Polycistronic Messenger Research Articles

Sequential transcription and translation in the lactose operon of Escherichia coli

After induction of a culture of Escherichia coli by a thiogalactoside, β-galactoside transacetylase (A) appears after a lag about 1 min longer than that for β-galactosidase (Z) although the induction of both messengers appears to be immediate. As the translation time for A, as defined by labeling with an amino acid analogue, is shorter than that for Z, the extra lag is interpreted as being due to sequential reading of an integral operon-messenger. Also, the amount of A synthesized after deinduction is higher on a time equivalent basis than the amount of Z, the excess being interpreted as due to ribosomes on the polycistronic messenger which have terminated the translation of Z at the time of deinduction. The polar effect of radiation damage on the Z and A coding capacity of the messenger confirms the sequential model. Large deletions in Z and β-galactoside permease (Y) shorten the total lag of A correspondingly. Transcription is sequential in the same direction as translation since, in a culture induced in the presence of 5-fluorouracil, deinduction by dilution of the culture into uracil restores some of the A coding capacity but none of the Z coding capacity. Finally, the determination of the half-life of A coding capacity as the time constant of the decreasing rate of synthesis after a pulse indicates that the A part of the messenger can be inactivated independently from the Z part, thus accounting for the decreased molar yield of the A cistron product. A model consistent with colinear transcription, translation and breakdown processes is proposed.

Synthesis, utilization and degradation of lactose operon mRNA in Escherichia coli.

Abstract The action of inducer, and the kinetics of transcription, utilization, and degradation of lactose operon messenger RNA have been studied. Cells of Escherichia coli were made sensitive to actinomyein by brief treatment with EDTA, and then induced. The levels of β-galactosidase and thiogalactoside transacetylase were assayed at various times before and after the termination of induction by removal of inducer or addition of actinomyein. The results support the following conclusions. 1. (1) β-Galactosidase messenger RNA and transacetylase messenger RNA are degraded at approximately the same rate. If it is assumed that this operon produces a polycistronic messenger, then both ends are degraded at approximately the same rate. 2. (2) Inducer does not affect the rate of translation or the rate of degradation of β-galactosidase messenger RNA. 3. (3) After induction, 2.5 minutes (at 30 °C) are required for β-galactosidase induction to become insensitive to actinomyein. A total of four minutes is required for transacetylase induction to become insensitive to actinomyein. These results suggest that inducer action and the transcription of β-galactosidase take 2.5 minutes, while an additional 1.5 minutes is required to complete transcription of the entire operon. 4. (4) After the appropriate cistron has been transcribed, the remaining events culminating in appearance of either β-galactosidase or transacetylase take approximately 1.5 to 2 minutes. The results suggest that synthesis of β-galactosidase proceeds while transacetylase messenger RNA is still being formed. If this messenger RNA is polycistronic, it can be postulated that it is transcribed, translated and degraded simultaneously.

Regulation of Protein Synthesis: An Alternative to the Repressor-Operator Hypothesis

REGULATION OF PROTEIN SYNTHESIS: AN ALTERNATIVE TO THE REPRESSOR-OPERATOR HYPOTHESIS* M. GRUBER and R. N. CAMPAGNE\ The synthesis ofproteins is often regulated by specific "effectors," i.e., inducers and "co-repressors." These substances—usually compounds of low molecular weight—can elicit or inhibit the co-ordinated synthesis of a group of metabolically related proteins, e.g., the enzymes of a metabolic pathway. This, and genetic evidence, ledJacob and Monod [i, 2] to propose the Operon, a stretch of DNA comprising several structural genes, thus coding for a number of polypeptide chains. At one end of this Operon, eitherjust outside or just within the first structural gene, an operator was assumed switching the operon on and off. The effectors of protein synthesis, or possibly their immediate derivatives, were assumed to interact with a "repressor," preventing the transcription ofthe operon into RNA. The stereospecific interaction with the effector would change the repressor by an allosteric transition [3], thus inducing or destroying its affinity for the operator and thereby preventing or allowing the transcription ofthe operon. The repressor was assumed to be the product of a regulator gene which is not necessarily spatially or structurally related to the structural genes ofthe operon. This concept and its elaboration had a very stimulating effect on research in the field of regulated protein synthesis and created order in a mass of seemingly unrelated phenomena. One of the predictions of the analysis byJacob and Monod was the existence ofmessenger RNA, now a concept well established in molecular biology. Subsequent studies by many authors have shown that the operon is a reality. In a number ofcases, the clustering on the chromosome and the * An earlier version was published in Koninkl. Ned. Akad. Wetenschap., Proc, Series C, 68: i, 1965. t Professor and Associate Professor, respectively, Biochemistry Department of the University, Groningen, The Netherlands. 125 common regulation of genes for enzymes acting in the same metabolic pathway have been demonstrated [4]. The operon is probably transcribed into a single polycistronic messenger RNA which has only one starting point for the ribosome (polarity effects). It has been shown that the order offormation ofthe different enzymes ofan operon upon induction occurs sequentially beginning at the operator end [5]. Further, evidence has been obtained that regulation by effectors occurs at the level of the transcription of DNA into RNA, which does not mean that additional regulation at the level ofpolypeptide formation is excluded or that there cannot exist a coupled regulation at both levels [6]. Nature ofthe Repressor The nature [7] ofthe repressor, however, or indeed its existence in the sense ofJacob and Monod's hypothesis, has remained uncertain. Whereas genetic analysis has unequivocally demonstrated the existence of regulator genes, i.e., genes which through a cytoplasmic product control the induction or repression process, the identification of this product as a repressor directly interacting with an operator region has not been possible experimentally. Such a repressor should have two properties which, in our opinion, cannot be ascribed to any single class of substances: (1) The repressor should interact stowspecifically with the effector, and by this interaction should undergo conformational changes. (2) The repressor should interact se^HeMce-specifically with a rather long polynucleotide sequence. The first property requires that the repressor is a protein, since only proteins are known to possess such spatial specificity; it excludes DNA and RNA. The second property requires that the repressor is a polynucleotide, since proteins of a reasonable molecular weight cannot interact specifically with more than a few bases (or base pairs). In E. coli each operator should consist ofat least ten base pairs in order to specify each individual Operon;1 in higher organisms the number should be even larger. Such numbers of 1 The number for E. coli can be derived from either of the following calculations: (i) n base pairs are needed to discriminate between each individual operator and any other base sequence in the chromosome: 4 X io6 (total number ofbase pairs in the E. coli chromosome) ^ 411 (number of possible combinations with four bases at 11 sites); (2) five base pairs for an unequivocal signal indicating "starting point ofan operon" (any sequence offour base pairs will occasionally occur among neighboring...

Control of viral RNA translation as the mechanism of interferon action.

One of the most remarkable molecules of biological origin implicated in the control of gene expression is interferon. Discovered in 1957 by Isaacs and Lindenmann, this molecule, a slightly acidic protein with a molecular weight of about 30,000 (Merigan, 1964), is produced by the cell in response to an appropriate stimulus, usually viral nucleic acid. Interferon can induce in animal cells a state of refractoriness to infection by an enormously broad spectrum of viruses, including both RNA and DNA types. If virus infection is viewed as the introduction into susceptible cells of a new polycistronic messenger, the viral genome of RNA viruses and the transcribed genome of DNA viruses, then interferon presumably acts on the cell to prevent expression of most or all of the viral genome. Thus, information concerning the molecular basis of interferon action should provide insight into the basic problems concerned with the expression and control of...

Polarity in the tryptophan operon of E. coli.

Polarity appears to be a general characteristic of all operons that are transcribed into polycistronic messenger RNA (Ames and Martin, 1964; Newton; Martin et al., this volume) and is defined as the reduction in the relative rates of synthesis of those polypeptides specified by the genes of an operon on the operator-distal side of a mutated gene. Mutational changes which produce polar effects appear to have as a common consequence the introduction of a chain-terminating codon (Newton et al., 1965; Henning et al., 1965; Yanofsky and Ito, 1966; Martin et al., 1966). This is believed to result in the production of a polypeptide fragment of the normal chain, corresponding to the nucleotide sequence up to the chain-terminating codon (see Sarabhai et al., 1964), in addition to causing polar effects. Mutants of the amber, ochre and frameshift types, but not CRM+ mutants, show polarity (Newton et al., 1965; Henning et al.,...

A polycistronic messenger RNA associated with beta-galactosidase induction.

A polycistronic messenger RNA associated with beta-galactosidase induction.

The read-out of turnip yellow mosaic virus ribonucleic acid as a polycistronic messenger in cell-free systems of Escherichia coli

1. Association of turnip yellow mosaic virus ribonucleic acid and varying numbers of isolated 70-S ribosomes from Escherichia coli occurs at 0° under appropriate ionic conditions and in the absence of soluble enzymes and cofactors required for protein synthesis. 2. This association leads to the formation of polysomal aggregates of varying sizes up to octamers and decamers. The average size of these aggregates is substantially influenced by the ribosome-ribonucleic acid ratio. A saturation-point is reached in the range of 10–15 ribosomes added per messenger molecule. 3. Maximal incorporation of amino acids is observed in reaction mixtures containing ribosomes and viral ribonucleic acid in similar ratios. 4. A Mg 2+ content of 1.8·10 −2 M, which was found to be optimal for polypeptide synthesis, appeared highly favourable also for polysome formation. 5. Association of ribosomes with turnip yellow mosaic virus ribonucleic acid apparently occurs in the complete absence of protein synthesis. The latter process may be initiated by adding amino acids, soluble enzymes and cofactors. 6. Turnip yellow mosaic virus ribonucleic acid may be considered as a polycistronic messenger. It is suggested that many if not all cistrons are accessible before the onset of polypeptide formation. However, all cistrons are not necessarily transcribed with the same efficiency. 7. Cell-free extracts of E. coli rapidly inactivate turnip yellow mosaic virus ribonucleic acid following first-order kinetics.