Gene Regulation

What are the molecular determinants and mechanisms of gene regulation?

• The most common mechanism of gene regulation is the decision to initiate transcription; gene expression is also regulated at the level of RNA processing, RNA degradation, protein synthesis, protein modifications, and protein degradation.

• Gene regulation is mediated by proteins called transcription factors (trans-acting factors) that bind to specific sites on DNA called cis-acting sites. Transcription factor binding to cis-acting sequences occurs through noncovalent interactions of amino acid side chains on the protein with the nucleotide bases in the major groove of DNA.

• The activity of transcription factors can be modulated allosterically by the binding of small-molecule ligands, by posttranslational modification, and by regulated degradation in a controlled manner.

• The two major types of gene regulation are positive control and negative control. In positive control, transcriptional activator proteins stimulate the transcription of target genes, whereas in negative control, transcriptional repressor proteins inhibit transcription.

• When a protein represses its own expression, the resulting negative feedback regulation maintains a constant level of protein. When a protein activates its own expression, the resulting positive feedback regulation drives the system into either a fully off or fully on state.

• Epigenetic states involve modifications to DNA or histone proteins that are inherited without changes to the DNA sequence. Two examples are cytosine methylation, which inhibits gene expression, and histone acetylation, which stimulates gene expression. Epigenetic states are maintained through multiple generations.

• Analysis of gene expression using reporter genes is an example of a biochemical application that uses recombinant DNA technology to investigate mechanisms of gene regulation.

How is prokaryote gene regulation mediated by control of operon transcription?

• Gene regulation in prokaryotes almost always occurs by the action of transcription factors working locally at the promoter of the gene being regulated. Prokaryotic transcription factor proteins operate directly on the RNA polymerase to activate or repress its action.

• Prokaryotic genes encoding proteins with related functions, such as a biosynthetic pathway, are often organized into single transcription units called operons. Expression of the entire set of genes can then be regulated by action at a single promoter.

• A regulon is a set of genes that are coordinately regulated but not physically linked to each other as in an operon.

• Genes encoding proteins required for catabolic pathways are expressed only when substrates are available; genes encoding proteins required for biosynthetic pathways are expressed only when the end product is at low levels in the cells.

How are the lac, SOS, and trp operons of E. coli regulated?

• The lac operon is expressed only when lactose is present in the environment and when glucose is absent.

• In the presence of lactose, the lac repressor dissociates from a site near the promoter, allowing RNA polymerase access to the promoter. In the absence of glucose, the level of cAMP is high, cAMP binds to the CRP activator, and the cAMP–CRP complex binds near the lac promoter to stimulate its expression.

• The SOS regulatory circuit of E. coli controls the response of the cell to conditions that damage DNA or inhibit DNA replication. During normal growth, the LexA repressor protein decreases expression of the SOS regulon.

• DNA damage activates the RecA protein to a form that stimulates LexA cleavage, inactivating LexA and turning on the SOS genes.

• The trp operon of E. coli has two regulatory controls. The first is negative control by the Trp repressor, which binds tryptophan and turns off transcription of the operon. The second mechanism is attenuation, which functions after transcriptional initiation to prevent transcription of the structural genes when the encoded biosynthetic enzymes are unnecessary.

• The mechanism of trp operon attenuation involves the formation of alternate mRNA structures as a consequence of coupled transcription and translation. One mRNA structure is a terminator, which causes RNA polymerase to terminate; the other is an antiterminator, which prevents the terminator from forming. The antiterminator forms when charged tryptophanyl-tRNA is unavailable, leading to read-through into the rest of the operon.

How do eukaryotic activator proteins regulate genes packaged in chromatin?

• Eukaryotic DNA is packaged into DNA–protein structures called nucleosomes, in which 147 bp of DNA wrap around a protein core called a histone octamer. Nucleosomes can be further compacted into higher-order structures, some of which inhibit gene expression.

• The default state of eukaryotic genes is that they are not expressed because they are packaged into condensed chromatin. Expression of eukaryotic genes is an active process that modifies chromatin in various ways to allow access by the transcription machinery.

• Eukaryotic transcriptional activator proteins do not interact directly with RNA polymerase II. Instead, they act to recruit other proteins to the genes being regulated. RNA polymerase II becomes part of the pre-initiation complex at a late stage.

• Transcriptional activator proteins have two major roles in transcription. First, they antagonize the repressive effects of chromatin by recruiting modifying proteins. Second, they recruit protein components that function in the general transcription machinery.

• One major class of nucleosome modification is chemical modification of the N-terminal histone tails. Common modifications are acetylation of lysines, phosphorylation of serines, and methylation of lysines. Enzymes that carry out these modifications are recruited by transcriptional activator and repressor proteins.

• Lysine acetylation is carried out by histone acetyltransferases (HATs). This modification is associated with actively transcribed genes. Deacetylation by histone deacetylases (HDACs) accompanies transcriptional repression of genes.

• Lysine methylation has different effects on transcription, depending on which residue is methylated. For instance, methylation at one particular site leads to a highly compacted structure for the chromatin.

• In highly compacted chromatin, the DNA itself is often methylated on cytosine residues. The resulting 5-methylcytosine is usually found in the palindromic sequence CG, and both strands are methylated. This pattern of methylation can readily be passed on during DNA replication and is highly stable.

• Nucleosomes can move laterally along the DNA or be removed from DNA by chromatin remodeling complexes, which are recruited to genes by activator proteins. These large multiprotein complexes are ATP-dependent and function to expose cis-acting sites for other transcription factors.

• Transcriptional activator proteins often bind at cis-acting sites called enhancers, which can function at variable distances from the promoter and in either orientation. They interact with the promoter by looping the DNA to assemble the pre-initiation complex.

• In multicellular eukaryotes, most genes are turned off in cells in which their products are not needed, usually by mechanisms that permanently silence them by placing them in highly condensed chromatin.

How can just four transcription factors induce pluripotency in differentiated cells?

• In single-celled eukaryotes, the expression of genes is typically controlled by environmental signals. One example of an environmental signal in yeast is the presence or absence of the sugar galactose. When galactose is present, it derepresses the activator protein GAL4.

• The GAL4 activator has a modular organization with a series of functional and structural domains. GAL4 contains a DNA binding domain and a separate activation domain.

• In Drosophila development, a large number of transcription factors is expressed in a defined sequence of events; in each case, the combination of factors dictates which transcription factors are turned on and off at later times.

• The expression of the Drosophila even-skipped (eve) gene occurs in a series of seven stripes along the axis of the embryo, where each stripe has a separate set of regulatory controls. The eve stripe 2 enhancer region contains cis-acting sites for four different transcription factors (Bicoid, Hunchback, Giant, and Krüppel) that function together to restrict expression of the eve structural gene to a small number of cells.

• Induced pluripotent stem (iPS) cells are generated by introducing DNA for four transcription factor genes (Oct4, Sox2, Klf4, and c-Myc), which function together to convert differentiated cells into pluripotent cells. Special culture conditions can be used to differentiate iPS cells into a number of distinct cell types.

• The discovery that differentiated cells can be converted into pluripotent cells using iPS technology led to three main conclusions: (1) the differentiated state is maintained by epigenetic mechanisms that can be reversed in tissue culture by the exogenous addition of four transcription factor genes, (2) the conversion of differentiated cells into iPS cells is a general process and not limited to specific differentiated cell types, and (3) induction of the pluripotent state requires expression of endogenous genes such as Nanog in addition to the exogenous addition of the genes Oct4, Sox2, Klf4, and c-Myc.