Stimulation of in vitro RNA synthesis by ribosomes and ribosomal proteins

Abstract

RNA synthesis by purified RNA polymerase on bacteriophage T4 DNA in vitro was stimulated two- to threefold when 70 s ribosomes from Escherichia coli were present. This activity was retained in the ribosomes washed with NH 4Cl and thus free of translational initiation factors. Both ribosomal subunits, 30 s and 50 s, stimulated RNA synthesis. Stimulation by 50 s subunits ruled out the possibility that the stimulation of RNA synthesis is due to the ribosomes removing nascent RNA from the RNA-transcribing complex by specifically binding to the RNA. Instead, we found that the stimulation of RNA synthesis by ribosomes is related to the initiation process of RNA synthesis by RNA polymerase. Ribosomes stimulated the initial rate of RNA synthesis but had no effect on the rate of RNA chain elongation or the size of RNA produced. Stimulation of RNA synthesis by ribosomes required the presence of sigma factor. Delayed addition of ribosomes to the already initiated RNA-synthesizing system diminished the stimulatory effect of ribosomes. The stimulatory activity of ribosomes was recovered in the split protein fraction but not in the core material when 70 s ribosomes, 30 s and 50 s ribosomal subunits were banded in a CsCl equilibrium gradient. The activity was found only in acidic ribosomal proteins but not in basic ribosomal proteins. A few stimulatory acidic proteins were fractionated from both 30 s and 50 s ribosomal subunits by DE52 cellulose chromatography. The mode of stimulation of RNA synthesis by active ribosomal protein was similar to that by ribosomes and appears to be related to the initiation of RNA synthesis. Involvement of ribosomes or ribosomal proteins in the control of RNA synthesis through a positive control mechanism in conjunction with sigma factor is suggested.