Screening of a genomic library to identify interacting partners of a “bait” protein is one of the common uses of the two-hybrid assay. To complete the screening system, you make a genetic library of “prey” fusion proteins by splicing a cDNA library containing random genes from the organism (the source of the bait protein) with a gene encoding a transcription-activation domain (e.g., the activation domain of Gal4p). You transfer the prey plasmid library into yeast, along with the bait fusion plasmid. Expression of β-galactosidase (as reporter gene) in the transformed yeast could be measured by the presence of blue colonies on plates containing X-gal (see Chapter 7); this will occur only in cells where the prey fusion protein interacts with the bait fusion protein. You can then sequence the prey gene to identify the interacting partner.

Because the 18 bp site is a near palindrome, the activator probably functions as a dimer. Given that both protein A and protein B are required for activating gene X, they may form a heterodimer that has specificity for the binding site. This could be tested in an electrophoretic mobility shift assay or any other assay that measures DNA binding. Alternatively, the 18 bp site might be bound by a third, unidentified protein, and proteins A and B might bind different DNA sites. To test this, the site could be used in a functional DNA-binding assay to follow the binding protein during purification, allowing identification of the correct protein. Footprinting could be used as an assay (see Chapter 20), and the DNA sequence could be used to make an affinity chromatography resin to aid purification (see Chapter 7). A final possibility is that protein A and/or protein B interact with a different protein to bind the 18 bp site. This could be tested by purification using a functional assay such as footprinting. The purified active protein would reveal the additional protein.

S-19

There are more hydrogen-bond donor and acceptor groups on the nucleotide bases in the major groove than on those in the minor groove, providing much better discrimination between bases.

Heterochromatin is highly condensed and transcriptionally inert, because the histone proteins make promoters inaccessible. The less-condensed euchromatin has undergone a structural remodeling, allowing some regions to be transcribed. The alterations include covalent modification (such as acetylation) of histones and displacement of nucleosomes, creating exposed regions of DNA that are probably binding sites for regulatory proteins.

The primary transcript of an miRNA that is an stRNA is about 70 nucleotides long, with self-complementary internal sequences that form hairpin structures. The precursor is cleaved into 20- to 25-nucleotide partial duplexes, one strand of which can bind complementary stretches in cellular mRNAs. This binding can inhibit gene expression by blocking translation or facilitating mRNA degradation.

Positive regulation. Positive and negative regulation are defined in terms of the type of protein involved in the regulation. Regulation by an activator is positive regulation; regulation by a repressor is negative regulation.

A leucine zipper motif. The motif contains Leu (L) residues at every seventh position. It often functions in transcription factors to form a dimer interface by forming a coiled-coil.

The regulon may be subject to combinatorial control, in which subsets of the regulon genes are needed in certain circumstances. The 13 genes may be subject to regulation by another regulatory protein—either another repressor that needs to be removed or an activator that needs to be present for transcription to occur.

Most repressors with helix-turn-helix motifs function as oligomers, many as homodimers. When the plasmid- encoded mutant repressor is synthesized at high levels in the cell, most of the wild-type repressor molecules synthesized are incorporated into less functional heterodimers with a mutant repressor subunit.

One of the best-characterized transcription-activation motifs in eukaryotes is a string of acidic amino acid residues. The acidic peptide segments fused to LexA functioned as activation motifs or domains. The LexA fusion proteins could bind to the correct site adjacent to gene X via the LexA operator and activate transcription of gene X by means of the acidic fused peptides.

Regulatory proteins with helix-turn-helix, helix-loop- helix, or homeodomain motifs generally function as dimers and bind to sequences with inverted repeats. Proteins with zinc finger motifs can function as monomers and have no constraint to bind inverted repeats. Thus, strings of zinc finger motifs can be linked together to bind almost any sequence, whether it contains repeated elements or not.

The process is called RNA interference. The enzyme Dicer cleaves the double-stranded RNA into small interfering RNAs (siRNAs), which can bind to the mRNA and prevent its translation.

On binding a hormone molecule, the steroid hormone receptor dimerizes and the hormone-receptor complex is transported into the nucleus.

There are several possible explanations. Many eukaryotic genes are regulated by more than one activator protein, and another activator may be needed. Many eukaryotic genes are encapsulated (and silenced) in heterochromatin, and remodeling of the chromatin may be required in the region where the gene is located to allow activation. The protein may need to be modified, and the modifying enzyme (e.g., kinase) may not be present in the cell. Finally, perhaps the activator cannot be transported into the nucleus.

(a) The regulatory sequences for the chromosomal GAL1 gene were known to respond only to Gal4p, and the DNA-binding elements of Gal4p had been removed in the fusion protein. (b) Given the finding that the fusion protein functioned as a repressor in E. coli, the researchers knew that the DNA-binding elements were properly folded and bound to the normal LexA-binding sites. (c) The LexA-Gal4 fusion protein is functional and stimulates gene expression to a level similar to that stimulated by Gal4p at UAS_G. (d) LexA by itself does not activate gene expression in this system. (e) Positioning of the LexA operator 577 bp rather than 178 bp away from the transcription start site lowers transcription activation by about 25%. (f) When the upstream sequences contain UAS_G or the 17mer, expression depends on the cellular Gal4p, which in turn is expressed only in the presence of galactose. (g) The LexA protein itself does not activate transcription; instead, a segment of Gal4p (that does not include the DNA-binding elements) is required. Thus the LexA part of the LexA-Gal4 fusion protein is not altering the DNA structure in any way that facilitates transcription. Instead, the Gal4p portion must be directly interacting with RNA polymerase.