Suppose you are planning to use the yeast two-hybrid assay to identify proteins that interact with a particular target protein (see Chapter 7). The assay makes use of the ability to separate the DNA-binding domain of a typical eukaryotic activator protein from its activation domain. You genetically fuse the gene encoding the protein you are studying (the “bait”) to the gene encoding the DNA-binding domain of the bacterial protein LexA, so that they are expressed as a single fusion protein. You place the binding site for LexA upstream from lacZ (encoding β-galactosidase) as a reporter gene—its expression can be selected for and easily detected. How might you design the rest of this genetic screen to identify the genes encoding proteins that interact with your bait protein?

Activator proteins A and B are required to express gene X. Analysis of the DNA upstream from the gene X promoter identified an 18 bp sequence with near twofold symmetry that is required for activation. Purification of the gene A and gene B products showed that both proteins form homodimers, but neither the A nor the B homodimer binds the 18 bp site. What are the possible functions of the A and B activators with respect to the 18 bp site? Propose a test of one of your ideas.

Most proteins that regulate gene expression bind at specific DNA sequences, recognizing those sequences primarily through protein-DNA interactions within in the major groove of the DNA. Why is the major groove used for sequence recognition more often than the minor groove or the phosphoribose backbone?

Briefly describe the relationship between chromatin structure and transcription in eukaryotes.

MicroRNAs known as small temporal RNAs (stRNAs) have been discovered in higher eukaryotes. Describe their characteristics and general function.

An effector molecule binds to an activator protein, changing the activator’s conformation so that it is no longer active. Transcription of the gene is thus shut down. Is this positive or negative regulation?

A transcription activator contains the following sequence:

IARLEEKVKTLKAQNSELASTANMLTEQVAQLKQ

The sequence includes a motif that may be used by certain transcription factors. What is this motif called? How does it function?

In one bacterial species, investigators find a regulon that coordinates the expression of 17 genes and identify a repressor that binds a defined site upstream from all the regulon genes. When the investigators inactivate the repressor protein, transcription of 4 of the genes increases. However, no transcription of the other 13 genes is observed, despite the presence of good promoters for RNA polymerase binding. Suggest a reason for the lack of transcription of these genes.

A repressor protein effectively blocks transcription from bacterial gene X. A mutant form of the repressor is engineered with an altered DNA-binding site in the helix-turn-helix motif. This mutant repressor does not repress transcription from gene X. When the mutant repressor is expressed at high levels on a plasmid that is introduced into the bacterial cell, transcription of X is increased even though the wild-type repressor (capable of binding its normal DNA binding site and shutting down transcription) is present in the same cell. Explain.

A eukaryotic transcription activator typically has separate DNA-binding and transcription-activation domains. The LexA protein is a bacterial repressor that binds to a particular LexA operator sequence. A researcher fuses random gene sequences from the E. coli genome to the gene encoding LexA. In yeast, the researcher replaces a binding site for a gene activator for gene X with the LexA operator. A small subset (about 1%) of the randomly constructed fused genes encode modified LexA proteins that activate gene X. Most of the “extra” gene material fused to the lexA gene encodes peptide segments that have high concentrations of amino acids with negatively charged side chains at neutral pH. Explain how the protein products of these gene fusions activate gene X. (Note: This problem reflects a classic published experiment.)

Zinc finger motifs have been appropriated for use in biotechnology. Several of these motifs can be strung together in an engineered protein, together with a fused nuclease domain, to create what has been dubbed a zinc finger nuclease. Such nucleases can be constructed to recognize and cleave almost any DNA sequence with high specificity. Explain why zinc finger motifs, rather than helix-turn-helix, helix-loop-helix, or homeodomain motifs, have been adapted for this purpose.

One of the classic ways to determine the function of a gene is to eliminate its function and determine the effect on the cell. Many approaches to eliminating gene function involve mutating or deleting the gene. In nematode worms, it is possible to shut down the function of almost any gene by synthesizing a double-stranded RNA complementary to the mRNA of the target gene and including it in the nematode’s food. Explain how this works and identify the cellular system involved.

Steroid hormone receptors are located in the cytoplasm, where they can interact with incoming hormones. However, steroid hormones act by regulating gene function, and genes are in the nucleus. How is this regulation achieved?

Expression of the CRP transcription activator in E. coli readily leads to transcription of the lactose metabolism genes when lactose is present and glucose is not. If a particular eukaryotic activator is expressed in the appropriate eukaryotic cell, introduced on an engineered virus or plasmid, it often does not trigger transcription of its target gene. Explain.