Helmut Ponta

Control of gene expression in bacteriophage T7: Transcriptional controls

SummaryTwo transcriptional control mechanisms of T7 can be distinguished both affecting the transcription by E. coli RNA polymerase: An early control and an “early-late” control. In wild type infections, both transcriptional control proteins appear at approximately the same time. Mutations in the early control gene have, therefore, little effect on transcription, if tested in the presence of virus RNA polymerase. Using mutants in T7 RNA polymerase, the appearance of the “early-late” control is delayed. Then, the effect of the early control gene is dramatic, its deficiency leading to an overproduction of host and early T7 RNA. The early RNA control appears to be exerted by the T7 protein kinase, the “early-late” control protein is most likely identical with the transcriptional inhibitor, which has been isolated and purified (Ponta et al., 1974). Both control proteins inhibit the initiation of RNA synthesis by E. coli RNA polymerase.

E. coli membranes become permeable to ions following T7-virus-infection.

SummaryInfection of E. coli with the viruses T7 or T3 leads to a dramatic efflux of potassium ions. This ion efflux is caused by the virus particle since no concomitant protein synthesis is required. T7 mutants carrying deletions in the M-gene (Schweiger et al., 1975), however, yield virus particles disturbed in the ion release.

Early T7 gene expression: rates of RNA synthesis and degradation, protein kinase dependent termination of transcription, and efficiency of translation.

SummaryThe mode of transcription of early T7 genes starting from one promotor region and generating a unique polycistronic RNA species suggests the appearance of equimolar amounts of the monocistronic species after RNA processing. Rate measurements revealed, however, a disproportion in the generation of the individual early RNA species. The rate of appearance of the promotor-proximal M gene message (nomenclature see in Table 1) is 4–5x the rate of appearance of all other species. This rate pattern is caused by termination behind the M gene because i) the rate of RNA degradation is fairly similar for most RNA species and ii) termination behind the M gene is released in a T7 mutant lacking protein kinase or in wild type infections in the absence of protein synthesis. Then, the RNA species are produced in equimolar amounts.The rate of degradation is similar for all early RNA species except for the protein kinase message. As measured by two independent methods, the physical halflives of M, POL, 1.1, and LIG (nomenclature see Table 1) message were 7–8 min (30°), while KIN RNA was degraded with a halflife of 4 min. The functional halflives were around 50% of the physical halflives. There is apparently no relationship between size of RNA and halflife, and the data suggest specific signals on each RNA which determine the rate of degradation.The monocistronic RNA species are utilized with different rates in translation. The M gene is not only transcribed more often, it is also translated with highest efficiency. The in vivo translation of the POL gene message occurred with the lowest rate.

A virus-specified mechanism for the prevention of multiple infection—T7- and T3-mutual and superinfection exclusion

SummaryCo-and superinfection of cells with T3/T7 result in exclusion (mutual or superinfection exclusion). The exclusion mechanism is also directed against homologous (or identical) virus. Exclusion is established after the adsorption but before the genome becomes available for gene expression or replication, that is only one virus per cell develops. The exclusion is triggered by a constituant of the viral particle. An early T7 gene (M gene) (Schweiger et al., 1975) is essential for the formation of exclusion competent virions.

Control of gene expression in bacteriophage T7

SummaryA transcriptional inhibitor is isolated and purified from phage T7 infected E. coli cells. The inhibitor is a protein of the molecular weight 14000. It interferes with the transcription by E. coli RNA polymerase no matter which template is used but it does not affect the action of T7 RNA polymerase. Inhibition of holoenzyme is more severe than that of core enzyme. The target of inhibition is the RNA polymerase which is blocked in initiation.

Development ofE. coli virus T1: The pattern of gene expression

SummaryT1 infected bacteria exhibit a distinct pattern of gene expression. The control of this expression is accessible to biochemical analysis. T1 induces the synthesis of 31 proteins inE. coli. The virion contains 15 proteins. By means of T1 amber mutants, 10 gene products have been assigned to specific T1 genes. Three classes of T1 proteins are defined by the kinetics of their syntheses: early, early-late and late proteins. The regulation of protein synthesis involves at least three mechanisms: for cessation of host gene expression, for discontinuation of the early class during the late phase and for induction of the late T1 proteins. The positive control of late gene expression is not coupled to replication. The host RNA-polymerase transcribes the viral genome throughout the infectious cycle. No virus coded RNA-polymerase is induced.

Membrane alteration induced by T7 virus infection.

To survive and multiply successfully in a host cell, an infecting virus has to induce immediate changes. In coli virus T7 infections a mechanism involving the viral M gene product avoids the digestion of its DNA by host restriction nucleases [ 1,2] . The same gene product is also responsible for the killing of the host cell [3] for ion efflux after infection [4] and for exclusion of competing viruses [5] . Previously, we have discussed the evidence indicating that these events are based on a very early membrane alteration induced by the virus [4]. Here we present direct evidence for just such a rapid membrane alteration after T7 infection, using pyrene as a fluorescence probe in the cell membrane.

Protein kinase ofAcetabularia

SummaryAcetabularia cells contain a protein kinase activity which transfers phosphate from ATP to serine or threonine residues of proteins. The enzyme does not respond to cAMP or cGMP. The specific activity increased during development and reached a maximum just before beginning of cap formation. The kinase appears to be a chloroplast associated enzyme.

BIOCHEMISTRY OF DEVELOPMENT OF E.COLI VIRUSES T7 AND T1

Publisher Summary This chapter elaborates the biochemistry of development of E . coli viruses T7 and T1. T7 proteinkinase functions as a kybernetic feedback regulator of early transcription. The gene for proteinkinase is the second early T7 gene. Mutations in the kinase genes are disturbed in the pattern of transcriptions. To uncover the effect of proteinkinase on transcription, the transcription of late genes by T7 RNA polymerase has to be eliminated. Double mutants in the genes for proteinkinase and for T7 RNA polymerase are deficient in the reduction of host RNA polymerase mediated transcription at both signals. Host RNA polymerase, then, transcribes through the whole genome with increased efficiency. In addition to inefficient termination, the described mutants turn off early T7 transcription and host transcription as measured by ribosomal RNA synthesis with considerable delay. It is found that proteinkinase restricts transcription in preparation of more efficient RNA synthesis by virus-coded RNA polymerase.

Messenger-selective inhibitor for the initiation of translation in Escherichia coli: Nitrofurantoin

Nitrofurantoin (S-nitro-2-furfurylidene-l-aminohydantoin) interferes with the synthesis of inducible proteins such as P-galactosidase or galactokinase much more than with the synthesis of most Escherichia coli proteins or of phage proteins [ 11. We have previously shown, mainly from experiments in vitro, that this inhibition is exerted at the initiation of translation. In vitro, the synthesis of galactokinase was inhibited by nitrofurantoin to the same extent whether DNA or RNA was used as template. The synthesis became resistant, however, if polysomes were permitted to read out from the same mRNA [ 11. In order to broaden the basis of these conclusions, a series of in vivo experiments has been performed. The new data not only confirm the selective action of nitrofurantoin on mRNA, but are suggestive of a direct interaction of nitrofurantoin with mRNA sequences.