The synthesis and function of ribosomes in a new mutant of Escherichia coli.

MacDonald, Russell E.; Turnock, G.; Forchhammer, Jes

doi:10.1073/pnas.57.1.141

Cited by 46 publications

(25 citation statements)

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“…Escherichia coli strain 15-28 is a mutant containing an abnormally high concentration of the immediate precursor to the 50 s ribosomal subunit (MacDonald, Turnock & Forchhammer, 1967). The strain grows much less quickly than the wild-type and a genetic analysis (Turnock, 1969) showed that at least two mutations are responsible for its complex phenotype.…”

Section: Introductionmentioning

confidence: 99%

A Mutant of Escherichia coli with a Defect in Energy Metabolism

Turnock

Erickson

Ackrell

et al. 1972

Journal of General Microbiology

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A mutant of Escherichia coli with a decreased growth efficiency has been investigated. The results of growth studies with different substrates and of measurement of P/O ratios in membrane preparations suggest that the strain is defective in the ability to couple synthesis of ATP to electron transport. I N T R O D U C T I O NEscherichia coli strain 15-28 is a mutant containing an abnormally high concentration of the immediate precursor to the 50 s ribosomal subunit (MacDonald, Turnock & Forchhammer, 1967). The strain grows much less quickly than the wild-type and a genetic analysis (Turnock, 1969) showed that at least two mutations are responsible for its complex phenotype. One of these mutations (b-; Turnock, 1969) confers upon the cell an altered response to the antibiotic streptomycin and a decreased efficiency in the utilization of the growth substrate, compared to the parent strain. The latter property is examined in this paper; the results obtained suggest that the mutation reduces the efficiency of energy metabolism, probably that associated with oxidative phosphorylation. The significance of this finding and ways in which other mutants defective in oxidative phosphorylation might be selected are discussed. METHODSStrains of Escherichia coli. The two strains, b-and b f , used in tlus paper were obtained by mating Hfr 15-5 with strain 1 5 -2 8~ (Turnock, 1969). They both carry the same str and thy alleles, but do not contain the mutation responsible for the high concentration of ribosome precursor in strain 15-28.Media and growth conditions. Unless otherwise stated, liquid cultures were grown with aeration by shaking at 37'. Culture population densities were measured spectrophotometrically at 450 nm with a Gilford microsample spectrophotometer (light path, I cm). The minimal salts medium used was that of Turnock (1969), supplemented with thymine (10 mg/l) and glucose (2 g/l) or carbon source as indicated. For the preparation of cell-free extracts the bacteria were grown in minimal medium with glucose as carbon source supplemented with I % vitamin-free Casamino acids.For growth under anaerobic conditions, inocula were grown in screw-capped bottles filled to the neck with medium and diluted into growth flasks bubbled with95 % N2, 5% CO,. When NO3-(as 0-1 % KNO,) was used as the terminal electron acceptor, the flasks were bubbled with N,, and NaHCO, (25 mM) included in the medium (Cox et al. 1970).Growth yields. The extinction of the culture, measured at 450 nm, is proportional to the cell mass (Schaechter, Maalere & Kjeldgaard, 1958) and so this parameter was employed to

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Section: Introductionmentioning

confidence: 99%

A Mutant of Escherichia coli with a Defect in Energy Metabolism

Turnock

Erickson

Ackrell

et al. 1972

Journal of General Microbiology

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“…The 70s ribosomes of strain TP28 have a normal complement of proteins yet function rather poorly in protein synthesis [8,23]. The reasons for this are not known; perhaps an altered pathway of assembly generates ribosomes of altered conformation.…”

Section: Discussionmentioning

confidence: 99%

“…The values of Ai for L28 and L33 thus set an upper limit to the content of native 50s subunits in the mutant. If free ribosomes regulate rates of RNA synthesis [4], a subunit deficiency might explain why the RNA content of the mutant is, in a given medium, about twice that of its parent [8].…”

Section: Discussionmentioning

confidence: 99%

“…The parent strain, Eschevichia coli 15 thy pro (15TP), and the mutant, TP28, derived from it [8] were grown with aeration at 30°C in a Tris-based minimal medium [I21 supplemented with glucose (0.2%), L-proline (50 pg/ml) and thymine (10 pg/ml). Doubling times were 60 -65 min (1 5TP) and 160 -180 min (TP28).…”

Section: Bacteria and Growth Conditionsmentioning

confidence: 99%

“…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.…”

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confidence: 99%

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Ribosomal protein synthesis by a mutant of Escherichia coli

Butler

Wild

1984

Eur J Biochem

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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...

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The assembly of ribosomes

et al. 1969

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Functionally active 30s ribosomes can be reconstituted i n vitro from 16s RNA and a mixture of 305 ribosomal proteins under certain defined conditions. Our previous studies on the specificity and kinetics of reconstitution are summarized and discussed. The reconstitution reaction is first-order with respect to formation of active 305 ribosomes. The rate-limiting reaction is probably unimolecular, and it represents the structural rearrangement of an intermediate. Presumed reconstitution intermediates, or RI particles, have been isolated from reconstitution mixtures incubated at low temperature. It has been concluded that the reconstitution takes place in stepwise fashion: 16s RNA. --?->RI particles-+ RI* particles ------+ 305 ribosomes. New experimental results that show the highly cooperative nature of the assembly reaction are described; the number of sites, per RNA chain, which can bind ribosomal proteins independently from each other (i.e., without cooperativity) is at most two to three. Finally, another approach to the study of ribosome assembly i n vivo is described. It utilizes cold-sensitive E. coli mutants that are defective in ribosome biosynthesis at low temperature and accumulate incomplete "intermediate" ribosomal particles.+ RI proteins heating + S proteins

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The synthesis and function of ribosomes in a new mutant of Escherichia coli.

Cited by 46 publications

References 19 publications

A Mutant of Escherichia coli with a Defect in Energy Metabolism

A Mutant of Escherichia coli with a Defect in Energy Metabolism

Ribosomal protein synthesis by a mutant of Escherichia coli

The assembly of ribosomes

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