A single glutamic acid residue plays a key role in the transcriptional activation function of lambda repressor

Bushman, Frederic D.; Shang, Charles; Ptashne, Mark

doi:10.1016/0092-8674(89)90514-x

Cited by 95 publications

(90 citation statements)

References 34 publications

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“…This a-helix was found to be amphipathic with hydrophilic residues exposed on one side of the helix and hydrophobic residues on the other. Such a-helices are often form strong activation regions in transcription factors (Bushman et al, 1989).…”

Section: Discussionmentioning

confidence: 99%

The MYB305 Transcription Factor Regulates Expression of Nectarin Genes in the Ornamental Tobacco Floral Nectary

et al. 2009

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We have isolated and characterized the cDNA encoding the ornamental tobacco (Nicotiana langsdorffii X N. sanderae) homolog of the antirrhinum (Antirrhinum majus) MYB305. This transcription factor was robustly expressed at Stage 12 of nectary development but was only weakly expressed in the earlier Stage 6 nectaries. The ornamental tobacco MYB305 contains a conserved R2R3 MYB DNA binding domain with 76 amino acids in the activation domain. A green fluorescent protein-MYB305 fusion localized to nucleus of tobacco protoplasts and yeast one-hybrid assays demonstrated that it functions as a transcription activator. A conserved 23-amino acid C-terminal domain is required to activate gene expression. The coding region of the myb305 cDNA was expressed in Escherichia coli as a glutathione S-transferase fusion protein and was purified to homogeneity. This protein shows binding to two consensus MYB binding sites on the ornamental tobacco Nectarin I (nec1) promoter as well as to the single site located on the Nectarin V (nec5) promoter. Deletions of either of the binding sites from the nec1 promoter significantly reduced expression in nectary tissues. Temporally, MYB305 expression precedes that of nec1 and nec5, as would be expected if the MYB305 factor regulates expression of the nec1 and nec5 genes. Ectopic expression of MYB305 in foliage was able to induce expression of both nec1 and nec5, as well as two flavonoid biosynthetic genes in the foliage. Finally, RNA interference knockdown of MYB305 resulted in reduced expression of both nectarins and flavonoid biosynthetic genes. We conclude that expression of MYB305 regulates expression of the major nectarin genes in the floral nectary.

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Section: Discussionmentioning

confidence: 99%

The MYB305 Transcription Factor Regulates Expression of Nectarin Genes in the Ornamental Tobacco Floral Nectary

et al. 2009

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“…Detailed studies on the structure of the pCIGlu^°^ DNA-binding domain could provide insight into the mechanism of myb-mediated transcriptional activation. Single amino acid substitutions within the helix-turn-helix DNA-binding domain of bacteriophage X repressor have been shown to dramatically decrease transcriptional activation (Bushman et al 1989), possibly by alteration of an RNA polymerase direct contact point. Specific amino acid residues located within the MyoDl DNA-binding domain are also believed to be critical for transcriptional activation function (Davis et al 1990).…”

Section: Discussionmentioning

confidence: 99%

Identification of functional domains in the maize transcriptional activator C1: comparison of wild-type and dominant inhibitor proteins.

1991

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Genes encoding fusions between the maize regulatory protein CI and the yeast transcriptional activator GAL4 and mutant CI proteins were assayed for their ability to trans-activate anthocyanin biosynthetic genes in intact maize tissues. The putative DNA-binding region of CI fused to the transcriptional activation domain of GAL4 activated transcription of anthocyanin structural gene promoters in cl aleurones, cl Rscm2 embryos, and cl r embryogenic callus. Cells receiving these constructs accumulated purple anthocyanin pigments. The Cl acidic region fused to the GAL4 DNA-binding domain activated transcription of a GAL4-regulated promoter. An internal deletion of Cl also induced pigmentation; however, frameshifts in either the amino-terminal basic or carboxy-terminal acidic region blocked tra/is-activation, and the latter generated a dominant inhibitory protein. Fusion constructs between the wild-type Cl cDNA and the dominant inhibitor allele Cl-I cDNA were used to identify the amino acid changes in Cl responsible for the Cl-I inhibitory phenotype. Results from these studies establish that amino acids within the myb-homologous domain are critical for transcriptional activation.

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“…5a). These libraries are based on a λ cI optimized mutant (cI opt ) with a strong activation region 39 . The protocol presented here is optimized for the construction of combinatorial libraries by Gibson assembly.…”

Section: Experimental Designmentioning

confidence: 99%

Intracellular directed evolution of proteins from combinatorial libraries based on conditional phage replication

2017

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Directed evolution is a powerful tool to improve the characteristics of biomolecules. Here we present a protocol for the intracellular evolution of proteins with distinct differences and advantages in comparison with established techniques. these include the ability to select for a particular function from a library of protein variants inside cells, minimizing undesired coevolution and propagation of nonfunctional library members, as well as allowing positive and negative selection logics using basally active promoters. a typical evolution experiment comprises the following stages: (i) preparation of a combinatorial M13 phagemid (pM) library expressing variants of the gene of interest (GoI) and preparation of the Escherichia coli host cells; (ii) multiple rounds of an intracellular selection process toward a desired activity; and (iii) the characterization of the evolved target proteins. the system has been developed for the selection of new orthogonal transcription factors (tFs) but is capable of evolving any gene-or gene circuit function-that can be linked to conditional M13 phage replication. Here we demonstrate our approach using as an example the directed evolution of the bacteriophage l cI tF against two synthetic bidirectional promoters. the evolved tF variants enable simultaneous activation and repression against their engineered promoters and do not cross-react with the wild-type promoter, thus ensuring orthogonality. this protocol requires no special equipment, allowing synthetic biologists and general users to evolve improved biomolecules within ~7 weeks.© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved. the chances of propagating nonfunctional library members because of multiple infections. A GOI with the desired characteristics upregulates gene VI expression on the AP, completing the phage life cycle. For example, a randomized TF library member that activates an artificial promoter upstream of gVI will increase its own phage production (Fig. 4a). In this way, a protein with novel desired properties can be selected after several rounds of reinfection. Applications of the methodThe method has been used to evolve a set of dual activator-repressor switches for orthogonal logic gates, based on bacteriophage λ cI variants, and multi-input promoter architectures, and these switches have been successfully applied in downstream synthetic gene circuits 19 . In general, the method is capable of evolving any gene-or gene circuit function-on the PM that can be linked to pVI production. This is analogous to previous uses of phageassisted continuous evolution (PACE) 22 (Fig. 4). With PACE, a wide range of medically and biotechnologically relevant biomolecules, including polymerases 22 , proteases 23 and genome-editing proteins 10 , as well as protein-protein interactions 24 , were linked to conditional M13 phage propagation. In principle, any application in which directed evolution approaches have been...

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A single glutamic acid residue plays a key role in the transcriptional activation function of lambda repressor

Cited by 95 publications

References 34 publications

The MYB305 Transcription Factor Regulates Expression of Nectarin Genes in the Ornamental Tobacco Floral Nectary

The MYB305 Transcription Factor Regulates Expression of Nectarin Genes in the Ornamental Tobacco Floral Nectary

Identification of functional domains in the maize transcriptional activator C1: comparison of wild-type and dominant inhibitor proteins.

Intracellular directed evolution of proteins from combinatorial libraries based on conditional phage replication

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