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Studies on the low molecular weight RNA bound to e. coli ribosomes

University of British Columbia

Ribosomes were readily prepared by various procedures from coli B cells grown to the mid-log or late log phase. The ability of these ribosomes to support protein synthesis varied with the method of preparation. The low molecular weight RNA (LMWRNA) bound to these ribosomes was studied and it, too, varied with the type of preparation. After these initial studies, the procedure which entailed seven extractions with 0.5-1.0 M NH₄Cl in the presence of varying levels of magnesium concentration was used in all further experiments for the preparation of ribosomes (WRib). Attempts were made to remove all the tRNA bound to WRib. Treatment with HIO₄, resulted in a complete disruption of ribosomal structure and was abandoned. Puromycin (PM) treatment failed to remove all the bound tRNA but resulted in a more active preparation. Incubation of these PM-treated WRib with 0.1 mM Mg⁺⁺ not only removed all the bound tRNA but also resulted in inactive preparations. Treatment of the WRib with 0.1 mM Mg⁺⁺ alone gave identical results. Ribosomal subunits were found to be devoid of bound tRNA. Attempts to substitute these subunits in a protein-synthesizing system in place of whole ribosomes (WRib) failed. The LMWRNA bound to WRib was fractionated by a number of techniques - Sephadex G-100 chromatography, DEAE-Sephadex A-50, and preparative gel electrophoresis. The optical density patterns of the fractionations showed only small differences, however, acrylamide gel electrophoresis studies of the peak fractions indicated that chromatography on Sephadex G-100 was the method of choice for the separation of LMW-RNA. The tRNA bound to WRib was fully characterized. E. coli WRib have bound tRNA which has acceptor activity for all the amino acids tested. The amount of charging varied from one tRNA species to another - those for tryptophan and methionine were bound in the largest amounts. The significance of these results cannot, as yet, be explained. The total amount of tRNA bound to WRib amounted to approximately 1.3-1.7% of the total ribosomal RNA (rRNA). Similarly the amount of 5S RNA bound represented 1.5-2.8% of the total rRNA. These percentages are equivalent to 0.9-1.3 molecules of tRNA bound per molecule of 5S RNA. Another species of rRNA, 4.5S RNA was found bound to the WRib but in small amounts. Most of the 5S RNA preparations contained this RNA. The tRNA bound to WRib, prepared from a pyrimidine-requiring mutant (ATCC13135) grown in the presence of ³H-uracil, was exchanged with unlabeled tRNA. After exchange 5.7 molecules of tRNA were bound per molecule of 5S RNA and virtually all of the labeled tRNA had been removed. These exchanged WRib were no longer active in a protein-synthesizing system. The tRNA from these WRib was fractionated on a BD-cellulose column. The radioactivity was evenly spread throughout the salt gradient but a peak was isolated in the ethanol gradient. The radioactivity in this peak region may be accounted for by the presence of 4.5S RNA. The results suggest that all tRNAs are bound to the WRib to the same degree and indirectly supports the results reported in the literature of the non-existence of a specific chain-terminating tRNA.

Text

2011-03-21

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http://hdl.handle.net/2429/32652

Biochemistry and Molecular Biology

University of British Columbia

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