The DNA sequence within each somatic cell contains the information required to synthesize thousands of different proteins and RNA molecules. Typically, a cell expresses only a fraction of its genes. Different cell types in multicellular organism arise because each type expresses a different set of genes. Moreover, cells can change the pattern of genes in response to changes in their environment, such as signals from other cells. Although all the steps involved in expressing the gene can, in principle, be regulated, for most genes, the initiation of RNA transcription is the most important point of control.
STEPWISE REGULATION OF GENE EXPRESSION
There are several potential sites for regulation starting with DNA and transcription to posttranslational modification of a newly synthesized protein (Figure 10.1). While epigenetic changes to the genome involve both chemical and structural modifications to the chromatin and DNA, processing and transport of the newly synthesized mRNA into the cytoplasm are also regulated. In the cytoplasm, the stability of the mRNA can be controlled, as well as its translatability. Most proteins are modified after translation and this can control their activities, compartmentalization, and half-lives.
FIGURE 10.1.Regulation of gene expression can occur at different levels.
When and how often a gene sequence is copied into RNA is termed transcriptional control and this occurs at two levels:
- Structural-chemical modifications (i.e., acetylation of histones and demethylation of CpG nucleotides, see Chapter 6) convert compacted chromatin into a less tightly coiled DNA structure (Figure 10.2), allowing access by transcription factors required for gene expression.
FIGURE 10.2.Transcription requires DNA to be decondensed.
- DNA-binding proteins, known as transcription factors, modulate gene expression to turn transcription on or off. There are two categories of transcription factors, general (or basal) and specific.
1. General (basal) transcription factors: General transcription factors are abundant proteins that assemble on all genes transcribed by RNA polymerase II (see Chapter 8). These transcription factors are important for basal activity of the promoter and to position and activate RNA polymerase II at the start of a protein-coding sequence (Figure 10.3)
FIGURE 10.3.General transcription factors are needed for priming transcription.
2. Specific transcription factors: Specific transcriptional factors or gene regulatory proteins are present in very few copies in the individual cells and perform their function by binding to a specific DNA nucleotide sequence and allowing the genes that they control to be activated or repressed. These proteins recognize short stretches of double-stranded DNA of defined sequence and thereby determine which of the thousands of genes in a cell will be transcribed. Many unique regulatory proteins have been identified, each with unique structural motifs, and most bind to the DNA as homodimers or heterodimers (Figure 10.4). The precise amino acid sequence of the motif determines the DNA sequence(s) recognized. Specific transcription factors are important for tissue-specific gene expression and for cell growth and differentiation, and some lipid-soluble hormones regulate the transcription factors in their target cells.
FIGURE 10.4.Specific transcription factors have a modular design.
Some examples of transcription factors and the sequences recognized by these proteins on DNA are represented in Table 10.1.
TABLE 10.1.Specific Transcription Factors are Designed to Bind to Specific DNA Sequences and Regulate Gene Transcription
|Transcription Factor||Sequence Recognized|
|Myc and Max||CACGTG|
|Fos and Jun||TGACTCA|
|TR (thyroid hormone receptor)||GTGTCAAAGGTCA|
|RAR (retinoic acid receptor)||ACGTCATGACCT|
RNA Polymerase II catalyzes the synthesis of RNA from a DNA template at a singular rate of about 30 to 40 nucleotides per second. Although the rate of synthesis (transcription) is constant, the numbers of polymerases that simultaneously synthesize RNA from a give gene sequence determine the absolute gene transcription rate. Specific transcription factors modulate the number of RNA polymerase molecules actively synthesizing RNA from a given segment of DNA (Figure 10.5).
For this reason, transcription factors have a modular design consisting of at least two distinct domains (DNA-binding and transcription-activating domains). One domain consists of the structural motif that recognizes specific DNA sequences (DNA binding—discussed above) and the other domain contacts the transcriptional machinery and accelerates the rate of transcription initiation by accelerating the assembly of the general transcription factors at the promoter site (transcription activating) (see Figure 10.5).
FIGURE 10.5.Specific transcription factors influence the number of RNA polymerases that bind to DNA and initiate transcription.
RNA processing control
The primary transcript is produced as heterogeneous nuclear RNA containing introns which are eventually spliced out to create the mature mRNA (see Chapter 8). This process occurs in the nucleus and the subsequent processing is necessary to control the number of mRNA molecules that are eventually translated.
- mRNA capping: The addition of the 5? cap structure (see Chapter 8) is critical for an mRNA to be translated in the cytoplasm and is also needed to protect the growing RNA chain from degradation in the nucleus by 5? exonucleases.
- Poly(A) tail: The second modification of an mRNA transcript occurs at its 3? end, the addition of a poly(A) tail (approx. 200 adenine nucleotide residues are added). The polyadenylation reaction is an important regulatory step because the length of poly(A) tail modulates both mRNA stability and translation efficiency. The poly(A) tail protects the mRNA from premature degradation by 3?exonucleases.
- Removal of introns: Following the modification of the 5? and 3? ends of the primary transcript, the noninformational intron segments are removed and the coding exon sequences joined together by RNA splicing (Figure 10.6). The specificity of exon joining is conferred by the presence of signal sequences marking the beginning (5? donor site) and the end (3? acceptor site) of the intron segment. As these signal sequences are highly conserved, alterations in these sequences can lead to aberrant mRNA molecules.
FIGURE 10.6.RNA processing reactions.
4. Alternative splicing: The ability of the genes to form multiple proteins by joining different exon segments in the primary transcript is called alternative splicing. Alternative splicing is made possible by changing the accessibility of the different splice sites to the splicing machinery by RNA binding proteins. These proteins could mask preferred splice sites or change local RNA structure to promote the splicing of alternate sites. In addition, cell specific regulation can determine the type of alternate transcript and eventually the protein product produced. RNA splicing also allows switching between the production of nonfunctional and functional proteins, membrane-bound protein versus secreted protein, etc. The ability to make more than one protein product from a gene may also explain why the human genome has fewer genes than expected (Figure 10.7).
FIGURE 10.7.Alternative splicing of genes to generate multiple proteins.
Alternative splicing of the calcitonin gene
Alternative splicing produces two different proteins from the calcitonin gene. In the parafollicular cells of the thyroid gland, the calcitonin gene produces an mRNA that codes for the calcium-regulating hormone calcitonin, which counteracts the action of parathyroid hormone. Calcitonin is used in the treatment of postmenopausal osteoporosis in women when estrogen is contraindicated. In neural tissues, the same calcitonin gene is spliced differently and uses a different polyadenylation site to give rise to a neuropeptide, calcitonin gene–related peptide (CGRP), which plays a central role in the pathophysiology of migraine. The serum levels of CGRP are elevated in patients during all forms of vascular headaches, including migraines and cluster headaches. These findings suggest that CGRP-receptor antagonists might be effective in treating migraine by blocking CGRP activity.
RNA transport into the cytoplasm
The fully processed mRNAs represent only a small proportion of the RNA found in the nucleus. Damaged and misprocessed RNAs are retained within the nucleus and degraded. A typical mature mRNA carries a collection of proteins that identifies it as being the mRNA destined for transport. Export takes place through nuclear pore complex but mRNAs and their associated proteins are large and require active transport. Energy for export is supplied by the hydrolysis of GTP.
The length of time mRNAs remain in the cytosol determines the amount of protein product produced by translation. All transcripts have a finite lifetime in the cell. The steady state level of individual RNA species in a cell is determined by both the rate of transcription and the rate of decay.
1. mRNA half-life: In eukaryotic cells, mRNAs are degraded at different selective rates. A measure of degradative rate for a particular mRNA is called the half-life (the period it takes to degrade an RNA population to half its initial concentration). The unstable mRNAs usually code for regulatory proteins whose production levels change rapidly within cells (e.g., growth factors and gene regulatory proteins) (half-life—minutes to hours). The stable mRNAs usually code for house keeping proteins (half-life in days).
2. mRNA 3?UTRs: The mRNA contains regions that are not translated and these include the 5? untranslated region (5?UTR) and the 3? untranslated region (3?UTR). The stability of an mRNA can be influenced by signals inherent in an RNA molecule. The sequence AUUUA, when found in the 3?UTR, is a signal for early degradation (and therefore short lifetime). The more times the sequence is present, the shorter the lifespan of the mRNA. Because it is encoded in the nucleotide sequence, this is a set property of each different mRNA. Sequences in the 3?UTR form a stem-loop structure that allows for protein binding and protection from degradation (Figure 10.8).
FIGURE 10.8. Untranslated regions on mRNA.
Regulation of iron levels
Iron deficiency continues to be one of the most prevalent nutritional deficiencies throughout the world. The diagnosis of iron deficiency is based primarily on laboratory measurements. Since free iron is reactive and therefore toxic to the body, most of the iron exists within the body bound to proteins. Free iron is bound to storage proteins (ferritin) or is transported bound to transferrin. Although ferritin is mostly confined to the intracellular compartment, trace amounts are secreted into the blood. Because plasma ferritin is proportional to that stored intracellularly, consequently, plasma ferritin is a valuable index of intracellular stores in iron deficiency anemia.
Transferrin receptor levels are also measured clinically as the rate of internalization of iron by cells is determined by its level of cell surface expression. Ferritin and transferrin receptor biosynthesis are inversely modified based on cellular iron levels. Transferrin receptor biosynthesis is increased when available iron levels are low and decreased when iron levels are high. As shown in Figure 10.9, this regulation is accomplished by altering the amount of transferrin receptor and ferritin mRNA. The iron response elements on the transferrin receptor and ferritin mRNA are bound by the iron response protein whose activity is dependent on the iron status of the cell.
FIGURE 10.9. Regulation of transferrin receptor and ferritin mRNA.
The second basic step in gene expression is the translation of mRNAs into protein. Translation occurs in the cytoplasm; however, not all mRNAs are translated on arrival. The RNA molecules in the cytoplasm are constantly associated with proteins, some of which may function to regulate translation. Mechanisms for translational repression are most widely operative. In the case of ferritin, its mRNA is maintained within the cytoplasm but inhibited from being translated until intracellular concentration of iron rises. The block in this case is mediated by the binding of a repressor protein (the same protein that binds transferrin receptor mRNA in the 3?UTR) to the 5? nontranslated end of the molecule (see Figure 10.9).
Once cytoplasmic ribosomal complexes have synthesized a protein, the functional capabilities of the protein are often not realized until the protein has been modified. Although these mechanisms, collectively known as posttranslational modifications, are not thought of as gene expression controls, it is recognized that the manifestation of gene expression is not complete until a protein is carrying out its function within the cell.
The presence of double-stranded (ds) RNA in a eukaryotic cell can trigger a process known as RNA interference or RNAi (also known as RNA silencing or RNA inactivation). RNAi consists of two main phases. First, the dsRNA is recognized by an endonuclease (Dicer) and cleaved into smaller molecules of 21 to 24 nucleotides called short interfering RNA (siRNA). In the second phase, a single strand (the guide or antisense strand) of the siRNA associates with proteins to form an RNA-induced silencing complex, or RISC. The guide strand component of RISC then hybridizes with a complementary sequence of a full-length target mRNA. An endonuclease (Slicer) in the RISC degrades the target mRNA (Figure 10.10). RNAi is thought to be a part of the body’s natural immune system evolved as a defense against retroviruses, such as the human immunodeficiency virus (HIV), that store their genetic information in dsRNA.
FIGURE 10.10.RNA interference or RNAi.
Antisense RNA oligonucleotides and inhibitory RNA (RNAi) as potential chemotherapeutic agents
Antisense RNA is a single-stranded RNA that is complementary to an mRNA designed to inhibit specific mRNA translation by its ability to base pair with the mRNA and physically obstruct the translation machinery from accessing the mRNA. This approach was developed in the 1990s to overcome the limitations of cytotoxic chemotherapy using DNA intercalating drugs and antimetabolites, which do not discriminate between normal and cancer cells. Historically, this approach has not yielded results due to the effects of RNA interference, a process that has more recently been elucidated.
But this technology has been effective in the creation of the first antisense RNA drug Vitravene (fomivirsen) used to treat cytomegalovirus-induced retinitis in immune-compromised AIDS patients. The drug is a periodically administered intravitreal injection and is claimed to cause only mild side effects as compared to some other antiviral drugs. RNA interference of exogenously supplied double-stranded RNA offers substantial therapeutic potential. Applications that are in clinical trials include siRNA treatment for age-related macular degeneration (AMD), siRNAs against the respiratory syncytial virus, and a phase I trial to treat patients infected with HIV.
- Eukaryotic gene expression can be regulated at different levels of gene expression, including transcription, processing, mRNA stability, and translation.
- Transcription can be controlled by several transcription factors and is an important level of control. While general transcription factors are required for basal expression, specific transcription factors increase the transcription of a gene over basal levels when bound to enhancers and other response elements. They are also important for tissue-specific gene expression.
- Some RNA molecules can undergo differential splicing, producing different mRNA molecules encoding slightly different polypeptides.
- Transcription factors have one functional domain for DNA binding and one for transcription activation. Transcription factors can be classified according to the structure of their DNA-binding domains; these include zinc finger proteins, helix-turn-helix proteins, leucine zipper proteins, helix-loop-helix proteins, and steroid receptors. Transcription factors affect the number of RNA polymerases binding DNA.
- In the cytoplasm, translation can be controlled by both the ability of ribosomes to bind mRNA and by the stability of the mRNA.
- Sequences in the 3?UTR control stability and sequences in the 5?UTR control translation efficiency.
- Proteins are also made active by posttranslational modifications that include phosphorylation, gamma carboxylation, etc.