Molecular Genetics 4: Gene Expression
Learning Objectives
- Explain how genetic information is expressed through the processes of transcription, translation, and protein synthesis
- Compare the structures and functions of the major RNA molecules involved in gene expression
- Describe the molecular mechanisms of translation and the roles of codons, ribosomes, and transfer RNAs in protein production
- Explain how gene expression is regulated at multiple levels, including transcriptional, post-transcriptional, translational, and post-translational regulation
- Evaluate the influence of transcription factors, epigenetic modifications, and chromatin structure on patterns of gene expression
- Describe how differential gene expression contributes to cell differentiation, development, and adaptation to environmental change
Chapter Introduction
Every cell in an organism contains the same genetic information, the same set of genes. Therefore, different sets of genes are required for the various functions of different cells or tissues, as well as for plant responses to environmental stimuli or stresses. This is achieved by regulating the activity of genes according to the physiological demands of a particular cell type, developmental stage, or environmental condition. This regulation of activity is known as gene expression.
The term expression can be used in different ways that are sometimes confusing. Typically, if a gene product is produced, the gene is considered “expressed”. However, it sometimes occurs that a transcript might be produced but not a protein, or that a protein is produced but it is in an inactive state. In such cases, although a gene product is produced, the biological activity encoded by that gene is not present. For the purposes of this section, the key point is how the biological activity encoded by a gene is regulated.
The expression of genes in specific plant cells, tissues, and organs and the timing of this expression require a precise level of regulation. Expression, or genetic function, can potentially be regulated at any of the steps from transcription, RNA processing, translation, through post-translational protein modification. Regulation can be qualitative (i.e. gene expression is either “on” or “off”) or quantitative (i.e. expression levels can be modulated “up” or “down”). Fluctuations in the intensities of external stimuli coupled to changes that occur at the genomic level result in different developmental outcomes or physiological states.
Regulation of gene expression at the level of transcription can be brought about through chromatin and histone modifications. Also, a gene sequence can be differentially spliced to produce mRNA products of variable lengths leading to new protein products with novel functions. Some genes do not encode proteins but short forms of RNAs with regulatory functions such as induction of flowering. Finally, proteins products can be subjected to modifications such as phosphorylation or dephosphorylation to alter their functions or can be completely degraded to turn off a gene.
Mechanisms of Gene Expression
As outlined in the previous chapter, the flow of genetic information in cells from DNA to mRNA to protein is described by the central dogma (Figure 10.1), which states that genes specify the sequence of mRNAs, which in turn specify the sequence of amino acids making up all proteins. The decoding of one molecule to another is performed by specific proteins and RNAs. Because the information stored in DNA is so central to cellular function, it makes intuitive sense that the cell would make mRNA copies of this information for protein synthesis, while keeping the DNA itself intact and protected.
The copying of DNA to RNA is relatively straightforward, with one nucleotide being added to the mRNA strand for every nucleotide read in the DNA strand. The translation to protein is a bit more complex because three mRNA nucleotides correspond to one amino acid in the polypeptide sequence. However, the translation to protein is still systematic and colinear, such that nucleotides 1 to 3 correspond to amino acid 1, nucleotides 4 to 6 correspond to amino acid 2, and so on.
Comparison of DNA and RNA
DNA’s structure allows its function as information storage. A second nucleic acid, called ribonucleic acid or RNA, is a single-stranded molecule similar in structure to DNA, but with some key differences. Like DNA, RNA is composed of nucleotides that are linked by phosphodiester bonds. Each nucleotide has the same basic structure: a sugar attached to a base and one or more phosphates (Figure below).
However, there are some key differences between RNA and DNA. First, while DNA is typically double-stranded, RNA is typically single-stranded. Second, DNA is made up of deoxyribonucleotides whereas RNA is composed of ribonucleotides. The distinction is the type of sugar that is attached to the base (ribose in RNA, deoxyribose in DNA). Third, DNA contains the bases Adenine (A), Thymine (T), Cytosine (C), and Guanine (G). RNA does not contain Thymine (T), and in its place uses Uracil (U) instead. Thus, the base pairs in RNA are A-U and C-G.
There are four major types of RNA: messenger RNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), and microRNA (miRNA). The first, mRNA, carries the information encoded in DNA. If a cell requires synthesising a certain protein, the gene for this product turns “on” and the mRNA is synthesised based on the sequence of nucleotides in the DNA in the nucleus. The mRNA base sequence is complementary to the DNA sequence from which it has been copied. However, in RNA, the base T is absent and U is present instead. For example, if the DNA strand has a sequence AATTGCGC, the sequence of the complementary RNA is UUAACGCG.
Ribosomal RNA (rRNA) is a major constituent of ribosomes, the protein synthesis machines of the cell. Ribosomes are a complex of molecules that use the information encoded in mRNA to connect amino acids in the correct sequence. A key part of this process is the translation of the nucleotide sequence into an amino acid sequence. There are only 4 nucleotides and 20 amino acids, so a grouping of 3 nucleotides is used to determine the correct amino acid (more on this when we dive into the details of translation). This is done by transfer RNA (tRNA). There are many different types of tRNA, each of which carries a particular amino acid and its corresponding nucleotide triplet.
MicroRNA are the smallest RNA molecules and their role involves regulating gene expression by interfering with the expression of certain mRNA messages.
| Base found in DNA | Base inserted into RNA |
|---|---|
| A | U |
| T | A |
| C | G |
| G | C |
Table 10.1. The base pairing rules of transcription. The base-pairing rules slightly differ from that of DNA replication. Because RNA uses uracil (U) instead of thymine (T) as one of its pyrimidines, A in the DNA template pairs with U in RNA. Source: Limeri, L.; & Reid, J. Central dogma, In Introductory biology I, used under a Creative Commons Attribution NonCommercial 4.0 International licence.
Though both RNA and DNA contain the nitrogenous bases adenine, guanine, and cytosine, RNA contains the nitrogenous base uracil instead of thymine. Uracil pairs with adenine in RNA, just as thymine pairs with adenine in DNA. Uracil and thymine have very similar structures; uracil is an unmethylated form of thymine.
The nucleotide sequence of RNA, which is complementary to the DNA sequence, allows RNA to encode genetic information. RNA though carries the genetic information of just one gene. Hence, compared to DNA, RNA molecules are relatively small.
Translation
As with mRNA synthesis, protein synthesis can be divided into three phases: initiation, elongation, and termination. The process of translation is similar in prokaryotes and eukaryotes. Here we’ll explore how translation occurs in E. coli, a representative prokaryote, and specify any differences between prokaryotic and eukaryotic translation.
Triplet code/codon/anticodon
To summarise what we know to this point, the cellular process of transcription generates messenger RNA (mRNA), a mobile molecular copy of one or more genes with an alphabet of A, C, G, and uracil (U). Translation of the mRNA template converts nucleotide-based genetic information into a protein product. Protein sequences consist of 20 commonly occurring amino acids; therefore, it can be said that the protein alphabet consists of 20 letters. Each amino acid is defined by a three-nucleotide sequence called the triplet codon. The relationship between a nucleotide codon and its corresponding amino acid is called the genetic code.
Given the different numbers of “letters” in the mRNA and protein “alphabets,” combinations of nucleotides corresponded to single amino acids. Using a three-nucleotide code means that there are a total of 64 (4 × 4 × 4) possible combinations; therefore, a given amino acid is encoded by more than one nucleotide triplet (Figure below).
Three of the 64 codons terminate protein synthesis and release the polypeptide from the translation machinery. These triplets are called stop codons. Another codon, AUG, also has a special function. In addition to specifying the amino acid methionine, it also serves as the start codon to initiate translation. The reading frame for translation is set by the AUG start codon near the 5' end of the mRNA. The genetic code is universal. With a few exceptions, virtually all species use the same genetic code for protein synthesis, which is powerful evidence that all life on Earth shares a common origin.
mRNA
Because the information stored in DNA is so central to cellular function, it makes sense that the cell would make temporary copies of the information, in the form of mRNA, while keeping the DNA itself intact and protected in the nucleus of the cell. Additionally, the production of mRNA also amplifies the information; one gene gives rise to thousands of copies of mRNA templates, each of which may be used to produce large amounts of protein quickly through the actions of ribosomes. After transcription, eukaryotic pre-mRNAs must undergo several processing steps to become mature mRNA before they can be translated.
The additional steps involved in eukaryotic mRNA maturation create a molecule with a longer half-life; eukaryotic mRNAs last for several hours. Eukaryotic (and prokaryotic) tRNAs and rRNAs also undergo processing before they can function as components in the protein-synthesis machinery. Pre-mRNAs are first coated in RNA-stabilising proteins; these protect the pre-mRNA from degradation while it is processed and exported out of the nucleus. The three most important steps of pre-mRNA processing are the addition of stabilising and signalling factors at the 5’ and 3’ ends of the molecule, and the removal of the non-coding DNA segments (called introns) through splicing. In rare cases, the mRNA transcript can be “edited” after it is transcribed (see the evolution connection below).
Ribosomes
Even before an mRNA is translated, a cell must invest energy to build each of its ribosomes. In E. coli, there are between 10,000 and 70,000 ribosomes present in each cell at any given time. A ribosome is a complex macromolecule composed of structural and catalytic rRNAs, and many distinct polypeptides. In eukaryotes, the nucleolus is completely specialised for the synthesis and assembly of rRNAs.
Ribosomes exist in the cytoplasm of prokaryotes and in the cytoplasm and rough endoplasmic reticulum of eukaryotes. Mitochondria and chloroplasts also have their own ribosomes in the matrix and stroma, which look more similar to prokaryotic ribosomes (and have similar drug sensitivities) than the ribosomes just outside their outer membranes in the cytoplasm. Ribosomes dissociate into large and small subunits when they are not synthesising proteins and reassociate during the initiation of translation. In E. coli, the small subunit is described as 30S, and the large subunit is 50S, for a total of 70S (recall that Svedberg units are not additive).
Mammalian ribosomes have a small 40S subunit and a large 60S subunit, for a total of 80S. The small subunit is responsible for binding the mRNA template, whereas the large subunit sequentially binds tRNAs. Each mRNA molecule is simultaneously translated by many ribosomes, all synthesising protein in the same direction: reading the mRNA from 5' to 3' and synthesising the polypeptide from the N terminus to the C terminus. The complete mRNA/poly-ribosome structure is called a polysome.
tRNAs
The tRNAs are structural RNA molecules that were transcribed from genes by RNA polymerase III. Depending on the species, 40 to 60 types of tRNAs exist in the cytoplasm. Transfer RNAs serve as adaptor molecules. Each tRNA carries a specific amino acid and recognises one or more of the mRNA codons that define the order of amino acids in a protein. Aminoacyl-tRNAs bind to the ribosome and add the corresponding amino acid to the polypeptide chain. Therefore, tRNAs are the molecules that actually “translate” the language of RNA into the language of proteins.
Of the 64 possible mRNA codons—or triplet combinations of A, U, G, and C—three specify the termination of protein synthesis and 61 specify the addition of amino acids to the polypeptide chain. Of these 61, one codon (AUG) also encodes the initiation of translation. Each tRNA anticodon can base pair with one or more of the mRNA codons for its amino acid. For instance, if the sequence CUA occurred on an mRNA template in the proper reading frame, it would bind a leucine tRNA expressing the complementary sequence, GAU. The ability of some tRNAs to match more than one codon is what gives the genetic code its blocky structure.
As the adaptor molecules of translation, it is surprising that tRNAs can fit so much specificity into such a small package. Consider that tRNAs need to interact with three factors: 1) they must be recognised by the correct aminoacyl synthetase (see below); 2) they must be recognised by ribosomes; and 3) they must bind to the correct sequence in mRNA.
RER
Ribosomes transfer their newly synthesised proteins into the RER's lumen where they undergo structural modifications, such as folding or acquiring side chains. These modified proteins incorporate into cellular membranes—the ER or the ER's or other organelles' membranes. The proteins can also secrete from the cell (such as protein hormones, enzymes). The RER also makes phospholipids for cellular membranes.
If the phospholipids or modified proteins are not destined to stay in the RER, they will reach their destinations via transport vesicles that bud from the RER’s membrane (figure below showing ER lumen).
Since the RER is engaged in modifying proteins (such as enzymes, for example) that secrete from the cell, you would be correct in assuming that the RER is abundant in cells that secrete proteins. This is the case with liver cells, for example.
Golgi
We have already mentioned that vesicles can bud from the ER and transport their contents elsewhere, but where do the vesicles go? Before reaching their final destination, the lipids or proteins within the transport vesicles still need sorting, packaging, and tagging so that they end up in the right place. Sorting, tagging, packaging, and distributing lipids and proteins takes place in the Golgi apparatus (also called the Golgi body), a series of flattened membranous sacs (see Figure below).
The side of the Golgi apparatus that is closer to the ER is called the cis face. The opposite side is the trans face. The transport vesicles that formed from the ER travel to the cis face, fuse with it, and empty their contents into the Golgi apparatus' lumen. As the proteins and lipids travel through the Golgi, they undergo further modifications that allow them to be sorted. The most frequent modification is adding short sugar molecule chains. These newly modified proteins and lipids then tag with phosphate groups or other small molecules in order to travel to their proper destinations.
Finally, the modified and tagged proteins are packaged into secretory vesicles that bud from the Golgi's trans face. While some of these vesicles deposit their contents into other cell parts where they will be used, other secretory vesicles fuse with the plasma membrane and release their contents outside the cell.
In another example of form following function, cells that engage in a great deal of secretory activity (such as salivary gland cells that secrete digestive enzymes or immune system cells that secrete antibodies) have an abundance of Golgi.
Ribosomes exist in the cytoplasm of prokaryotes and in the cytoplasm and rough endoplasmic reticulum of eukaryotes. Ribosomes dissociate into large and small subunits when they are not synthesising proteins and reassociate during the initiation of translation. In E. coli, the small subunit is described as 30S, and the large subunit is 50S, for a total of 70S (recall that Svedberg units are not additive). Mammalian ribosomes have a small 40S subunit and a large 60S subunit, for a total of 80S. The small subunit is responsible for binding the mRNA template, whereas the large subunit sequentially binds tRNAs. Each mRNA molecule is simultaneously translated by many ribosomes, all synthesising protein in the same direction: reading the mRNA from 5' to 3' and synthesising the polypeptide from the N terminus to the C terminus. The complete mRNA/poly-ribosome structure is called a polysome.
Posttranslational Modifications
During and after translation, individual amino acids may be chemically modified, signal sequences appended, and the new protein “folded” into a distinct three-dimensional structure as a result of intramolecular interactions. A signal sequence is a short sequence at the amino end of a protein that directs it to a specific cellular compartment. These sequences can be thought of as the protein’s “train ticket” to its ultimate destination and are recognised by signal-recognition proteins that act as conductors. For instance, a specific signal sequence terminus will direct a protein to the mitochondria or chloroplasts (in plants). Once the protein reaches its cellular destination, the signal sequence is usually clipped off.
Many proteins fold spontaneously, but some proteins require helper molecules, called chaperones, to prevent them from aggregating during the complicated process of folding. Even if a protein is properly specified by its corresponding mRNA, it could take on a completely dysfunctional shape if abnormal temperature or pH conditions prevent it from folding correctly.
The number of distinct primary structures of proteins (proteoforms) from 20,000 human genes is amplified again through posttranslational modifications (PTMs) of proteins, which produce up to a million different variants. PTMs are present in both eukaryotes and prokaryotes, but are more common in eukaryotic cells, in which about 5% of the genome is dedicated to enzymes that carry out posttranslational modifications of proteins.
Protein posttranslational modifications result from the enzymatic or nonenzymatic attachment of specific chemical groups to amino acid side chains. Such modifications occur either following protein translation or concomitant with translation. Posttranslational modifications influences both protein structure and physiological and cellular functions. Examples of enzymatic PTMs include phosphorylation, glycosylation, acetylation, methylation, sumoylation, palmitoylation, biotinylation, ubiquitinylation, nitration, chlorination, and oxidation/reduction. Nonenzymatic PTMs include glycation, nitrosylation, oxidation/reduction, acetylation, and succinylation. Some rare and unconventional PTMs, such as glypiation, neddylation, siderophorylation, AMPylation, and cholesteroylation, are also known to influence protein structure and function. Note that many of these modifications are not made in isolation. It is common for proteins to have several different types of modifications and that these modifications can differ depending on the tissue type and environmental circumstances present. The major PTMs in eukaryotes, their target amino acid residue(s), and the types of enzyme(s) or protein(s) involved are shown below.
| Posttranslational modification | Target amino acid residue(s) | Enzyme(s) or proteins involved |
|---|---|---|
| Phosphorylation | Tyrosine, serine, threonine | Kinases, phosphatases |
| Glycosylation N-linked | Asparagine | Glycosyltransferases, deglycosylases |
| Glycosylation O-linked | Serine/threonine | Glycosyltransferases, deglycosylases |
| Acetylation | Lysine | Acetyltransferases, deacetylases |
| Methylation | Lysine, arginine | Methyltransferases, demethylases |
| Ubiquitination | Lysine | Ubiquitin-activating enzymes, ubiquitin-conjugating enzymes, ubiquitin ligases, deubiquitinases |
| Sumoylation | Lysine | Ubiquitin-activating enzymes, ubiquitin-conjugating enzymes, ubiquitin ligases, deubiquitinases |
| Myristoylation | Glycine | N-Myristoyltransferases |
| Prenylation | Cysteine | Farnesyltransferases, geranyl geranyltransferases |
| Palmitoylation | Cysteine | DHHC protein acyltransferases, acyl-protein thioesterases |
| Sulfation | Tyrosine | Sulfatases, desulfatases |
| S-Nitrosylation | Cysteine, methionine | |
| Glycation | Lysine | |
| Nitration | Tyrosine | Denitrases |
| Chlorination | Tyrosine | Myeloperoxidases |
| Oxidation/reduction | Cysteine | Peroxidases, oxidases, glutathione, thioredoxin |
| Carbonylation | Lysine, proline, arginine, threonine |
Table 10.2. Common protein post-translational modifications, their target amino acid residues, and the enzyme(s) or protein(s) involved. Source: Santos, Ana L., Lindner, Ariel B., (2017). Protein posttranslational modifications: roles in aging and age-related disease, Oxidative medicine and cellular longevity. https://doi.org/10.1155/2017/5716409. Used under a Creative Commons Attribution 4.0 International licence.
Posttranslational modifications can exert their effect through many different structural changes, including opening and closing the active site, changing the conformation and electrostatic properties of binding sites, changing the flexibility of the chain, increasing or decreasing intrinsically disordered regions, altering protein-protein interactions, etc. These effects can also arise from the binding of small molecules.
Regulation of Gene Expression
To develop a multicellular organism, cells must differentiate to specialise for different functions. Three basic categories of cells make up the mammalian body: germ cells, somatic cells, and stem cells. Each of the approximately 100 trillion cells an adult human has its own copy or copies of the genome except certain cell types, such as red blood cells, that lack nuclei in their fully differentiated state. Most cells are diploid; they have two copies of each chromosome. The process of cellular differentiation is regulated by transcription factors and growth factors, and results in expression or inhibition of various genes between the cell types, thereby resulting in varying proteomes between cell types. The variation in proteomes between cell types is what drives differentiation and thus, specialisation of cells. The ability of transcription factors to control whether a gene will be transcribed or not that contributes to specialisation and growth factors to aid in the division process are key components of cell differentiation.
Somatic cells are diploid cells that make up most of the human body, such as the skin and muscle. Germ cells are any line of cells that give rise to gametes — eggs and sperm — and thus are continuous through the generations. Stem cells, on the other hand, have the ability to divide for indefinite periods and to give rise to specialised cells. They are best described in the context of normal human development.
Embryonic Development
Development begins when a sperm fertilises an egg and creates a single cell that has the potential to form an entire organism. In the first hours after fertilisation, this cell divides into identical cells. In humans, approximately four days after fertilisation and after several cycles of cell division, these cells begin to specialise, forming a hollow sphere of cells, called a blastocyst. The blastocyst has an outer layer of cells, and inside this hollow sphere, there is a cluster of cells called the inner cell mass. The cells of the inner cell mass go on to form virtually all of the tissues of the human body. Although the cells of the inner cell mass can form virtually every type of cell found in the human body, they cannot form an organism. These cells are referred to as pluripotent.
Pluripotent stem cells undergo further specialisation into multipotent progenitor cells that then give rise to functional cells. Examples of stem and progenitor cells include:
- Hematopoietic stem cells (adult stem cells) from the bone marrow that give rise to red blood cells, white blood cells, and platelets
- Mesenchymal stem cells (adult stem cells) from the bone marrow that give rise to stromal cells, fat cells, and types of bone cells;
- Epithelial stem cells (progenitor cells) that give rise to the various types of skin cells
- Muscle satellite cells (progenitor cells) that contribute to differentiated muscle tissue
A pathway that is guided by the cell adhesion molecules is created as the cellular blastomere differentiates from the single-layered blastula to the three primary layers of germ cells in mammals, namely the ectoderm, mesoderm and endoderm (listed from most distal, or exterior, to the most proximal, or interior). The ectoderm ends up forming the skin and the nervous system, the mesoderm forms the bones and muscular tissue, and the endoderm forms the internal organ tissues.
Transcriptional gene regulation
Since transcription is the first step in gene expression, it makes sense in terms of cellular economy, to regulate expression at this point, and in fact this is one of the most important regulatory points. We already described the involvement of RNA polymerase in the transcription process, but in fact there are other protein factors that are required. Proteins involved in transcriptional regulation are known as transcription factors. It is the interaction of these transcription factors with specific DNA sequences that regulate the process of gene transcription.
The concept of differential (regulated) gene expression. As just described, not every gene is expressed all the time. When a gene displays different levels of expression in different circumstances, this is known as differential expression. Circumstances that might apply include, but are not limited to, different plant tissues (root vs. leaf), different developmental stages (germination vs. reproductive development), or in response to different environmental stimuli (cold stress or pathogen attack).
The term differential expression can also be used to compare the expression of different genes. If two genes show different expression patterns (among plant tissues or in response to environmental stimuli), they are considered differentially expressed, whereas genes that showed very similar patterns of expression would be considered co-expressed.
Promoters. As mentioned, transcription is regulated through the interactions of proteins, transcription factors, with specific DNA sequences. Most regulatory DNA sequences governing gene transcription are located on the 5′ border of the transcribed region. This region is called the gene promoter.
Promoters contain a core, which is required for the binding of the “basal transcriptional machinery”, including RNA polymerase. The “TATA” box, with a consensus sequence TATAA, is located within the core promoter, usually 25-30 nucleotides upstream of the transcription initiation site.
Promoters also contain regulatory sequences that determine when, where, and to what level genes are transcribed. Promoters can vary in length from a hundred to a few thousand nucleotides.
The full promoter sequences of different genes that are expressed in a similar manner may be different. However, such promoters often contain short sequence “motifs” that are similar, referred to as cis elements. Early work (Benfy and Chua, 1990) to understand the function of different promoter elements in regulating gene expression in plant cells and tissues revealed that various combinations of cis elements are able to be interpreted by the cell and control gene expression. Sometimes cis elements promote gene transcription and sometimes they function to restrict transcription of genes in particular cells and tissues.
Promoter analysis is facilitated through the use of reporter genes. The reporter gene produces effects that are easily identifiable and quantifiable, which can be used to determine the function of a regulatory region of another gene (promoter, promoter elements, or enhancers) in cells, tissues, or organs. Such analyses are critical to crop biotechnology where targeted expression of genes in particular tissues is often desirable. To test whether a promoter is effective in conferring the expression of a gene to a particular tissue, scientists fuse the putative promoter to a reporter gene and introduce the promoter-gene fusion into plants. An example of a reporter is the GUS gene, which encodes a GUS enzyme (beta-Glucuronidase) that, when expressed, produces a blue colour upon addition of a substrate.
Enhancers are DNA sequences that increase the rate of transcription of a gene when they are present, although alone they cannot cause transcription to occur. Enhancers are usually position and orientation independent. Although they are normally located upstream of the promoter, they can also be located on the 3’ region of the gene or even with the coding region. Enhancers can increase the transcription when added to genes that they are not normally associated with. This is a useful property for biotechnology, allowing promoters to be manipulated for increased levels of transcriptional regulation. Some enhancers function at all times in all cells and tissues and they are referred to as constitutive. Other enhancers function in specific tissues at specific developmental stages. Some are active only in response to environmental signals. The AACCA enhancer on the promoter of soybean β-conglycinin gene encoding a seed storage protein functions specifically in seeds. Enhancers are thought to interact with specific nuclear proteins involved in transcription. For example, enhancers might facilitate the binding of transcription factors and direct these factors along the DNA strand to the direction of the promoter. Alternatively, enhancers may facilitate changes in DNA structure such as modification of the chromatin structure.
The counterpart of an enhancer is a silencer. Silencers have all the properties just described for enhancers, except they function to dampen, or decrease, the levels of transcription controlled by a promoter.
Transcription factors
RNA polymerase binds the promoter at the TATA box and in cooperation with other proteins drives gene transcription. The proteins that interact with RNA polymerase to facilitate its binding to the promoter and to regulate its activity are known as transcription factors. Some transcription factors, known as “basal transcription factors” are fundamental to RNA polymerase binding and function, and are expressed in all living cells.
Other transcription factors bind to cis regulatory DNA sequences of promoters, enhancers or silencers. These proteins interact in complex ways with the basal transcription machinery, to regulate the activity of RNA polymerase, and therefore gene transcription. Thus, transcription factors regulate when or where individual genes are expressed, and to what level.
Transcription factors can function as either positive or negative regulators. That is, they can either function to induce (increase) gene transcription or to repress it. The consequence of many transcription factors depends on their interactions with other proteins. Two factors together may be required for gene activity, and exclusion of one of the factors in space and time offers a mechanism for differential gene expression. Some transcription factors might function as a positive regulator in one context but as a negative regulator in another context, depending on what other cis elements and/or transcription factors might be present.
If DNA encoding a specific gene is to be transcribed into RNA, the nucleosomes surrounding that region of DNA can slide down the DNA to open that specific chromosomal region and allow for the transcriptional machinery (RNA polymerase) to initiate transcription. Nucleosomes can move to open the chromosome structure to expose a segment of DNA but do so in a very controlled manner.
Active open regions of chromatin are called euchromatin (Figure 10.14). Regions of the genome that are transcriptionally active are typically euchromatic. Tightly wound regions of chromatin are called heterochromatin. Heterochromatic regions of the genome are typically silenced and transcriptionally inactive.
Modifications to DNA and histones
How the histone proteins move, and whether the DNA is wrapped loosely or tightly around them, is dependent on signals found on both the histone proteins and on the DNA. These signals are chemical tags added to histone proteins and DNA that tell the histones if a chromosomal region should be open or closed. These tags are not permanent but may be added or removed as needed. They are chemical modifications (phosphate, methyl, or acetyl groups) that are attached to specific amino acids in the protein or to the nucleotides of the DNA. The tags do not alter the DNA base sequence, but they do alter how tightly wound the DNA is around the histone proteins.
This type of gene regulation is called epigenetic regulation. Epigenetic means “around or above genetics.” The changes that occur to the histone proteins and DNA do not alter the nucleotide sequence and are not permanent. Instead, these changes are temporary, although they can and often do persist through multiple rounds of cell division. They alter the chromosomal structure (open euchromatin or closed heterochromatin) as needed, but do not change the sequence of bases within the DNA.
A gene can be turned on or off depending upon the location and modifications to the histone proteins and DNA. If a gene is to be transcribed, the histone proteins and DNA are modified surrounding the chromosomal region encoding that gene. This opens the chromosomal region (it becomes euchromatic) to allow access for RNA polymerase and other proteins, called transcription factors, to bind to the promoter region, located just upstream of the gene, and initiate transcription. If a gene is to remain turned off, or silenced, the histone proteins and DNA have different modifications that signal a closed chromosomal configuration. In this closed configuration (heterochromatin), the RNA polymerase and transcription factors do not have access to the DNA and transcription cannot occur.
DNA Methylation
A common type of epigenomic modification is called methylation. Methylation involves attaching small molecules called methyl groups, each consisting of one carbon atom and three hydrogen atoms, to DNA nucleotides or the amino acids that make up the histone proteins.
When DNA is methylated, the methyl group is typically added to cytosine nucleotides. This occurs within very specific regions called CpG islands. These are stretches with a high frequency of cytosine and guanine dinucleotide DNA pairs (CG) found in the promoter regions of genes. When this configuration exists, the cytosine member of the pair can be methylated (a methyl group is added). This modification changes how the DNA interacts with proteins, including the histone proteins that control access to the region. When methyl groups are added to a particular gene, that gene is turned off or silenced, and no protein is produced from that gene.
“Histone Code” Hypothesis
The histone code hypothesis is the hypothesis that transcription of a gene is in part regulated by modifications made to histone proteins, primarily on their somewhat floppy ends (their “tails”). Many of the histone tail modifications correlate very well to chromatin structure and both histone modification state and chromatin structure correlates well to gene expression levels. The most important concept in the histone code hypothesis is that the histone modifications serve to recruit other proteins by specific recognition of the modified histone, rather than through simply stabilising or destabilising the interaction between histone and the underlying DNA. These recruited proteins then act to alter chromatin structure actively or to promote transcription.
The histone code has the potential to be massively complex. There are at least 20 modifications that are made to histone tails that have been relatively well characterised, and there is the potential for many more that we have not discovered. Each histone can be modified on multiple amino acids, with multiple different chemical modifications. The information that can be stored in the histone code dwarfs the amount that is stored in the order of the bases in the human genome.
Types of Regulation
Cells in multicellular organisms are specialised; cells in different tissues look very different and perform different functions. For example, a muscle cell is very different from a liver cell, which is very different from a skin cell. These differences are a consequence of the expression of different sets of genes in each of these cells. All cells have certain basic functions they must perform for themselves, such as converting the energy in sugar molecules into energy in ATP. Each cell also has many genes that are not expressed and expresses many that are not expressed by other cells, such that it can carry out its specialised functions.
In addition, cells will turn on or off certain genes at different times in response to changes in the environment or at different times during the development of the organism. Unicellular organisms, both eukaryotic and prokaryotic, also turn on and off genes in response to the demands of their environment so that they can respond to special conditions.
The control of gene expression is extremely complex. Malfunctions in this process are detrimental to the cell and can lead to the development of many diseases, including cancer.
How Steroid Hormones Regulate Transcription
Steroid hormones cross the cell membranes to have their effects. Common steroid hormones include testosterone, estrogens, progesterone, glucocorticoids, and mineral corticoids. Once inside the target cell, such hormones bind to a steroid-hormone receptor protein to form a steroid-hormone-receptor complex.
The receptor may be in the cytoplasm or in the nucleus, but in the end the hormone receptor complex must bind to DNA-regulatory elements of a gene, either to enhance or to silence transcription. Therefore, a steroid hormone must cross the plasma membrane and may also need to cross the nuclear envelope. You can follow the binding of a steroid hormone to a cytoplasmic receptor in Figure 10.16. Here the hormone (depicted as a red triangle in the illustration) enters the cell. An allosteric change in the receptor releases a protein subunit called Hsp90 (the black rectangle). The remaining hormone-bound receptor enters the nucleus.
The fascinating thing about Hsp90 is that it was first discovered in cells subjected to heat stress. When the temperature gets high enough, cells shut down most of transcription and instead transcribe Hsp90 and/or other special heat-shock genes. The resulting heat-shock proteins seem to protect the cells against metabolic damage until temperatures return to normal. Of course, most cells never experience such high temperatures, so the evolutionary significance of this protective mechanism is unclear. But as we now know, heat-shock proteins have critical cellular functions, in this case blocking the DNA-binding site of a hormone receptor until a specific steroid hormone binds to it.
No longer associated with the Hsp90 protein, the receptor bound to its hormone cofactor now binds to a cis-acting transcription-control element in the DNA, turning the transcription of a gene on or off. The hormone receptors for some steroid hormones are already in the nucleus of the cell, so the hormone must cross not only the plasma membrane but also the nuclear envelope in order to access the receptor. As for steroid hormone functions, we’ve seen their roles in activating genes for gluconeogenesis. Steroid hormones also control sexual development and reproductive cycling in females, sexual development and sperm maturation in males, salt and mineral homeostasis in the blood, metamorphosis in arthropods, and more, all by regulating gene expression.
Prokaryotic versus Eukaryotic Gene Expression
To understand how gene expression is regulated, we must first understand how a gene becomes a functional protein in a cell. The process occurs in both prokaryotic and eukaryotic cells, just in slightly different fashions.
Because prokaryotic organisms lack a cell nucleus, the processes of transcription and translation occur almost simultaneously. When the protein is no longer needed, transcription stops. As a result, the primary method to control what type and how much protein is expressed in a prokaryotic cell is through the regulation of DNA transcription into RNA. All the subsequent steps happen automatically. When more protein is required, more transcription occurs. Therefore, in prokaryotic cells, the control of gene expression is almost entirely at the transcriptional level.
The differences in the regulation of gene expression between prokaryotes and eukaryotes are summarised in Table 10.3 (below).
| Prokaryotic organisms | Eukaryotic organisms |
|---|---|
| Lack nucleus | Contain nucleus |
| RNA transcription and protein translation occur almost simultaneously | RNA transcription occurs prior to protein translation, and it takes place in the nucleus. RNA translation to protein occurs in the cytoplasm.
RNA post-processing includes addition of a 5' cap, poly-A tail, and excision of introns and splicing of exons. |
| Gene expression is regulated primarily at the transcriptional level | Gene expression is regulated at many levels (epigenetic, transcriptional, post-transcriptional, translational, and post-translational) |
Table 10.3. Differences in the regulation of gene expression of prokaryotic and eukaryotic organisms. Source: How genes are regulated, In Concepts of biology, 1st Canadian Edn, by Charles Molnar and Jane Gair, used under a Creative Commons Attribution 4.0 International License.
RNA-level regulation
Regulation of gene expression occurs at many levels, including post-transcriptionally. From the standpoint of the expression level for a given gene, a critical factor is the level of fully processed, mature mRNA. When we consider the level of the mature form a particular transcript at any given time reflects the steady state balance between synthesis, processing and degradation. The post-transcriptional regulation of RNA occurs through several mechanisms.
Translational regulation
As described, for most genes it is the protein product that performs the biological function. As such, regulating the amount of protein production effectively regulates gene expression. There are several known mechanisms by which the process of translation is known to be regulated. A detailed consideration of these mechanisms is beyond the scope of this course, but in general it is important to bear in mind their importance. For example, under certain stress conditions, “normal” translation is halted and only mRNAs related to stress tolerance are selectively permitted to be translated into proteins. This selective translation is important to allow plants to quickly respond to stress and to conserve energy under stress conditions.
Translation of mRNAs can play an important role in determining their overall stability. Mutations that add premature stop codons often lead to rapid degradation of the mRNA. Ribonucleases (figure 9) or other factors involved in degradation may recognise the number or spacing of ribosomes on an mRNA and degrade those that are not produced properly. This may help prevent synthesis of proteins with incorrect functions which will negatively impact cellular processes. Errors during transcription could add or omit nucleotides which would alter the proper codon sequence creating mutant proteins.
RNA stability and degradation
The control of mRNA degradation rate, or turnover, is an important regulatory mechanism in gene expression. As mentioned, the level of a particular transcript at any given time reflects the steady state balance between synthesis and degradation. Thus, even though a gene may be transcribed at a high rate, a high rate of RNA turnover could effectively shut off the gene. The stability of mRNA can be regulated globally, to affect all or most transcripts, or it can be very specific to a particular mRNA. Regulated mRNA turnover may be particularly important in plants, since plants cannot move to avoid harsh environmental conditions and stresses are known to induce specific changes in RNA stability. Other factors such as light and plant hormones are also known to regulate mRNA stability.
The degradation of mRNA is catalysed by enzymes called ribonucleases. Ribonucleases include exonucleases, which degrade only from one end of the transcript, and endonucleases, which can attack the mRNA molecule internally.
Therefore, controlling ribonuclease access to the mRNA substrate is an important regulatory mechanism in gene expression. This control is achieved through various mechanisms including, controlling the amount of nucleases present, regulating the activity of the nuclease, sequestering the nuclease to particular cellular locations to restrict its access to RNA, and to control the stability of the mRNA by decreasing accessibility to nuclease.
Four regions of an mRNA are important for its overall stability. These include, the 5′ untranslated region and the cap, coding region, 3’ untranslated region, and the poly(A) tail. The cap at the 5′ end protects the mRNA from exonucleases that degrade RNA from the 5′ to the 3′ direction. The 3′ untranslated region may contain short sequences that influence mRNA stability. For example, the repetition of the sequence AUUUA in the 3′ untranslated region of many animal and plant genes is associated with mRNAs with short half-lives (the time it takes for half the RNA to be degraded, after transcription is stopped).
The poly(A) tail increases mRNA stability but is not sufficient alone. It only provides stability when bound by proteins such as poly(A) binding protein (PABP). Test-tube experiments have shown that, removing PABP from a stable mRNA destabilises it, while adding back purified PABP restores stability.
Protein Regulation
Clearly the amount of a protein within a cell (or organism) is a function of the number of mRNAs encoding the protein, the rate that these mRNAs are recognised and translated, and the rate at which functional protein is formed, which in turn depends upon folding rates and their efficiency. It is generally the case that once translation begins, it continues at a more or less constant rate. In the bacterium E. coli, the rate of translation at 37ºC is about 15 amino acids per second. The translation of a polypeptide of 1500 amino acids therefore takes about 100 seconds. After translation, folding and, in multisubunit proteins, assembly, the protein will function (assuming that it is active) until it is degraded.
Many proteins within the cell are necessary all of the time. Such proteins are termed “constitutive” or house-keeping proteins. Protein degradation is particularly important for controlling the levels of “regulated” proteins, whose presence or concentration within the cell may lead to unwanted effects in certain situations. The regulated degradation of a protein typically begins when the protein is specifically marked for degradation. This is an active and highly regulated process, involving ATP hydrolysis and a multi-subunit complex known as the proteosome. The proteosome degrades the polypeptide into small peptides and amino acids that can be recycled. As a mechanism for regulating protein activity, however, degradation has a serious drawback, it is irreversible.
Proteins can be chemically modified with the addition of groups including methyl, phosphate, acetyl, and ubiquitin groups. The addition or removal of these groups from proteins regulates their activity or the length of time they exist in the cell. Sometimes these modifications can regulate where a protein is found in the cell—for example, in the nucleus, in the cytoplasm, or attached to the plasma membrane.
Chemical modifications occur in response to external stimuli such as stress, the lack of nutrients, heat, or ultraviolet light exposure. These changes can alter epigenetic accessibility, transcription, mRNA stability, or translation—all resulting in changes in expression of various genes. This is an efficient way for the cell to rapidly change the levels of specific proteins in response to the environment. Because proteins are involved in every stage of gene regulation, the phosphorylation of a protein (depending on the protein that is modified) can alter accessibility to the chromosome, can alter translation (by altering transcription factor binding or function), can change nuclear shuttling (by influencing modifications to the nuclear pore complex), can alter RNA stability (by binding or not binding to the RNA to regulate its stability), can modify translation (increase or decrease), or can change post-translational modifications (add or remove phosphates or other chemical modifications).
The addition of a ubiquitin group to a protein marks that protein for degradation. Ubiquitin acts like a flag indicating that the protein lifespan is complete. These proteins are moved to the proteasome, an organelle that functions to remove proteins, to be degraded (10.17). One way to control gene expression, therefore, is to alter the longevity of the protein.
Common types of post-translational modifications
The first mechanism is covalent modification by the addition of various chemical groups. A wide variety of groups can be involved, including small organic groups (methylation, acetylation) lipids (myristoylation, farnesylation, palmitylation), carbohydrates (glycosylation, glucosylation), small proteins (ubiquitination, sumoylation) and inorganic molecules (phosphorylation, sulfation). Such covalent modifications are generally accomplished by the activity of enzymes specialised to perform these modifications. Many of these modifications are also reversible; that is these groups can be added to a protein and subsequently removed. Phosphorylation is a particularly noteworthy reversible modification that is common in the regulation of many proteins. Enzymes that add phosphate groups to other proteins are called protein kinases, and those that remove phosphates are called phosphatases. As such, protein kinases and phosphatases are central to many cellular regulatory systems.
A second common mechanism of protein modification is through proteolytic cleavage. Proteolysis occurs as part of the general turnover of cellular proteins, which is required to eliminate damaged proteins and recycle amino acids. Proteolysis is important for the processing of certain proteins, for example in the removal of the signal peptide of proteins targeted to specific cellular compartments. It can also occur in a highly specific manner whereby particular proteins are targeted for degradation or for cleavage at a specific site within the protein. Proteolytic modifications are non-reversible.
Proteins can undergo modification through complex formation. Such complexes can occur among proteins or between a protein and a cofactor. Proteins can also be modified according to conditions of the cellular environment. The redox state can result in oxidation or reduction of proteins, particularly of sulfhydryl side groups. Cellular pH can affect the charge of ionizable side groups.
Common types of protein regulation
All of the above mentioned types of protein modification can alter protein conformations and thus have regulatory consequences on protein function or activity. Protein regulation is highly complex and there are a myriad of different ways by which this occurs. Again we will just briefly consider a few of the more common mechanisms.
One major way protein modification can regulate protein function is by altering their activity. This can be true for many types of proteins including, but not limited to, enzymes, transcription factors, signalling proteins, and structural proteins.
Cells are compartmentalised into several membrane bound organelles, including the nucleus, chloroplasts, mitochondria, peroxisomes, endoplasmic reticulum (ER), golgi, and vacuoles. Each of these compartments performs unique metabolic functions that require a set of proteins. Compartmentation is regulated for some proteins. For example, upon exposure to light the phytochrome protein moves into the nucleus where it affects the expression of light-regulated genes.
Proteolytic processing is involved in several important regulatory processes. Many proteins are synthesised in an inactive form that requires proteolytic cleavage for activation. The full-length translation product before processing is often called the precursor, or a preprotein. As mentioned, cells contain membrane-bound compartments. Since membranes are impermeable to most proteins, an active mechanism is necessary to move a protein across a membrane. For proper delivery to their organellar destinations, specific amino acid sequences, called target signals must be present in a protein (to serve as an “address”). For example, entry into the chloroplast is achieved by the presence of a target signal called the transit peptide. This is at the amino terminal end of the protein and is proteolytically cleaved during import.
Another example of proteolytic processing is seen in a plant defence response in members of the solanaceae (for example, tomato and potato). An 18-amino acid peptide hormone called systemin is secreted by plant cells that are damaged by insects or mechanical wounding. Systemin production by wounded cells is required to induce the synthesis of proteins involved in defence. Systemin induces defence responses in wounded cells, and throughout the plant. Analogous to animal peptide hormones (e.g., insulin), systemin is initially synthesised as a much larger (200 amino acids) precursor called pro-systemin. Pro-system is inactive; however, upon wounding it undergoes proteolytic cleavage to produce activated systemin.
Targeted protein degradation
The amount of protein present in a cell or tissue is determined by both its rate of synthesis and its rate of degradation. Therefore, protein degradation is an important mechanism by which the plant can regulate biological activity (i.e., a genetic function). For example, one way to shut down a metabolic pathway is by degrading one of the key enzymes controlling the rate of the entire pathway. Therefore, protein degradation is an essential component of gene regulation to meet cellular demands for growth, development, and defence.
Protein degradation must be carefully controlled to fine tune gene expression to allow plants to adapt to new environmental conditions. Often, cells will adopt several complex mechanisms for proteolytic degradation of proteins. Enzymes that cleave or degrade proteins are referred to as proteases. For example, plant vacuoles are rich in proteases that play a similar function in protein degradation as that of lysosomes in animal cells. Protease activity must be tightly regulated to prevent accidental degradation of essential proteins. Sequestering proteases in particular organelles like the vacuole separates them from other organelles and is one of to control their activity.
An important mechanism by which specific proteins are targeted for degradation is through ubiquitin-mediated proteasomal degradation. The proteasome is a large complex of multiple protein subunits that have a protease activity to degrade proteins. Proteins get marked for proteasomal degradation with a small protein called ubiquitin. Ubiquitin is covalently attached to specific proteins in response to environmental or developmental signals. This allows plants to quickly adapt to changing conditions by eliminating proteins whose functions are not advantageous under the new conditions. For example, the photoreceptor phytochrome mentioned above becomes targeted for degradation when light is no longer available. This allows plants to change their physiological functions going from daylight to night conditions. Protein degradation by the proteasome system is also an important regulatory mechanism for plant hormone signalling, for example the signalling of the defence hormone jasmonic acid, and growth hormone gibberellic acid.
Proteins have lifetimes that range from a few minutes to weeks or more. Cells continuously make proteins from and break them down to amino acids. One of the functions of protein degradation is to eliminate aberrant or damaged proteins which could harm the cell. The second function is to facilitate the recycling of amino acids. For example, most of the amino acids required for growth of the seedling are derived from the degradation of seed storage proteins. Conversely, in annual crop plants, many of the amino acids in seed storage proteins are derived from proteins degraded in leaves and other plant parts during senescence.
Real-World Applications
The study of gene expression extends beyond describing how genetic information flows from DNA to protein; it also provides opportunities to intervene in this process when expression becomes dysregulated. Many diseases arise not only from mutations in DNA, but from imbalances in gene expression, where proteins are produced at inappropriate levels or times. As a result, controlling gene expression at the level of RNA has become an important strategy in both research and clinical contexts.
One mechanism that illustrates this principle is RNA interference (RNAi), a conserved cellular pathway that regulates gene expression post-transcriptionally. In this process, small RNA molecules guide the RNA-induced silencing complex (RISC) to specific messenger RNAs (mRNAs), leading to their degradation or preventing their translation into protein. When the RNA sequence is highly complementary to its target, the mRNA is cleaved, effectively reducing or eliminating production of the encoded protein. This system allows cells to fine-tune protein output and maintain internal balance.
Advances in molecular biology have enabled the deliberate use of this natural pathway to modulate gene expression in controlled ways. Synthetic small interfering RNAs (siRNAs) can be designed to match the sequence of a specific gene, allowing targeted reduction of its expression (Figure 10.20). This has become a widely used approach in experimental biology to study gene function, but it also has broader implications for disease intervention. By reducing the expression of genes that contribute to pathology, it is possible to alter disease progression at a molecular level.
The application of this concept can be seen in conditions where harmful proteins accumulate due to imbalanced gene expression. In hereditary transthyretin-mediated amyloidosis (hATTR), the protein transthyretin (TTR) misfolds and forms aggregates that damage tissues. Rather than attempting to correct the protein after it has been produced, RNAi-based approaches target the TTR mRNA within liver cells, reducing overall protein synthesis. In this context, siRNA incorporated into RISC binds with high complementarity to a conserved sequence within the 3′ untranslated region of the TTR transcript, leading to its degradation before translation (Traber & Yu, 2023). This reduces the abundance of both normal and mutant protein forms and demonstrates how controlling mRNA stability can influence disease outcomes.
The ability to selectively reduce or modify gene expression highlights a shift in biomedical science from observation to intervention. Approaches that operate at the level of RNA offer a means of addressing diseases at their molecular origin, particularly in cases where protein-targeting strategies are limited. As knowledge of gene regulation continues to expand, the capacity to manipulate gene expression with precision is likely to play an increasingly important role in the development of diagnostics, experimental models, and therapeutic strategies.
The foundational principle of genetics stating that genetic information flows in a one-way direction in cells: from DNA to RNA to protein.
Ribonucleic acid; a typically single-stranded nucleic acid composed of ribonucleotides containing ribose sugar and the bases A, U, C, and G.
Messenger RNA; a mobile, processed RNA transcript that carries protein-coding instructions from DNA in the nucleus to ribosomes in the cytoplasm.
The structural and catalytic RNA molecules that comprise the major functional components of ribosomes.
Small adaptor RNA molecules that carry specific amino acids and recognise mRNA codons using a complementary anticodon during translation.
Small, non-coding RNA molecules (miRNAs) that regulate gene expression post-transcriptionally by binding complementarily to target mRNAs to degrade or silence them.
An unmethylated pyrimidine nitrogenous base unique to RNA that complementarily base-pairs with adenine.
Consecutive triplets of nucleotides in mRNA that specify a particular amino acid or translation termination signal during protein synthesis.
Three codons (UAA, UAG, UGA) in mRNA that terminate translation by recruiting a release factor instead of a tRNA.
The codon (AUG) that initiates translation, setting the reading frame and encoding methionine.
An organelle of the endomembrane system consisting of stacked membranous disks (dictyosomes) that modifies, tags, packages, and distributes lipids and proteins received from the ER.
A short, N-terminal amino acid sequence on a nascent protein that targets it to a specific cellular compartment or organelle.
The distinct physical protein molecular variants that arise from a single gene due to splicing, mutations, or post-translational modifications.
Varying levels of gene transcription and expression observed across different tissues, developmental stages, or environmental conditions.
Genes that display highly synchronised, similar transcription and expression patterns across various biological states.
A regulatory DNA sequence upstream of a coding region that recruits RNA polymerase and transcription factors to initiate transcription.
Genes producing easily visible and quantifiable products (such as GUS or green fluorescent protein) fused to promoters in research to study gene regulation.
A regulatory DNA sequence that binds negative regulators to decrease or damp the level of gene transcription controlled by a promoter.
