Synthetic control of gene expression is critical for metabolic engineering efforts Specifically, precise control of key pathway enzymes (heterologous or native) can help maximize product formation The fundamental level of transcriptional control takes place at promoter elements that drive gene expression Endogenous promoters are limited in that they do not fully sample the complete continuum of transcriptional control, and do not maximize the transcription levels achievable within an organism To address this issue, several attempts at promoter engineering have shown great promise both in expanding the cell-wide transcriptional capacity of an organism and in enabling tunable levels of gene expression Thus, this review highlights the recent advances and approaches for altering gene expression control at the promoter level Furthermore, we propose that recent advances in the understanding of transcription factors and their DNA-binding sites will enable rational and predictive control of gene expression

Promoter engineering: recent advances in controlling transcription at the most fundamental level.

Genomic Analysis of Bacteriophages SP6 and K1-5, an Estranged Subgroup of the T7 Supergroup

Genome of Xanthomonas oryzae bacteriophage Xp10: an odd T-odd phage.

Calcium plays a dual role in the activation of protein C: it inhibits protein C activation by alpha-thrombin, whereas it is required for protein C activation by the thrombomodulin-thrombin complex. Available information suggests that these calcium effects are mediated through calcium induced structural changes in protein C. In this paper, we demonstrate that substitution of Asp167 (located in the activation peptide of human protein C, occupying position P3 relative to the peptide bond Arg169-Leu170 which is susceptible to hydrolysis by thrombin) by either Gly or Phe results in protein C derivatives which are characterized by an altered response to calcium. At 3 mM calcium, alpha-thrombin activated the derivatives 5- to 8-fold faster compared with the wild-type, an effect which was shown to be caused by a decreased inhibitory effect of calcium on the reaction. These same single amino acid substitutions enhanced the affinity of the thrombomodulin-thrombin complex for the substrate at 3 mM calcium 3-(Gly-substitution) to 6-(Phe-substitution) fold, either without influencing kcat (Gly-substitution) or with a 2.5-fold decrease of kcat. For both derivatives, the calcium concentrations resulting in half maximal inhibition of activation by alpha-thrombin and in half maximal stimulation of activation by the thrombomodulin-thrombin complex increased from 0.3 mM to 0.6 mM. It is concluded that Asp167 is involved in the calcium induced inhibition of protein C activation by thrombin. Moreover, our studies demonstrate that it is feasible to enhance the efficiency of enzymatic reactions by introducing point mutations in the substrate.

https://www.embopress.org/doi/pdf/10.1002/j.1460-2075.1990.tb07411.x

Recombinant human protein C derivatives: altered response to calcium resulting in enhanced activation by thrombin.

Identification of a region of the bacteriophage T3 and T7 RNA polymerases that determines promoter specificity.

Recombinant phages between T7 and T3 have been isolated that grow well on strains of Escherichia coli that contain the F factor. One phage that has been characterized physically and genetically is predominately of the T7 genotype. Within this hybrid phage, two separate regions of T3 DNA have been located which are necessary for the phenotype of productive growth on F-containing strains. One of these, designated M1, contains the right part of gene 1 and continues through gene 1.3; the second, M2, appears to lie between gene 3 and gene 4.

Recombinants Between Bacteriophages T7 and T3 Which Productively Infect F-Plasmid-Containing Strains of Escherichia coli

The in vivo function of the gene 2 protein of bacteriophage T7 has been examined. The gene 2 protein appears to modulate the activity of the gene 3 endonuclease in order to prevent the premature degradation of any newly-formed DNA concatemers. This modulation is not however a direct interacton between the two proteins. In single-burst experiments rifamycin can substitute for the gene 2 protein, allowing formation of fast-sedimenting replicative DNA intermediates and progeny phage production. This suggests that the sole function of the gene 2 protein is inhibition of the host RNA polymerase and that the latter enzyme directs or promotes the endonucleolytic action of the gene 3 protein.

/pdf/the-role-of-bacteriophage-t7-gene-2-protein-in-dna-yb84vtu3dz.pdf

The role of bacteriophage T7 gene 2 protein in DNA replication

The T7-T3 recombinant phage BO2, whose isolation and physical properties are described in the accompanying paper (Molineux et al., J. Virol. 46:881-894, 1983), has been characterized by physiological means after infection of male (F plasmid-containing) and female strains of Escherichia coli. Single-step growth analyses have shown that the hybrid phage gives a burst about two-thirds that of T7 or T3 on females and about one-half that of T3 on males. This reduced burst size can be correlated with altered kinetics of macromolecular synthesis, probably at the level of transcription. The T3 insertions of BO2, designated M1 and M2, that are essential for growth of the hybrid phage on male strains, have been shown to be trans acting and can rescue T7 from F-mediated restriction. The nature of the gene products encoded by the M1 and M2 regions and their role in overcoming the abortive infection of males by T7 are discussed.

Physiological properties of a T7-T3 recombinant bacteriophage that productively infects strains of Escherichia coli that harbor the F plasmid.

Tandem promoter/enhancer units create a versatile regulatory element for the expression of genes in mammalian cells

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Recombinants Between Bacteriophages T7 and T3 Which Productively Infect F-Plasmid-Containing Strains of Escherichia coli

The role of bacteriophage T7 gene 2 protein in DNA replication

Physiological properties of a T7-T3 recombinant bacteriophage that productively infects strains of Escherichia coli that harbor the F plasmid.

Tandem promoter/enhancer units create a versatile regulatory element for the expression of genes in mammalian cells