Unabalanced Ribosomal Protein Synthesis in a Strain of Eascherichia coli Containing a Cloned, Truncated 16-S Ribosomal RNA Gene

Abstract

An Escherichiu coli K12 strain, carrying the promotor and proximal portion of the 16-S rRNA gene from rrnB cloned in the high-copy-number plasmid psF2124, has been examined for abnormalities in ribosome biogenesis. Both ribosomal RNA accumulation and ribosome content are depressed in this strain as compared to the control strain carrying the plasmid vector alone. The rate of total protein synthesis, however, appears to be normal. In contrast, the rate of ribosomal protein synthesis, relative to total protein synthesis, is elevated. The rates of synthesis of individual ribosomal proteins were determined and found to vary greatly, ranging from severe under-synthesis (displayed especially by proteins L7/L12) to massive over-synthesis (displayed particularly in the case of protein S7). Analysis of the rates of synthesis of other proteins coded for by the S12 operon revealed that protein S22 was moderately overproduced, but elongation factors EF-G and EF-Tu appear to be synthesized at the same rate as EF-Ts, all three being moderately under-synthesized relative to total soluble proteins.One of the central questions in molecular biology concerns the mechanisms that regulate the coordinate production of the three species of RNA and more than fifty different proteins that make up the ribosome particle in Escherichia coli. The genes coding for the ribosomal proteins (r-proteins) are scattered throughout the genome and are grouped into transcriptional units coding for from one to eleven proteins (ribosomal, elongation factor and RNA polymerase subunits). There are seven rRNA cistrons, each coding for the threc species of rRNA, which are clustered near the origin of chromosome replication. Despite this complexity each component is synthesized in the exact quantity necessary to produce the required number of completed particles. Since the discovery that ribosomal protein synthesis can be regulated at the translational level [I, 21, the hypothesis has been advanced that the primary site controlling ribosome biogenesis is at the level of ribosomal RNA transcription [I]. Regulation of ribosomal protein production is postulated to occur largely by feedback inhibition, either at the translational [I] or transcriptional [3] levels, by any r-protein in excess of that which is required by the available rRNA. A number of 'repressor' r-proteins (S4, S7, S8, L1, L4, L10) have been identified [3-61. Nevertheless, the mechanisms studied so far have not been completely successful in explaining the observed regulation of these cistrons it2 vivo, as has recently been discussed by Lindahl and Zengel [7].An additional system in which the coordinate regulation of rRNA and r-protein can be investigated would be helpful in unravelling this problem. The discovery that the presence of a cloned truncated 16-S rRNA gene on a high-copy-number plasmid causes net over-production of r-proteins provides such a system. The experiments described below indicate that in this strain there is a net over-production of ribosomal protein, some of which is subseq...