Regulation of Gene Expression

Cellular function is influenced by cellular environment. Adaptation to specific environments is achieved by regulating the expression of genes that encode the enzymes and proteins needed for survival in a particular environment. Factors that influence gene expression include nutrients, temperature, light, toxins, metals, chemicals, and signals from other cells. Malfunctions in the regulation of gene expression can cause various human disorders and diseases.

Regulation in Prokaryotes

Bacteria have a simple general mechanism for coordinating the regulation of genes that encode products involved in a set of related processes. The gene cluster and promoter, plus additional sequences that function together in regulation are called an operon.

The Arabinose Operon

Arabinose is a five-carbon sugar that can serve as an energy and carbon source for E. coli. Arabinose must first be converted into ribulose-5-phosphate before it can be metabolized. The arabinose operon has three genes,araB, araA and araD that encode for three enzymes to carry out this conversion. A fourth gene, araC, which has its own promoter, encodes a regulatory factor called the C protein.

The regulatory sites of the ara operon include four sites that bind the C protein and one CAP binding site. The araO₁ and araO₂ sites are upstream of the promoter and CAP binding sites. The other two C protein binding sites called araI₁ and araI₂ are located between the CAP binding site and the promoter.

In the absence of arabinose, dimers of the C protein bind to araO₂, araO₁ and araI₁. The C proteins bound to araO₂ and araI₁ associate with one another causing the DNA between them to form a loop effectively blocking transcription of the operon.

The C protein binds arabinose and undergoes a conformational change that enables it to also bind the araO₂ and araI₂ sites. This results in the generation of a different DNA loop that is formed by the interaction of C proteins bound to the araO₁ and araO₂ sites.

The formation of this loop stimulates transcription of the araC gene resulting in additional C protein synthesis, thus the C protein autoregulates its own synthesis. In the absence of glucose, cAMP-CAP is formed which binds to the CAP site. C protein bound at the araI₁ and araI₂ sites interacts with the bound CAP enabling RNA polymerase to initiate transcription from the ara operon promoter.

Regulation of Gene Expression in Eukaryotes

The genetic information of a human cell is a thousand fold greater than that of a prokaryotic cell. Things are further complicated by the number of cell types and the fact that each cell type must express a particular subset of genes at different points in an organisms development. Regulating gene expression so that a particular subset of genes is expressed in a specific tissue at specific points of development is very complicated. This increased complexity in regulation lends itself to malfunctions that cause disease. Three ways that eukaryotes regulate gene expression will be discussed: alteration of gene content or position, transcriptional regulation and alternative RNA processing.

1. Alteration of Gene Content or Position

The copy number of a gene or its location on the chromosome can greatly effect its level of expression. Gene content or location can be altered by gene amplification, diminution or rearrangement.

The expression of a particular gene can be augmented by amplifying its copy number. Histone proteins and rRNA are needed in large quantities by almost all eukaryotic cells therefore the genes encoding histones and rRNA exist in a permanently amplified state. Gene amplification can present problems with the use of chemotherapeutic drugs. Methotrexate inhibits dihydrofolate reductase, the enzyme responsible for regenerating the folates used in nucleotide synthesis. Tumor cells often become resistant to the drug because the gene encoding dihydrofolate reductase is amplified by several hundred fold resulting in more enzyme production then the drug can handle.

A gene whose expression is only needed at a particular developmental point or in a particular tissue may be shut off by gene diminution. As reticulocytes mature into red blood cells all of their genes are lost as the nucleus is degraded.

Gene rearrangement is used to generate each of the genes encoding the millions of different antibodies that are produced by B cells. Sometimes bad gene rearrangements occur that lead to improper gene regulation. This frequently occurs in cancer cells. Translocation of a segment from chromosome 8 to chromosomes that encode immunoglobulins leads to activation of a gene that transforms healthy B cells into Burkitt's lymphoma cells (unregulated proliferating B cells).

2. Transcriptional Regulation

Regions of each of the different chromosomes are either packaged as heterochromatin or euchromatin. In heterochromatin the DNA is very tightly condensed and rendered inaccessible to the transcriptional machinery, consequently heterochromatin is transcriptionally inactive. In human females one of each of the two X chromosomes is completely inactivated by being packaged into a heterochromatin to form a Barr body. The Cys residues in DNA in the heterochromatin are heavily methylated suggesting that methylation may play a role in the maintenance of heterochromatin. Drugs that interfere with methylation cause activation of previously inactive genes found in heterochromatin.

In euchromatin the DNA is not as condensed and is accessible to the transcription machinery. The regions of a chromosome that are maintained as hetero- and eu- chromatin may vary in a cell specific manner. This may enable the cells of specific tissues to express a particular subset of genes required for tissue function.

Proteins that participate in regulating gene expression are often called trans acting elements. At least 100 different proteins, many specific for the regulation of a particular gene, are known. Others play a more general role in regulating gene expression in a manner analogous to the activation of numerous prokaryotic genes by the CAP-cAMP complex. Trans-acting factors have multiple domains required for activity and may include DNA-binding, transcription-activating and ligand-binding domains.

DNA binding domains recognize specific DNA sequences in the regulatory regions of a gene. The DNA-binding domains of a regulatory protein generally consist of one of three motifs: helix-turn-helix, zinc finger or leucine zipper. DNA-binding proteins possessing these motifs bind with high affinity to their recognition sites and with low affinity to other DNA. A very small portion of the protein makes contact with the DNA through H-bonds and van der Waals interactions between amino acid side chains and the functional groups in the major groove and the phosphate backbone of the DNA. The remainder of the protein is involved in proper positioning of the DNA-binding domain and in making protein-protein contacts with other transcriptional proteins.

Proteins with this motif form symmetric dimers that recognize a symmetric palindromic DNA sequence. Each monomer of the dimer contains a region in which two a helices are held at 90 degrees to each other by a turn of four amino acids. One set of helices makes contact with about five base pairs in the major groove. The other set sits atop the phosphate backbone and helps to properly position the set of helices that fits into the major groove.

Proteins possessing this motif contain between 2 to 9 repeated domains that are each centered on a tetrahedrally coordinated zinc ion. Each zinc coordinated domain forms a loop containing an a-helix, this loop is called a zinc-finger. There are two types of zinc fingers: the C₂H₂ finger and the C_x finger.

C₂H₂ Finger:

Three fingers interact with the major groove and wrap around the DNA. Many transcription factors have this type of domain.

C_x Finger:

Proteins with this motif bind as dimers to the major groove of the DNA. Many steroid receptors have this type of domain.

Proteins with this type of motif have an amphipathic a-helix at their carboxyl terminus. One side of the helix consists of hydrophobic groups, usually leucine, that are repeated every seventh position for several turns of the helix. The other face consists of charged and polar groups.

These domains generally act separately and independently of the DNA-binding domains. Transcription-activating domains enhance transcription by physically ineracting with other regulatory proteins and/or with RNA polymerase. The actual mechanisms by which these domains activate or enhance transcription are not known.

Steroid hormones, thyroid hormones and retinoic acid are examples of ligands that activate transcription by binding to a specific domain on a receptor protein. Upon binding the receptor undergoes a conformational change that enables it to bind DNA. Once bound to the DNA a receptor protein can activate or repress transcription of the target gene.

Cis-acting elements are DNA sequences that are recognized and bound by the trans-acting elements that regulate transcription. There are two major types of cis-acting elements: promoters and regulatory elements.

Promoters are the sites where RNA polymerase must bind to the DNA in order to initiate transcription (see "RNA Synthesis and Processing" lecture). The rate or efficiency of promoter use by RNA polymerase is affected by the regulatory elements.

Regulatory elements are specific DNA sequences that are recognized and bound by the trans-acting elements that stimulate or inhibit the expression of a particular gene. There are two types: enhancers and response elements.

Enhancers are regulatory elements that increase or repress the rate of gene transcription.

Response Elements are regulatory sequences that facilitate the coordinated regulation of a group of genes. Certain ligands such as steroid hormones and cAMP bind to their receptors which in turn bind to their response element to activate or inhibit transcription.

3. Alternative Processing

Initiating transcription at an alternative start site places a different exon at the 5' end of the transcript. Examples of genes that use alternative start sites as a form of regulation include amylase, myosin and alcohol dehydrogenase.

Immunoglobin (antibody) heavy chains use an alternative polyadenylation site to affect the length of transcripts. The longer transcript encodes the m_m form which is localized to the cell membranes of lymphocytes, the shorter transcript encodes the secreted form, m_s.

Alternative splice sites are used to generate similar proteins with tissue specific functions called isoforms. Many peptide hormones exist as isoforms such as the calcitonin gene which is differentially spliced to produce calcitonin in the thyroid and calcitonin gene-related peptide in the neurons.

The stability of mRNA is quite variable form gene to gene. These variations in stability govern the length of time that mRNA is available for translation and hence the amount of protein that is synthesized. The half-lives of mRNA vary from 10 hours to minutes. Sequences in the 3' untranslated region of mRNA which serve as signals for rapid degradation have been identified in some mRNA's with very short half-lives. The length of the poly A tail also affects mRNA stability, with longer tails tending to have longer half-lives.

© Dr. Noel Sturm 2020