Regulation of Gene Expression Help
Regulation of Gene Expression
In contrast to bacteria, most eukaryotic cells (some algae, yeast, and protozoa are a few notable exceptions) are not free-living single cells. Multicellular eukaryotes usually show cellular differentiation. Differentiation allows cells to become specialized for certain tasks; e.g., liver cells are highly metabolic, muscle cells contract, nerve cells conduct impulses, red blood cells carry oxygen. The signals that cause eukaryotic cells to differentiate are largely endogenous (within the multicellular body). Eukaryotic cells cooperate with one another to maintain a fairly uniform internal environment despite variation in environmental conditions exterior to the organism; this regulatory phenomenon is known as homeostasis. Bacteria can turn their genes on or off repeatedly in response to various nutrients such as glucose or lactose in their environment. Switching genes on or off during development of eukaryotic cells, however, is usually a permanent change. Once a cell has started to differentiate, it can seldom be diverted to another developmental pathway.
Gene expression in eukaryotes involves six major steps that can each serve as a potential point for regulating protein production:
- Uncoiling of nucleosomes and chromatin remodeling
- Transcription of DNA into RNA
- Processing of the nuclear RNA (nRNA) or pre-mRNA
- Transport of mRNA from nucleus to cytoplasm
- Translation of mRNA into a polypeptide chain
- Processing of the polypeptide chain into functional proteins
These six areas can be generally divided into three main points of control: transcriptional regulation (1, 2), regulation of RNA processing and (3, 4), and translational control (5, 6). Additional control strategies, such as multicopy genes, and epigenetics will also be discussed.
Promoters and the genes that they control are generally adjacent; the promoter is the DNA sequence to which the RNA polymerase binds to begin transcription. Addition sites, called enhancers, may be several hundreds or thousands of base pairs either upstream or downstream from the promoters they stimulate. DNA binding proteins that bind to enhancer sequences are called activators or repressors. They interact with a series of other proteins that ultimately connects to the scaffold of proteins interacting at the promoter. Promoters and enhancers are also referred to as cis-acting elements because they are located on the same DNA strand as the gene they control. Enhancers can activate or repress the transcription of a gene. In yeast, enhancers are often referred to as upstream activating elements, or UASs, because they are usually found upstream of a gene.
In order for proteins to bind to the DNA at these regulatory sequences, and for the DNA to unwind to be transcribed, it must be liberated from its tightly coiled chromatin structure. Chemical modifications are made to histone proteins so their grip on DNA is loosened leading to the disassembly of nucleosome structures. After transcription, the nucleosomes quickly reform on the double-stranded DNA.
Unlike bacterial genes with similar functions, eukaryotic genes are not arranged under the control of one promoter. However, some genes are coordinately regulated despite their existence on completely different chromosomes. This coordinate regulation most likely occurs through a set of proteins called transcription factors. These proteins, encoded by separate and distinct genes, bind to specific DNA sequences in promoters to promote or repress the initiation of transcription. Thus, transcription factors belong to the class of proteins called DNA binding proteins. In this way, genes in different parts of the genome can still be coordinately controlled. These proteins are often referred to as trans-acting factors because they are encoded by genes at other locations in the genome (even on different DNA molecules).Most genes respond to more than one signal or combinations of signals, known as combinatorial gene regulation. Gene regulation can occur in response to either endogenous or exogenous signals.
- Exogenous Signals. Gene regulation in prokaryotes occurs mainly in response to exogenous signals such as the presence or absence of nutrients (e.g., glucose or lactose). Most gene regulation in eukaryotes occurs in response to endogenous signals produced by other cell types, but not exclusively so.
- Endogenous Signals. The best-known endogenous regulators of gene activity in eukaryotes are the hormones. These are substances produced by one cell type that have effects on other cell types. Hormones are usually transported throughout the organism (e.g., via the bloodstream in animals) but interact only with those cells that have the corresponding receptors on their cell surface. Some small hydrophobic molecules, such as steroids, may pass freely through the cell membrane and the hormone receptor could be in the cytoplasm or in the nucleus. The interaction of hormone and receptor eventually would cause a signal to be transmitted to the DNA at one or more specific sites to activate or repress the appropriate gene or set of genes.
EXAMPLE 13.6 When plants are grown in darkness for several days they start to lose their green color (etiolation) because of loss of the enzymes that catalyze chlorophyll synthesis. Within a few hours after exposure of an etiolated plant to sunlight, more than 60 photosynthetic enzymes, chloroplast rRNA, and chlorophyll synthesis occur. A protein called phytochrome is covalently bound to a light-absorbing pigment. In the dark, phytochrome is inactive; in sunlight, it becomes activated and is thought to become a transcription factor for production of an unknown number of photosynthetic enzymes.
EXAMPLE 13.7 Only the oviduct cells of the chicken respond to an injection of the steroid hormone estrogen by synthesizing ovalbumin mRNA. Other cell types fail to respond to estrogen because they lack the corresponding receptor. It is proposed that estrogen enters the cell by diffusion and binds to a cytoplasmic protein receptor. The hormone-receptor complex then migrates into the nucleus and initiates transcription of the ovalbumin gene.
A family of membrane proteins called G proteins are interposed between some signal molecules (e.g., hormones or neurotransmitters) and an "amplifier enzyme." If the hormone binds to a cell surface receptor, it induces a conformational change in the receptor. This change is transmitted through the cell membrane to a G protein, making it able to bind guanosine triphosphate (GTP); hence, the G in the name for these proteins. Binding of GTP causes a conformational change in the G protein that enables it to activate an amplifier enzyme. If the amplifier enzyme is adenyl cyclase, its activation results in the production of cyclic AMP(the second messenger). The AMP can then regulate the activity of one or more genes coordinately.
Hormones might promote transcription by any of the following mechanisms:
- The hormone could cause DNA to become uncoupled from histones (dissolution from nucleosomes) and thereby allow RNA polymerase to begin transcription.
- The hormone might act as an inducer by inactivating a repressor molecule.
- The hormone may bind directly to specific DNA sequences to facilitate binding of RNA polymerase or of a protein transcription factor.
- The hormone may activate an effector protein (comparable to the CRP protein of the bacterial lac operon) so that the complex can bind to a site on the DNA and thereby stimulate binding of RNA polymerase.
- The hormone could become attached to a protein already bound to DNA and thereby form an active complex that stimulates binding of RNA polymerase.
Modification of DNA nucleotides may play a role in regulating gene transcription. Some genes whose products are usually synthesized only in particular cell types (e.g., hemoglobin in erythrocytes; immunoglobulins in plasma cells) appear to be heavily methylated in cells that do not express the corresponding gene products and unmethylated in cells where those genes are expressed. The genes involved in general metabolism common to all cells are rarely methylated in or near their initiation regions. These genes are called housekeeping genes. This mechanism of transcriptional regulation is carried out by proteins, such as the mammalian MBD1 protein, that bind preferentially to methylated cytosine residues at CpG DNA sequence islands. Housekeeping genes utilize CpG-dependent promoter elements and TATA boxes, which most tissue-specific genes do not. The binding or nonbinding affects transcription in a similar manner to other transcription factors.
In the late 1990s, a new kind of gene regulation in eukaryotes, called RNA interference (RNAi), was discovered. RNAi exhibits its effects through the actions of small interfering RNAs (siRNA) and micro RNAs (miRNA). These small RNA molecules are 22 bp long and interfere with the expression of larger RNA molecules, such as cellular mRNAs or invading viral RNAs, through interactions with complementary sequence motifs in the two RNAs. Interfering RNAs may arise as degradation products from viral RNAs, transposons or as transcription products from genes within the genome of the organism. A protein called dicer creates 22 bp double-stranded RNA (dsRNA) fragments, which bind to proteins to form a complex called RISC (RNA-induced silencing complex). This complex interacts with larger target RNA molecules at complementary sequences. Once RISC binds to an RNA molecule, its translation into a protein is silenced either by physically blocking translation by ribosomes or by causing its degradation. The presence of dsRNA appears to be the primary initiating trigger for RNAi, but other triggers are being investigated. This mechanism may have evolved as a defense mechanism against infection by double-stranded RNA viruses. Another complex of proteins and siRNAs called RITS, or RNA-induced transcriptional silencing complex, has been implicated in chromatin silencing, indicating an important role in epigenetic phenomona (see later section in this chapter).
Regulation of mRNA PROCESSING
Eukaryotic genes contain introns (noncoding regions) interspersed among the coding regions (exons). Part of the process that converts primary transcripts to mature mRNA molecules involves removal of the introns and splicing of the exons together. Variations in the excision and splicing jobs can lead to different mRNAs and, following translation, to different protein products. This type of gene regulation is called alternative splicing. Alternative splicing can play a significant role in developmental processes, such as sex determination in the fruit fly and production of immunoglobulin genes in mammals.
Eukaryotic genes contain introns (noncoding regions) interspersed among the coding regions (exons). Part of the process that converts primary transcripts to maturem RNA molecules involves removal of the introns and splicing of the exons together. Variations in the excision and splicing jobs can lead to different mRNAs and, following translation, to different protein products. This type of gene regulation is called alternative splicing. Alternative splicing can play a significant role in developmental processes, such as sex determination in the fruit fly and production of immunoglobulin genes in mammals.
EXAMPLE 13.8 Immunoglobulins of class IgM have μ-type heavy chains that are produced in two varieties as a consequence of differences in intron/exon excision/splicing events. When processed in one way, the m-chains are longer, ending with a group of hydrophobic amino acids at their carboxyl ends. This ''water-fearing'' tail tends to lodge in the lipid membrane and extends into the cytoplasm. The amino terminus extends outside the cell where it participates with an L chain to form an antigen-combining site (see Example 13.21). Thus, it becomes a cell receptor for a specific antigen. An alternative splicing step removes from the primary transcript the sequence responsible for the hydrophobic tail. This shorter version of the μ-chain readily passes out of the cell and becomes part of the secretory antibody population found in the blood and other body fluids.
Regulation of Translation, Protein Stability, and Activity
There are three major methods by which eukaryotic cells are known to regulate protein production at the translational level: (1) by altering the half-life, or stability, of the mRNA, (2) by controlling the initiation and rate of translation, and (3) by modification of the protein after translation.
A typical eukaryotic mature mRNA consists of four major regions: (1) a 5' noncoding region (leader), (2) a coding region, (3) a 3' noncoding region (trailer), and (4) a poly-A tail. Each of the four segments may affect the half-life of mRNA molecules.
EXAMPLE 13.9 The mRNA transcribed from a normal human gene c-myc is relatively unstable, with a half-life of about 10 min. A mutant form of c-myc that is missing some of the 5' noncoding region of the normal c-myc produces an mRNA that is 3–5 times more stable than full-length mRNA.
EXAMPLE 13.10 Within the coding region of a histone gene, repositioning of the stop codon closer to the 5' end of its transcript not only produces abnormally short histone proteins but also at least doubles the half-life of the mutant mRNAs.
EXAMPLE 13.11 The mRNAs for human β-globin and δ-globin (Example 13.1) differ mainly in their 3' noncoding segments, yet δ-globin mRNA is degraded four times faster than β-globin mRNA.
EXAMPLE 13.12 Mature mRNA molecules do not normally exist as naked mRNAs but as ribonucleoprotein. One of the proteins normally bound to mRNAs is a poly (A)-binding protein (PABP). Experimental removal of PABP from normal mRNAs decreases their half-lives. Removal of poly-A tails from otherwise normal mRNAs greatly reduces their half-lives. Just how these changes in mRNA molecules influence their susceptibility to digestion by ribonuclease enzymes is not presently known.
EXAMPLE 13.13 Unfertilized sea urchin eggs store large quantities of mRNA complexed with proteins as ribonucleoprotein particles. In this inactive form it is called masked mRNA. Within minutes after fertilization, the mRNA somehow becomes ''unmasked'' and translation begins.
The 5' untranslated regions (UTR) of mRNA molecules (leader sequence) can serve as regulators of translational initiation. This process is generally mediated by the presence or absence of a particular nutrient or metabolite. For example, there is a sequence present in the 5' UTR of the human ferritin gene, called an iron responsive element (IRE), that responds to the presence and absence of iron. Ferritin is a molecule involved in iron storage. When iron is absent, a protein, called the IRE-BP, can bind to the IRE sequence in the 5' UTR of ferritin. This prevents efficient translation of the ferritin mRNA. However, when iron is present, the IRE-BP can no longer bind to the IRE and translation can proceed efficiently.
Posttranslational modifications, such as ubiquitination, can target proteins for proteolysis. Ubiquitin is a small protein that when covalently attached to target proteins signals their destruction by a complex of proteins known as the proteosome. Many genes involved in cell cycle regulation are quickly destroyed by this mechanism, allowing newly produced proteins to carry out the next step. Modifications such as phosphorylation are mechanisms that regulate protein activity that may lead to regulation of protein production. For example, some proteins are only active (i.e., can carry out their enzymatic or DNA binding capabilities) if they are phosphorylated on particular amino acid residues. Phosphorylation is carried out by enzymes called kinases. Phosphate residues can be removed by enzymes called dephosphorylases. In complex systems, there is often a cascade of kinases and dephosphorylases that activates a series of protein targets, leading ultimately to a transcription factor. The transcription factor then becomes activated (due to phosphorylation or dephosphorylation), resulting in the regulation of transcription of a particular gene or set of genes.
Another posttranslational control mechanism involves protein processing. Eukaryotes synthesize only monocistronic mRNAs, but the resulting single polypeptide chains may be cleaved into two or more functional protein components. A multicomponent protein such as this is termed a polyprotein.
EXAMPLE 13.14 A polyprotein called pro-opiomelanocortin is synthesized by the anterior lobe of the pituitary gland. A cut near the C (carboxyl) terminus first produces β-lipotropin. Then a cut near the N (amino) terminus produces adrenocorticotropic hormone (ACTH). In the intermediate lobe of the pituitary, β-lipotropin is further digested, releasing the C-terminal peptide β-endorphin; the ACTH is also cleaved to release α-melanotropin. Polypeptides that are destined to be released from the cell (after being processed in the Golgi apparatus) possess a signal peptide. This peptide usually consists of about 20 amino acids at or near the N terminus of a polypeptide chain. It serves to anchor the nascent polypeptide (as it is being synthesized) and its ribosome to the endoplasmic reticulum.
The abundance of a species of RNA molecules may be regulated at the gene level by several mechanisms that serve to amplify the copy number of the gene. In order to understand some processes of selective gene amplification, a distinction must be made between germ-line genes (those that are passed on to offspring) and somatic genes (not hereditary). Evolution progresses by sequential modifications of preexisting developmental patterns. Whatever mechanism that works initially to result in the solution to a biological problem tends to become so integrated into the overall developmental program with the passage of many generations that it cannot be changed. Thus, it is not surprising that different organisms may have evolved quite different mechanisms in response to common biological problems.
EXAMPLE 13.15 In the ciliate protozoans, there are two kinds of nuclei: a polyploid somatic macronucleus (controlling all transcription during vegetative growth and asexual reproduction) and a haploid micronucleus containing the germ line. Fusion of haploid nuclei from conjugation of opposite mating types produces a diploid zygotic nucleus. The old macronucleus then degenerates and the zygotic nucleus divides to produce a new haploid micronucleus and an immature macronucleus. The macronuclear genome then becomes polyploid like the polytene chromosomes of Drosophila. The macronuclear chromosomes, however, become highly fragmented, and most of the fragments (up to 95% in some species) are degraded. The surviving fragments contain the genes required for vegetative growth and asexual reproduction. The mechanisms controlling this selective degradation and the distribution of surviving fragments into progeny cells during cell division are essentially unknown.
EXAMPLE 13.16 Amphibian oocytes contain a hundred to a thousand times more rRNA genes than are found in somatic cells, almost all of the increase being due to large numbers of extra chromosomal nucleoli. Each nucleolus contains one or more circular DNA molecules having 1–20 tandemly arranged rRNA genes coding for the 45S rRNA precursor. Most of these nucleolar circles are produced by the rolling-circle mechanism (as discussed in Chapter 10). These circles contain somatic genes and cannot replicate themselves. Extra chromosomal rRNA genes must be derived from the tandemly repeated rRNA germ-line genes.
Unlike the amphibian rRNA genes in Example 13.16, the eggshell genes of Drosophila can be amplified without extra chromosomal replication. A large number of follicle cells surround the egg and produce the chorion. The genes encoding the chorionic proteins exist in two clusters (one on the X chromosome and one on an autosome). Only a single copy of each somatic gene is present. A developmentally controlled origin of replication, located within each gene cluster, is programmed to fire 3–6 times during interphase within the 5 h of choriogenesis. The process shown in Fig. 13-1 usually produces a 32- to 64-fold amplification of the eggshell genes.
Epigenetic phenomena influence gene activity through chemical modification of the DNA or chromatin without changing the nucleotide sequence. Cytosine methylation of DNA and alterations made to histone proteins through a variety of modifications, such as acetylation, phosphorylation, and ubiquitination are the most widely known epigenetic mechanisms. However, new roles for double-stranded RNA molecules, such as micro RNAs (miRNAs) and others, have recently been identified. Examples of epigenetic phenomena are X chromosome inactivation in female mammals, parental imprinting of gene expression, and position effect variegation in plants. While many epigenetic modifications are stable, and are thus heritable, others change within the genome of an organism over time or under different environmental conditions. This means that DNA sequence variation is not the sole genetic determinant of individual phenotypic variation.
Practice problems for these concepts can be found at:
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