Ribosomal protein synthesis by a mutant of Escherichia coli

Abstract

The mutant strain of Escherichia coli, TP28, synthesises ribosomes by an abnormal pathway and accumulates large quantities of 47s ribonucleoprotein particles. The protein complement of mutant 70s ribosomes is normal but 47s particles contain only traces of proteins L28 and L33 and have a significantly reduced content of four other proteins. The mutation reduces the rates of synthesis of L28 and L33 by about half but other widespread alterations ensue. In particular, ribosomal protein synthesis in the mutant strain becomes less well balanced than in its parent: some proteins, particularly those from promoter-proximal genes, are oversynthesized and their excess then degraded.The assembly of ribosomes by exponentially growing Escherichia coli is accompanied by the coordinated synthesis of ribonucleic acids and ribosomal proteins. Seven cistrons on the chromosome each code for the three ribosomal RNAs while the genes for the 50 or so ribosomal proteins are organized into transcription units that code for between one and eleven ribosomal proteins. Some units also contain genes for elongation factors for protein synthesis and subunits of RNA polymerase [I]. Assembly is coordinated so that under most conditions ribosome content matches growth rate and there are no appreciable pools of free RNA or of most ribosomal proteins [2]. One suggestion is that the nucleotide ppGpp is a major regulatory agent and reduces expression from ribosomal RNA (and perhaps some ribosomal protein) cistrons by interaction with RNA polymerase [3]. Another is that non-translating ribosomes feed back on ribosomal RNA synthesis; effects of ppGpp are then less direct [4]. In either case, a reduction in the rate of ribosomal RNA synthesis might lead to oversynthesis of ribosomal proteins. This is prevented by mechanisms in which ribosomal proteins bind to their mRNAs so as to inhibit translation or attenuate transcription [l, 51. These regulatory systems not only optimise the flow of components for ribosome synthesis but also enable assembly to continue under abnormal conditions, for example in organisms that contain plasmids incorporating truncated rRNA cistrons [6] or with multiple copies of ribosomal protein genes [7].In this paper we describe ribosomal protein synthesis in a mutant of E. coli with defects in ribosome synthesis and function. The mutant grows slowly but in a given medium has about twice the RNA content of its parent [8]. Mutant 70s ribosomes have about half the protein-synthetic activity and half the peptidyl-transferase activity of ribosomes from the parent strain and show reduced binding of analogues of peptidyl-tRNA and aminoacyl-tRNA. The proteins of 70s ribosomes from the mutant appear 'normal' (as judged by their mobilities and staining intensities on gels) and the reason for the reduced biological activity of the ribosomes is not Abbreviations. ppGpp, 3',5'-bis(diphosphate); SDS, sodium dodecyl sulfate. known [9, 101. Defects in ribosome assembly are also readily apparent because about half the 23s RNA of the mutant is in...

Rpl33, a Nonessential Plastid-Encoded Ribosomal Protein in Tobacco, Is Required under Cold Stress Conditions

“…The only experimental condition that elicited a strong phenotypic effect was chilling stress at 48C, suggesting that the lack of the chloroplast L33 protein renders plants sensitive to chilling. It is noteworthy in this respect that rpl33 loss-of-function mutants found in E. coli (Sims and Wild, 1976;Butler and Wild, 1984;Maguire and Wild, 1997) also displayed a cold-sensitive phenotype (Dabbs, 1991), suggesting that the cold stress-related function of L33 in translation has been evolutionarily conserved. However, E. coli rpl33 null mutants showed no measurable effect on translation in cells growing exponentially under normal conditions (Sims and Wild, 1976;Butler and Wild, 1984;Maguire and Wild, 1997), which is different from the situation in plants, where young expanding leaves show slightly reduced protein synthesis rates, as evidenced by a slightly delayed biogenesis of the photosynthetic complexes (Table 1).…”

Mutations in the rpmBG operon of Escherichia coli that affect ribosome assembly

“…A sample, resuspended in 1 ml of TMKSH buffer, was then mixed with one from 10 ml of a reference culture which had been labelled for two generations with 0.1 Ci of [ 14 C]lysine ml Ϫ1 . The mixed cells were broken, and carrier (nonradioactive) ribosomes from strain BM19 were added before proteins were extracted with acetic acid as described previously (2). Protein samples were analyzed by two-dimensional polyacrylamide gel electrophoresis in a system that combined the basic first dimension described by Madjar et al (15) with the second dimension described by Kenny et al (13).…”

The roles of proteins L28 and L33 in the assembly and function of Escherichia coli ribosomes in vivo

“…A mutant of E. coli with a defective rpmB,G operon is strain TP28 which, during slow exponential growth, oversynthesizes RNA and accumulates abnormal precursors ('47S particles') to 50S ribosomal subunits. This is because of a single base change in the Shine-Dalgarno sequence in the mRNA leader that halves the rates of synthesis of proteins L28 and L33 (Butler and Wild, 1984;Coleman et al, 1993). The 47S particles of strain TP28 lack proteins L28 and L33 and are deficient in three others.…”