Question

A biochemist replaces the DNA‐binding domain of the yeast Gal4 protein with the DNA‐binding domain of the Lac repressor and finds that the engineered protein no longer regulates transcription of the GAL genes in yeast.

A. Draw a diagram of the different functional domains you would expect to find in the Gal4 protein and in the engineered protein.

B. What might be done to the DNA‐binding site recognised by this chimeric protein to make it functional in activating transcription of the GAL genes?

Answer 1

SOLUTION-this protein is a positive regulator for the gene expression of the galactose-induced genes such as GAL1, GAL2, which code for enzymes used to convert galactose to glucose.it recognizes a 17 base pair in the yeast Saccharomyces cerevisiae regulation rest on a dosage-dependent functional interplay between the positive regulator of GLA4 protein and negative regulator, GLA80 protein.we have used this interplay to select in vitro generated fusions between the yeast ADH1promoter and GLA4 coding sequence the over product GLA4 PROTEIN, ALLOWING THE IDENTIFICATION OF gla4 PROTEIN IN CRUDE EXTRACTS FROM YEAST.one type of these constructions produces a GLA4 protein that lacks itsnormalNH2 terminus. this protein is unable to complement a GLA4 lesion but still remains a domain that functionally antagonizes the negative regulatory protein. the truncated protein also appears to affect changes in the relative transcriptional level of the structural genes.

SOLUTION-B- Fabry disease is an x-linked multi systematic lysosomal disorder caused by mutations of the alpha galactosides GLA gene. only a few of 450genetic lesions identified so far have been characterized by vitro expression studies

chimeric proteins are prepared by fusing the structural genes of the protein in question in the suitable expressions vector the DNA molecules to be fused can be short synthetic oligonucleotides or full-length structural gene

cell-penetrating peptides have been used to deliver nucleotide-based therapeutics to cells, but this approach has produced mixed results.

the cellular plasma membrane is a formidable protective barrier and considerable effort has been aimed at the development of delivery strategies for the introduction of exogenous biomacromolecules into mammalian cells both in vitro and Vivo.when the TAT peptides has been conjugated to polylysine the TAT functions exhibit significantly improved DNA delivery abilities compared to the TAT peptide alone taken together these results suggested that an optimal strategy may involve the certain of non-covenant protein/DNA complexes such that the DNA binding is mediated by a structured domain that is spatially separated from the cationic cell-penetrating domain.

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