Ribosomal Core Research Articles

A low molecular weight ribonucleic acid synthesized by Escherichia coli in the presence of chloramphenicol: Characterization and relation to normally synthesized 5 s ribonucleic acid

A low molecular weight RNA synthesized by Escherichia coli cells during exposure to chloramphenicol (100 μg/ml.) has properties which are similar, though not identical, to those of normally synthesized ribosomal 5 s RNA. We call it Cam-5 s RNA. Radioactive Cam-5 s RNA is annealed to E. coli DNA in a manner similar to that of normal 5 s RNA, and this hybridization can be inhibited in a competitive fashion by normal non-radioactive 5 s RNA. Cam-5 s RNA can replace normal 5 s RNA, when larger ribosomal subunits are reconstituted from lithium chloridetreated ribosomal cores and lithium chloride-split proteins. By thermal denaturation, the secondary structure of Cam-5 s RNA can be reversibly changed in a fashion similar to that of normal 5 s RNA, but none of the forms of Cam-5 s RNA coincide with the corresponding forms of normal 5 s RNA when they are co-chromatographed on columns of methylated albumin—kieselguhr. When 5 s RNA and Cam-5 s RNA are subjected to limited degradation by ribonuclease T 1 and fractionated by acrylamide gel electrophoresis, similar large partial digestion fragments are obtained from both RNA's. The over-all base compositions of Cam-5 s RNA and normal 5 s RNA are quite similar, and no methylated or other modified nucleotides could be detected in alkaline hydrolysates of Cam-5 s RNA which were fractionated in two dimensions by electrophoresis and descending chromatography. Two-dimensional fractionations of Cam-5 s RNA completely digested by pancreatic or T 1 ribonuclease demonstrate that the primary structure of Cam-5 s RNA is essentially identical to that of normal 5 s RNA except for the presence of different 5′-terminal nucleotide sequences: in Cam-5 s RNA, the normal 5′-terminal sequence pUpG- is absent and the following sequences are found, in a combined yield of one mole: pApUpUpUpG-, pUpUpUpG- and pUpUpG-. The RNA chains (Cam-5 s RNA) which have these 5′-terminal sequences are slightly longer than the 5′-terminus of normal 5 s RNA and probably represent precursor forms of mature 5 s RNA.

Ribosomal protein pool of Escherichia coli

Escherichia coli B cells, which were grown in a heavy medium ( 15NH 4Cl and 2H 2O) and then transferred to a light medium ( 14NH 4Cl and 1H 2O), contained an expanded pool of ribosomal proteins. The expansion was attributable to the partial breakdown of ribosomes formed in the heavy medium due to the density-transfer and the subsequent reutilization of the released intact ribosomal proteins for the synthesis of new ribosomes. Ribosomes were prepared from E. coli cells which were subjected to the density-transfer from a heavy medium to a light medium. Newly-formed light ribosomal cores were separated from pre-existing heavy ribosomal cores by CsCl equilibrium centrifugation. It was found that newly-formed light ribosomes contained radioactive proteins labeled only during growth in the heavy medium. These proteins were shown to be ribosomal and heavy, respectively, by acrylamide-gel electrophoresis and RbCl equilibrium centrifugation. The extent of incorporation of pulse-labeled proteins into ribosomes during a chase and also the extent of incorporation of newly-formed ribosomal RNA into ribosomes after the inhibition of protein synthesis by chloramphenicol were used to estimate the size of the soluble ribosomal protein pool. Normal cells contained a pool of about 10 % of the total ribosomal proteins. After density-transfer the size of the pool is expanded to approx. 20–25 % of the total ribosomal proteins. These results provided evidence that E. coli B cells can reutilize pre-existing ribosomal proteins for the synthesis of new ribosomes.

The requirement for tRNA for the shift in the optimum Mg ++ concentration during the synthesis of polyphenylalanine

Wheat germ contains a protein (tritin) that efficiently inhibits protein synthesis in cell-free extracts from animal cells but not from wheat germ. Tritin has been purified to apparent homogeneity and shown to block enzymatically polypeptide chain elongation. We have extended these studies to examine more closely the mechanism of tritin inactivation of animal cell ribosomes. Here we provide evidence suggesting that ATP and tRNA, previously thought to be tritin co-factors, function in the inhibition by altering the conformation of the ribosome to a form susceptible to tritin attack. Tritin treatment does not inhibit the binding of aminoacyl-tRNA to ribosomes, but it does partially reduce the ribosome binding of elongation factor 2. Tritin inhibition appears to affect the core ribosome and not ribosome-associated factors. However, the core ribosome itself is not a suitable substrate for tritin attack; a factor(s) is removed from ribosomes by high salt washing which is required for the tritin-induced inhibition. The ability to be inhibited is restored when ascites cell core ribosomes are supplemented with factors from either ascites cells or wheat germ. In contrast, neither ascites cell factors nor wheat germ factors will promote a significant tritin-induced inhibition of core ribosomes from wheat germ. This indicates that the specificity of tritin inhibition resides primarily at the level of the eukaryotic core ribosome.