl ◽  
E Coli ◽  
Exit Tunnel

ABSTRACT Ketolides represent the latest group of macrolide antibiotics. Tight binding of ketolides to the ribosome appears to correlate with the presence of an extended alkyl-aryl side chain. Recently developed 6,11-bridged bicyclic ketolides extend the spectrum of platforms used to generate new potent macrolides with extended alkyl-aryl side chains. The purpose of the present study was to characterize the site of binding and the action of bridged macrolides in the ribosomes of Escherichia coli. All the bridged macrolides investigated efficiently protected A2058 and A2059 in domain V of 23S rRNA from modification by dimethyl sulfate and U2609 from modification by carbodiimide. In addition, bridged macrolides that carry extended alkyl-aryl side chains protruding from the 6,11 bridge protected A752 in helix 35 of domain II of 23S rRNA from modification by dimethyl sulfate. Bridged macrolides efficiently displaced erythromycin from the ribosome in a competition binding assay. The A2058G mutation in 23S rRNA conferred resistance to the bridged macrolides. The U2609C mutation, which renders E. coli resistant to the previously studied ketolides telithromycin and cethromycin, barely affected cell susceptibility to the bridged macrolides used in this study. The results of the biochemical and genetic studies indicate that in the E. coli ribosome, bridged macrolides bind in the nascent peptide exit tunnel at the site previously described for other macrolide antibiotics. The presence of the side chain promotes the formation of specific interactions with the helix 35 of 23S rRNA.

scholarly journals In vitro reconstitution of the GTPase-associated centre of the archaebacterial ribosome: the functional features observed in a hybrid form with Escherichia coli 50S subunits

2006 ◽  
Vol 396 (3) ◽  
pp. 565-571 ◽  
Author(s):  
Takaomi Nomura ◽  
Kohji Nakano ◽  
Yasushi Maki ◽  
Takao Naganuma ◽  
Takashi Nakashima ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Ribosomal Proteins ◽  
Synthetic Activity ◽  
23S Rrna ◽  
Elongation Factors ◽  
Hybrid Form ◽  
Pyrococcus Horikoshii ◽  
E Coli ◽  
Functional Features

We cloned the genes encoding the ribosomal proteins Ph (Pyrococcus horikoshii)-P0, Ph-L12 and Ph-L11, which constitute the GTPase-associated centre of the archaebacterium Pyrococcus horikoshii. These proteins are homologues of the eukaryotic P0, P1/P2 and eL12 proteins, and correspond to Escherichia coli L10, L7/L12 and L11 proteins respectively. The proteins and the truncation mutants of Ph-P0 were overexpressed in E. coli cells and used for in vitro assembly on to the conserved domain around position 1070 of 23S rRNA (E. coli numbering). Ph-L12 tightly associated as a homodimer and bound to the C-terminal half of Ph-P0. The Ph-P0·Ph-L12 complex and Ph-L11 bound to the 1070 rRNA fragments from the three biological kingdoms in the same manner as the equivalent proteins of eukaryotic and eubacterial ribosomes. The Ph-P0·Ph-L12 complex and Ph-L11 could replace L10·L7/L12 and L11 respectively, on the E. coli 50S subunit in vitro. The resultant hybrid ribosome was accessible for eukaryotic, as well as archaebacterial elongation factors, but not for prokaryotic elongation factors. The GTPase and polyphenylalanine-synthetic activity that is dependent on eukaryotic elongation factors was comparable with that of the hybrid ribosomes carrying the eukaryotic ribosomal proteins. The results suggest that the archaebacterial proteins, including the Ph-L12 homodimer, are functionally accessible to eukaryotic translation factors.

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