Molecular Genetics 3: Gene Structure
Learning Objectives
- Explain the central dogma of molecular biology and the relationship between genes, RNA, and proteins
- Describe the structure and organisation of genes, including coding regions, regulatory sequences, introns, exons, and untranslated regions
- Compare the organisation and expression of genes in prokaryotic and eukaryotic cells
- Explain the processes of transcription, RNA processing, and alternative splicing in gene expression
- Describe how gene expression is regulated at transcriptional and post-transcriptional levels
- Evaluate the impact of genomics, including the Human Genome Project, on biomedical research, disease diagnosis, and personalised medicine
Chapter Introduction
Since the rediscovery of Mendel’s work in 1900, the definition of the gene has progressed from an abstract unit of heredity to a tangible molecular entity capable of replication, expression, and mutation. Genes are composed of DNA and are linearly arranged on chromosomes. Genes specify the sequences of amino acids, which are the building blocks of proteins. In turn, proteins are responsible for orchestrating nearly every function of the cell. Both genes and the proteins they encode are absolutely essential to life as we know it.
Central Dogma
The flow of genetic information in cells from DNA to mRNA to protein is described by the central dogma (Figure 9.1), which states that genes specify the sequences of mRNAs, which in turn specify the sequences of proteins.
Genes are DNA sequences that control traits in an organism by coding for proteins (Figure 9.2). Organisms such as plants and animals have tens of thousands of genes. The impact that a single gene’s information can have on an organism, however, is tremendous. Furthermore, organisms have all their genes in each of their cells, but they only need to use the information from a subset of these genes, depending on the type of cell and the cell’s stage of development. Therefore, the key to gene function is controlling its expression.
Gene structure and transcription
The flow of genetic information in cells from DNA to mRNA is accomplished by a process known as transcription (Figure 9.2). This first step in the central dogma of biology serves a few purposes. 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.
Gene Definition
The DNA sequence contains the information to control all biological functions, including the manifestation of traits important to agriculture (such as yield, drought tolerance and disease resistance) and health (such as cardiovascular health, sugar metabolism and development of cancer). How is the information contained in DNA sequences converted into the cellular activities necessary for plants and other organisms to function? DNA sequences are used to direct the synthesis of other molecules that actually perform these cellular functions.
Only particular regions of chromosomal DNA are transcribed. A gene can be considered as the region of transcribed DNA, along with associated regions of DNA important for the regulation of transcription. A gene has several parts that are each important to the function of the gene.
The regulatory region (also known as the promoter) contains DNA sequence involved in the control of where and when the genes will be turned on to produce mRNA. The coding region is the part of the gene that is used as template to produce RNA molecules in a process called transcription. Some RNA molecules perform cellular functions directly while many others (messenger RNAs) are used to direct the synthesis of proteins in a process called translation.
Gene Promoter
The signals for starting and stopping transcription are located within DNA sequences. Specific nucleotide segments called promoters are recognised by RNA polymerase to start RNA synthesis. After the transcription of full-length RNA strand is completed, a second segment of DNA called terminator invokes termination of RNA synthesis and the detachment of RNA polymerases from the DNA template.
Protein-coding region
The protein-coding region of a gene is composed of the sequence of nucleotides that codes for amino acids. As described further in the section on translation, the coding region begins with an ATG start codon (AUG in RNA) and then ends with one of three stop codons. These sequences include only exons, but not all exonic sequences are protein coding as they may include untranslated regions.
Untranslated regions (UTRs)
Mature transcripts contain some sequences that do not code for amino acid sequences in proteins. These are referred to as untranslated regions or UTRs. Most mRNA transcripts contain a 5′ and a 3′ UTR. The 5′ UTR contains sequences toward the 5′ end of the mRNA sequence, before the start codon. These sequences can often be important for translational regulation, and sometimes other functions. The sequences following the stop codon are the 3′ UTR. The 3′ UTR may also have important functions regulating transcript stability or directing transcript localisation within cells, or sometimes even transport (trafficking) between cells.
UTRs and introns are often useful in genetic studies. Protein coding regions are under strong selective pressure to produce functional proteins and so sequence variation is relatively rare. UTRs and introns on the other hand are under less stringent selection and are therefore sources of sequence variants that can be used to develop genetic markers.
Transcription
The genetic information of DNA is transferred to an intermediate molecule called RNA that is often translated to amino acid sequences used to build proteins. RNA is a nucleic acid, like DNA, but with some important differences. RNA contains a ribose sugar group instead of the deoxyribose found in DNA. RNA molecules are single stranded, instead of being double stranded. RNA contains a uridine (U) base and does not contain a thymidine base. The other bases (A, C, G) are contained in both RNA and DNA. U has the property of base-pairing with A.
RNA synthesis is directed by a DNA template in a process called transcription. A protein complex containing the enzyme RNA polymerase synthesises an RNA molecule by adding nucleosides to the 3′ end of a growing chain. The principle of base pairing is used again and each nucleotide base added is complementary to the corresponding base on the DNA template. Thus, the RNA is complementary in sequence to the template strand of DNA, which is also referred to as “antisense” or “negative” strand. The RNA is identical in sequence (except U replaces T) to the other strand, which is called the “sense” or “positive” strand. Because RNA molecules are produced by the process of transcription, they are often referred to as transcripts.
RNA processing
Coding (transcribed) region
This is the region that is transcribed by RNA polymerase, also known as the RNA coding region. As described below, it may include introns, sequences that are removed from the mature RNA molecule during RNA processing. The transcribed region is demarcated by promoter and terminator sequences.
Introns and exons
As mentioned, and described in detail below, introns are sequences that are removed from transcripts during RNA processing. Sequences that are retained in mature transcripts are called exons. The corresponding stretches of DNA are typically referred to with the same terms. Introns are commonly found in genes of eukaryotes but are rare in prokaryotic organisms.
Intron splicing
The process of transcription produces pre-mRNA that contains both introns and exons. The process of splicing involves removal of introns from pre-mRNA and joining together the exons. A complex group of proteins that form a spliceosome perform the splicing reaction.
Introns sometimes serve as boundaries for sequences encoding functional protein domains, leading to possibility for new and variant proteins by exon shuffling. Also, introns can provide possibility for productions of variant RNA forms through alternate splicing allowing more than one gene product from a single gene. Some introns result from the insertion of transposable elements and may be spliced perfectly of imperfectly, offering more possibility for new genetic diversity.
5′ Capping
The 5’ capping is the addition of a 7-methyl guanidine to the first nucleotide of mRNA molecule, usually and adenine or guanidine. The phosphodiester linkage between 7-methyl guanidine and the target nucleotide is 5′-5′ instead of 5′-3′, and 3 phosphates rather than 1 are retained in the linkage. The cap stabilises the 5′ end of the mRNA and plays a role in translation initiation.
Poly adenylation
The transcription of a gene may proceed beyond what ends up as 3′ end of mature mRNA. Thus the 3′ end of mRNA is formed after transcription. The enzyme poly(A) polymerase adds numerous adenosines to the 3′ end to result in what is called the poly(A) tail. The poly(A) tail is necessary for proper processing and transport of mRNA to the cytoplasm. The poly(A) tail is also important for the stability of mRNA, and initiation of translation in eukaryotic organisms.
For a cell to function properly, necessary proteins must be synthesised at the proper time and place. All cells control or regulate the synthesis of proteins from information encoded in their DNA. The process of turning on a gene to produce RNA and protein is called gene expression. Whether in a simple unicellular organism or a complex multi-cellular organism, the expression of individual genes, or groups of genes, is tightly regulated by the cell. For this to occur, there must be internal chemical mechanisms that control when a gene is expressed to make RNA and protein, how much of the protein is made, and when it is time to stop making that protein because it is no longer needed.
The regulation of gene expression conserves energy and space. It would require a significant amount of energy for an organism to express every gene at all times, so it is more energy efficient to turn on the genes only when they are required. In addition, only expressing a subset of genes in each cell saves space because DNA must be unwound from its tightly coiled structure to transcribe and translate the DNA. Cells would have to be enormous if every gene were expressed in every cell all the time. 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.
To understand how gene expression is regulated, we must first understand how a gene codes for a functional protein in a cell. The process occurs in both prokaryotic and eukaryotic cells, just in slightly different manners.
Prokaryotic Gene
Prokaryotic organisms are single-celled organisms that lack a cell nucleus, and their DNA therefore floats freely in the cell cytoplasm. To synthesise a protein, the processes of transcription and translation occur almost simultaneously. When the resulting protein is no longer needed, transcription stops. As a result, the primary method to control what type of protein and how much of each protein is expressed in a prokaryotic cell is the regulation of DNA transcription (Figure 9.7). All subsequent steps occur automatically. When more protein is required, more transcription occurs. Therefore, in prokaryotic cells, the control of gene expression is mostly at the transcriptional level.
The DNA of prokaryotes is organised into a circular chromosome, supercoiled within the nucleoid region of the cell cytoplasm. Genes for proteins that are needed for a specific function, or that are involved in the same biochemical pathway, are clustered together in blocks called operons. For example, all of the genes needed to use lactose as an energy source are coded next to each other in the lactose (or lac) operon and transcribed into a single mRNA.
In prokaryotic cells, there are three types of regulatory molecules that can affect the expression of operons: repressors, activators, and inducers. Repressors and activators are proteins produced in the cell. Both repressors and activators regulate gene expression by binding to specific DNA sites adjacent to the genes they control. In general, activators bind to the promoter site, while repressors bind to operator regions. Repressors prevent transcription of a gene in response to an external stimulus, whereas activators increase the transcription of a gene in response to an external stimulus. Inducers are small molecules that may be produced by the cell or that are in the cell’s environment. Inducers either activate or repress transcription depending on the needs of the cell and the availability of substrate.
The lac Operon: An Inducible Operon
One type of gene regulation in prokaryotic cells occurs through inducible operons, which have proteins that bind to activate transcription depending on the local environment and the needs of the cell. The lac operon is a typical inducible operon found in the bacterium Escherichia coli. When glucose concentrations are low, E. coli is able to use other sugars, like lactose, as energy sources. The lac operon encodes the genes necessary to acquire and process the lactose from the local environment. The Z gene of the lac operon encodes beta-galactosidase, which breaks lactose down to glucose and galactose.
Expression of the lac operon is turned off under normal conditions (high glucose) due to the binding of the lac repressor protein (Figure 9.8). Only when glucose is absent and lactose is present will the lac operon be transcribed (Figure 9.8). When lactose is present, its metabolite, allolactose, binds to the lac repressor and changes its shape so that it cannot bind to the lac operator to prevent transcription. RNA polymerase is now free to initiate transcription of the lac operon in response the presence of lactose.
The lac repressor stands between the cell and the enzymes needed for lactose processing. The expression of the operon continues until sufficient amounts of protein are produced. The allolactose is a victim of its own success and is targeted by enzymes and rendered inactive. The lac operon returns to its default state. This combination of conditions makes sense for the cell, because it would be energetically wasteful to synthesise the enzymes to process lactose if glucose was plentiful or lactose was not available. It should be mentioned that the lac operon is transcribed at a very low rate even when glucose is present and lactose absent.
The trp Operon: A Repressible Operon
Bacteria such as E. coli need amino acids to survive and are able to synthesise many of them. Tryptophan is one such amino acid that E. coli can either ingest from the environment or synthesise using enzymes that are encoded by five genes. These five genes are next to each other in what is called the tryptophan (trp) operon (Figure 9.9). The genes are transcribed into a single mRNA, which is then translated to produce all five enzymes. If tryptophan is present in the environment, then E. coli does not need to synthesise it and the trp operon is switched off by default. However, when tryptophan availability is low, the switch controlling the operon is turned on, the mRNA is transcribed, the enzyme proteins are translated, and tryptophan is synthesised.
The trp operon includes three important regions: the coding region, the trp operator and the trp promoter. The coding region includes the genes for the five tryptophan biosynthesis enzymes. Just before the coding region is the transcriptional start site. The promoter sequence, to which RNA polymerase binds to initiate transcription, is before or “upstream” of the transcriptional start site. Between the promoter and the transcriptional start site is the operator region.
The trp operaton contains the DNA code to which the trp repressor protein can bind. However, the repressor alone cannot bind to the operator. When tryptophan is present in the cell, two tryptophan molecules bind to the trp repressor, which changes the shape of the repressor protein to a form that can bind to the trp operator. Binding of the tryptophan–repressor complex at the operator physically prevents the RNA polymerase from binding to the promoter and transcribing the downstream genes.
When tryptophan is not present in the cell, the repressor by itself does not bind to the operator, the polymerase can transcribe the enzyme genes, and tryptophan is synthesised. Because the repressor protein actively binds to the operator to keep the genes turned off, the trp operon is said to be negatively regulated and the proteins that bind to the operator to silence trp expression are negative regulators.
The prokaryotes, which include Bacteria and Archaea, are mostly single-celled organisms that, by definition, lack membrane-bound nuclei and other organelles. A bacterial chromosome is a closed circle that, unlike eukaryotic chromosomes, is not organised around histone proteins. The central region of the cell in which prokaryotic DNA resides is called the nucleoid region. In addition, prokaryotes often have abundant plasmids, which are shorter, circular DNA molecules that may only contain one or a few genes. Plasmids can be transferred independently of the bacterial chromosome during cell division and often carry traits such as those involved with antibiotic resistance.
Transcription in prokaryotes (and in eukaryotes) requires the DNA double helix to partially unwind in the region of mRNA synthesis. The region of unwinding is called a transcription bubble. Transcription always proceeds from the same DNA strand for each gene, which is called the template strand. The mRNA product is complementary to the template strand and is almost identical to the other DNA strand, called the nontemplate strand, or the coding strand. The only nucleotide difference is that in mRNA, all of the T nucleotides are replaced with U nucleotides (Figure 9.10). In an RNA double helix, A can bind U via two hydrogen bonds, just as in A–T pairing in a DNA double helix.
The nucleotide pair in the DNA double helix that corresponds to the site from which the first 5' mRNA nucleotide is transcribed is called the +1 site, or the initiation site. Nucleotides preceding the initiation site are denoted with a “-” and are designated upstream nucleotides. Conversely, nucleotides following the initiation site are denoted with “+” numbering and are called downstream nucleotides.
Initiation of Transcription in Prokaryotes
Prokaryotes do not have membrane-enclosed nuclei. Therefore, the processes of transcription, translation, and mRNA degradation can all occur simultaneously. The intracellular level of a bacterial protein can quickly be amplified by multiple transcription and translation events that occur concurrently on the same DNA template. Prokaryotic genomes are very compact, and prokaryotic transcripts often cover more than one gene or cistron (a coding sequence for a single protein). Polycistronic mRNAs are then translated to produce more than one kind of protein.
Our discussion here will exemplify transcription by describing this process in Escherichia coli, a well-studied eubacterial species. Although some differences exist between transcription in E. coli and transcription in archaea, an understanding of E. coli transcription can be applied to virtually all bacterial species.
Prokaryotic RNA Polymerase
Prokaryotes use the same RNA polymerase to transcribe all of their genes. In E. coli, the polymerase is composed of five polypeptide subunits, two of which are identical. Four of these subunits, denoted α, α, β, and β', comprise the polymerase core enzyme. These subunits assemble every time a gene is transcribed, and they disassemble once transcription is complete. Each subunit has a unique role; the two α-subunits are necessary to assemble the polymerase on the DNA; the β-subunit binds to the ribonucleoside triphosphate that will become part of the nascent mRNA molecule; and the β' subunit binds the DNA template strand. The fifth subunit, σ, is involved only in transcription initiation. It confers transcriptional specificity such that the polymerase begins to synthesise mRNA from an appropriate initiation site. Without σ, the core enzyme would transcribe from random sites and would produce mRNA molecules that specified protein gibberish. The polymerase comprised of all five subunits is called the holoenzyme.
Prokaryotic Promoters
A promoter is a DNA sequence onto which the transcription machinery, including RNA polymerase, binds and initiates transcription. In most cases, promoters exist upstream of the genes they regulate. The specific sequence of a promoter is very important because it determines whether the corresponding gene is transcribed all the time, some of the time, or infrequently. Although promoters vary among prokaryotic genomes, a few elements are evolutionarily conserved in many species. At the -10 and -35 regions upstream of the initiation site, there are two promoter consensus sequences, or regions that are similar across all promoters and across various bacterial species (Figure 9.11). The -10 sequence, called the -10 region, has the consensus sequence TATAAT. The -35 sequence has the consensus sequence TTGACA. These consensus sequences are recognised and bound by σ. Once this interaction is made, the subunits of the core enzyme bind to the site. The A–T-rich -10 region facilitates unwinding of the DNA template, and several phosphodiester bonds are made. The transcription initiation phase ends with the production of abortive transcripts, which are polymers of approximately 10 nucleotides that are made and released.
Elongation and Termination in Prokaryotes
The transcription elongation phase begins with the release of the σ subunit from the polymerase. The dissociation of σ allows the core enzyme to proceed along the DNA template, synthesising mRNA in the 5' to 3' direction at a rate of approximately 40 nucleotides per second. As elongation proceeds, the DNA is continuously unwound ahead of the core enzyme and rewound behind it. The base pairing between DNA and RNA is not stable enough to maintain the stability of the mRNA synthesis components. Instead, the RNA polymerase acts as a stable linker between the DNA template and the nascent RNA strands to ensure that elongation is not interrupted prematurely.
Prokaryotic Termination Signals
Once a gene is transcribed, the prokaryotic polymerase needs to be instructed to dissociate from the DNA template and liberate the newly made mRNA. Depending on the gene being transcribed, there are two kinds of termination signals. One is protein-based and the other is RNA-based. Rho-dependent termination is controlled by the rho (ρ) protein, which tracks along behind the polymerase on the growing mRNA chain. Near the end of the gene, the polymerase encounters a run of G nucleotides on the DNA template and it stalls. As a result, the rho protein collides with the polymerase. The interaction with rho releases the mRNA from the transcription bubble.
Rho-independent termination is controlled by specific sequences in the DNA template strand. As the polymerase nears the end of the gene being transcribed, it encounters a region rich in C–G nucleotides. The mRNA folds back on itself, and the complementary C–G nucleotides bind together. The result is a stable hairpin that causes the polymerase to stall as soon as it begins to transcribe a region rich in A–T nucleotides. The complementary U–A region of the mRNA transcript forms only a weak interaction with the template DNA. This, coupled with the stalled polymerase, induces enough instability for the core enzyme to break away and liberate the new mRNA transcript.
Upon termination, the process of transcription is complete. By the time termination occurs, the prokaryotic transcript would already have been used to begin synthesis of numerous copies of the encoded protein because these processes can occur concurrently. The unification of transcription, translation, and even mRNA degradation is possible because all of these processes occur in the same 5' to 3' direction, and because there is no membranous compartmentalisation in the prokaryotic cell (Figure 9.12). In contrast, the presence of a nucleus in eukaryotic cells precludes simultaneous transcription and translation.
Eukaryotic Gene
Prokaryotes and eukaryotes perform fundamentally the same process of transcription, with a few key differences. The most important difference between prokaryote and eukaryote transcription is due to the latter’s membrane-bound nucleus and organelles. With the genes bound in a nucleus, the eukaryotic cell must be able to transport its mRNA to the cytoplasm and must protect its mRNA from degrading before it is translated. Eukaryotes also employ three different polymerases that each transcribe a different subset of genes. Eukaryotic mRNAs are usually monogenic, meaning that they specify a single protein.
Initiation of Transcription in Eukaryotes
Unlike the prokaryotic polymerase that can bind to a DNA template on its own, eukaryotes require several other proteins, called transcription factors, to first bind to the promoter region and then to help recruit the appropriate polymerase.
The features of eukaryotic mRNA synthesis are markedly more complex than those of prokaryotes. Instead of a single polymerase comprising five subunits, the eukaryotes have three polymerases that are each made up of 10 subunits or more. Each eukaryotic polymerase also requires a distinct set of transcription factors to bring it to the DNA template.
RNA polymerase I is located in the nucleolus, a specialised nuclear substructure in which ribosomal RNA (rRNA) is transcribed, processed, and assembled into ribosomes (Table 9.1). The rRNA molecules are considered structural RNAs because they have a cellular role but are not translated into protein. The rRNAs are components of the ribosome and are essential to the process of translation. RNA polymerase I synthesises all of the rRNAs from the tandemly duplicated set of 18S, 5.8S, and 28S ribosomal genes. (Note that the “S” designation applies to “Svedberg” units, a nonadditive value that characterises the speed at which a particle sediments during centrifugation).
| RNA Polymerase | Cellular Compartment | Product of Transcription | α-Amanitin Sensitivity |
|---|---|---|---|
| I | Nucleolus | All rRNAs except 5S rRNA | Insensitive |
| II | Nucleolus | All protein-coding nuclear pre-mRNAs | Extremely sensitive |
| III | Nucleolus | 5S rRNA, tRNAs, and small nuclear RNAs | Moderately sensitive |
Table 9.1. Locations, products and sensitivities of the three eukaryotic RNA polymerases. Source: Eukaryotic transcription, by, Mary Ann Clark, Matthew Douglas, and Jung Choi in Biology 2e, published by OpenStax, and used under a Creative Commons Attribution-NonCommercial-ShareAlike License v4.0.
RNA polymerase II is located in the nucleus and synthesises all protein-coding nuclear pre-mRNAs. Eukaryotic pre-mRNAs undergo extensive processing after transcription but before translation. For clarity, this module’s discussion of transcription and translation in eukaryotes will use the term “mRNAs” to describe only the mature, processed molecules that are ready to be translated. RNA polymerase II is responsible for transcribing the overwhelming majority of eukaryotic genes.
RNA polymerase III is also located in the nucleus. This polymerase transcribes a variety of structural RNAs that includes the 5S pre-rRNA, transfer pre-RNAs (pre-tRNAs), and small nuclear pre-RNAs. The tRNAs have a critical role in translation; they serve as the “adaptor molecules” between the mRNA template and the growing polypeptide chain. Small nuclear RNAs have a variety of functions, including “splicing” pre-mRNAs and regulating transcription factors.
A scientist characterising a new gene can determine which polymerase transcribes it by testing whether the gene is expressed in the presence of α-amanitin, an oligopeptide toxin produced by the fly agaric toadstool mushroom and other species of Amanita. Interestingly, the α-amanitin affects the three polymerases very differently (Table above). RNA polymerase I is completely insensitive to α-amanitin, meaning that the polymerase can transcribe DNA in vitro in the presence of this poison. RNA polymerase III is moderately sensitive to the toxin. In contrast, RNA polymerase II is extremely sensitive to α-amanitin. The toxin prevents the enzyme from progressing down the DNA, and thus inhibits transcription. Knowing the transcribing polymerase can provide clues as to the general function of the gene being studied. Because RNA polymerase II transcribes the vast majority of genes, we will focus on this polymerase in our subsequent discussions about eukaryotic transcription factors and promoters.
RNA Polymerase II Promoters and Transcription Factors
Eukaryotic promoters are much larger and more intricate than prokaryotic promoters. However, both have a sequence similar to the -10 sequence of prokaryotes. In eukaryotes, this sequence is called the TATA box, and has the consensus sequence TATAAA on the coding strand. It is located at -25 to -35 bases relative to the initiation (+1) site (Figure 9.13). This sequence is not identical to the E. coli -10 box, but it conserves the A–T rich element. The thermostability of A–T bonds is low and this helps the DNA template to locally unwind in preparation for transcription.
Instead of the simple σ factor that helps bind the prokaryotic RNA polymerase to its promoter, eukaryotes assemble a complex of transcription factors required to recruit RNA polymerase II to a protein coding gene. Transcription factors that bind to the promoter are called basal transcription factors. These basal factors are all called TFII (for Transcription Factor/polymerase II) plus an additional letter (A-J). The core complex is TFIID, which includes a TATA-binding protein (TBP). The other transcription factors systematically fall into place on the DNA template, with each one further stabilising the pre-initiation complex and contributing to the recruitment of RNA polymerase II.
Some eukaryotic promoters also have a conserved CAAT box (GGCCAATCT) at approximately -80. Further upstream of the TATA box, eukaryotic promoters may also contain one or more GC-rich boxes (GGCG) or octamer boxes (ATTTGCAT). These elements bind cellular factors that increase the efficiency of transcription initiation and are often identified in more “active” genes that are constantly being expressed by the cell.
Basal transcription factors are crucial in the formation of a preinitiation complex on the DNA template that subsequently recruits RNA polymerase II for transcription initiation. The complexity of eukaryotic transcription does not end with the polymerases and promoters. An army of other transcription factors, which bind to upstream enhancers and silencers, also help to regulate the frequency with which pre-mRNA is synthesised from a gene. Enhancers and silencers affect the efficiency of transcription but are not necessary for transcription to proceed.
Promoter Structures for RNA Polymerases I and III
The processes of bringing RNA polymerases I and III to the DNA template involve slightly less complex collections of transcription factors, but the general theme is the same. The conserved promoter elements for genes transcribed by polymerases I and III differ from those transcribed by RNA polymerase II. RNA polymerase I transcribes genes that have two GC-rich promoter sequences in the -45 to +20 region. These sequences alone are sufficient for transcription initiation to occur, but promoters with additional sequences in the region from -180 to -105 upstream of the initiation site will further enhance initiation. Genes that are transcribed by RNA polymerase III have upstream promoters or promoters that occur within the genes themselves.
Eukaryotic transcription is a tightly regulated process that requires a variety of proteins to interact with each other and with the DNA strand. Although the process of transcription in eukaryotes involves a greater metabolic investment than in prokaryotes, it ensures that the cell transcribes precisely the pre-mRNAs that it needs for protein synthesis.
Eukaryotic Elongation and Termination
Following the formation of the preinitiation complex, the polymerase is released from the other transcription factors, and elongation is allowed to proceed as it does in prokaryotes with the polymerase synthesising pre-mRNA in the 5' to 3' direction. As discussed previously, RNA polymerase II transcribes the major share of eukaryotic genes, so in this section we will focus on how this polymerase accomplishes elongation and termination.
Although the enzymatic process of elongation is essentially the same in eukaryotes and prokaryotes, the DNA template is considerably more complex. When eukaryotic cells are not dividing, their genes exist as a diffuse mass of DNA and proteins called chromatin. The DNA is tightly packaged around charged histone proteins at repeated intervals. These DNA–histone complexes, collectively called nucleosomes, are regularly spaced and include 146 nucleotides of DNA wound around eight histones like thread around a spool.
For polynucleotide synthesis to occur, the transcription machinery needs to move histones out of the way every time it encounters a nucleosome. This is accomplished by a special protein complex called FACT, which stands for “facilitates chromatin transcription.” This complex pulls histones away from the DNA template as the polymerase moves along it. Once the pre-mRNA is synthesised, the FACT complex replaces the histones to recreate the nucleosomes.
The termination of transcription is different for the different polymerases. Unlike in prokaryotes, elongation by RNA polymerase II in eukaryotes takes place 1,000 to 2,000 nucleotides beyond the end of the gene being transcribed. This pre-mRNA tail is subsequently removed by cleavage during mRNA processing. On the other hand, RNA polymerases I and III require termination signals. Genes transcribed by RNA polymerase I contain a specific 18-nucleotide sequence that is recognised by a termination protein. The process of termination in RNA polymerase III involves an mRNA hairpin similar to rho-independent termination of transcription in prokaryotes.
After RNA is transcribed, it must be processed into a mature form before translation can begin. This processing after an RNA molecule has been transcribed, but before it is translated into a protein, is called post-transcriptional modification. As with the epigenetic and transcriptional stages of processing, this post-transcriptional step can also be regulated to control gene expression in the cell. If the RNA is not processed, shuttled, or translated, then no protein will be synthesised.
Alternative RNA Splicing
In the 1970s, genes were first observed that exhibited alternative RNA splicing. Alternative RNA splicing is a mechanism that allows different protein products to be produced from one gene when different combinations of introns (and sometimes exons) are removed from the transcript (Figure 9.14). This alternative splicing can be haphazard, but more often it is controlled and acts as a mechanism of gene regulation, with the frequency of different splicing alternatives controlled by the cell as a way to control the production of different protein products in different cells, or at different stages of development. Alternative splicing is now understood to be a common mechanism of gene regulation in eukaryotes; according to one estimate, 70% of genes in humans are expressed as multiple proteins through alternative splicing.
How could alternative splicing evolve? Introns have a beginning and ending recognition sequence, and it is easy to imagine the failure of the splicing mechanism to identify the end of an intron and find the end of the next intron, thus removing two introns and the intervening exon. In fact, there are mechanisms in place to prevent such exon skipping, but mutations are likely to lead to their failure. Such “mistakes” would more than likely produce a nonfunctional protein. Indeed, the cause of many genetic diseases is alternative splicing rather than mutations in a sequence. However, alternative splicing would create a protein variant without the loss of the original protein, opening up possibilities for adaptation of the new variant to new functions. Gene duplication has played an important role in the evolution of new functions in a similar way — by providing genes that may evolve without eliminating the original functional protein.
Control of RNA Stability
Before the mRNA leaves the nucleus, it is given two protective “caps” that prevent the end of the strand from degrading during its journey. The 5′ cap, which is placed on the 5′ end of the mRNA, is usually composed of a methylated guanosine triphosphate molecule (GTP). The poly-A tail, which is attached to the 3′ end, is usually composed of a series of adenine nucleotides. Once the RNA is transported to the cytoplasm, the length of time that the RNA remains there can be controlled. Each RNA molecule has a defined lifespan and decays at a specific rate. This rate of decay can influence how much protein is in the cell. If the RNA decays more rapidly, translation has less time to occur, so less protein will be produced. Conversely, if RNA decays less rapidly, more protein will be produced. This rate of decay is referred to as the RNA stability. If the RNA is stable, it will be detected for longer periods of time in the cytoplasm. Binding of proteins to the RNA can influence its stability (Figure 9.16).
An antibody molecule is comprised of four polypeptides: two identical heavy chains (large peptide units) that are partially bound to each other in a “Y” formation, which are flanked by two identical light chains (small peptide units), as illustrated in Figure 9.17. Bonds between the cysteine amino acids in the antibody molecule attach the polypeptides to each other. The areas where the antigen is recognised on the antibody are variable domains and the antibody base is composed of constant domains.
In germ-line B cells, the variable region of the light chain gene has 40 variable (V) and five joining (J) segments. An enzyme called DNA recombinase randomly excises most of these segments out of the gene, and splices one V segment to one J segment. During RNA processing, all but one V and J segment are spliced out. Recombination and splicing may result in over 106 possible VJ combinations. As a result, each differentiated B cell in the human body typically has a unique variable chain. The constant domain, which does not bind antibody, is the same for all antibodies.
Antibody diversity is produced by the mutation and recombination of approximately 300 different gene segments encoding the light and heavy chain variable domains in precursor cells that are destined to become B cells. The variable domains from the heavy and light chains interact to form the binding site through which an antibody can bind a specific epitope on an antigen. The numbers of repeated constant domains in Ig classes are the same for all antibodies corresponding to a specific class. Antibodies are structurally similar to the extracellular component of the BCRs, and B cell maturation to plasma cells can be visualised in simple terms as the cell acquires the ability to secrete the extracellular portion of its BCR in large quantities.
Real-World Applications
Human Genome Project
Discovering the Human Genome
Scientists now know the sequence of all the DNA base pairs in the entire human genome. This knowledge was attained by the Human Genome Project (HGP), a $3 billion, international scientific research project that was formally launched in 1990. The project was completed in 2003, two years ahead of its 15-year projected deadline.
Determining the sequence of the billions of base pairs that make up human DNA was the main goal of the HGP. Another goal was mapping the location and determining the function of all the genes in the human genome. There are only about 20,500 genes in human beings.
A Collaborative Effort
Funding for the HGP came from the U.S. Department of Energy and the National Institutes of Health as well as from foreign institutions. The actual research was undertaken by scientists in 20 universities in the U.S., United Kingdom, Australia, France, Germany, Japan, and China. A private U.S. company named Celera also contributed to the effort. Although Celera had hoped to patent some of the genes it discovered, this was later denied.
Reference Genome of the Human Genome Project
In 2003, the HGP published the results of its sequencing of DNA as a human reference genome. The sequence of the human DNA is stored in databases available to anyone on the Internet. The U.S. National Center for Biotechnology Information (NCBI), part of the NIH, as well as comparable organisations in Europe and Japan, maintain the genomic sequences in a database known as Genbank. Protein sequences are also maintained in this database. The sequences in these databases are the combined sequences of anonymous donors, and as such do not yet address the individual differences that make us unique. However, the known sequence does lay the foundation to identify the unique differences among all of us. Most of the currently identified variations among individuals will be single nucleotide polymorphisms or SNPs. An SNP (pronounced "snip") is a DNA sequence variation occurring at a single nucleotide in the genome. For example, two sequenced DNA fragments from different individuals, GGATCTA to GGATTTA, contain a difference in a single nucleotide. If this, base change occurs in a gene, the base change then results in two alleles: the C allele and the T allele. Remember an allele is an alternative form of a gene. Almost all common SNPs have only two alleles. The effect of these SNPs on protein structure and function and any effect on the resulting phenotype are an extensive field of study.
A common-sense assumption about genomes would be that if genes specify proteins, then the more proteins an organism made, the more genes it would need to have, and thus, the larger its genome would be. Comparison of various genomes shows, surprisingly, that there is not necessarily a direct relationship between the complexity of an organism and the size of its genome. To understand how this could be true, it is necessary to recognise that while genes are made up of DNA, all DNA does not consist of genes (for purposes of our discussion, we define a gene as a section of DNA that encodes an RNA or protein product). In the human genome, less than 2% of the total DNA seems to be the sort of coding sequence that directs the synthesis of proteins. For many years, non-coding DNA in genomes was believed to be useless, and was described as “junk DNA” although it was perplexing that there seemed to be so much “useless” sequence. Recent discoveries have, however, demonstrated that much of this so-called junk DNA may play important roles in evolution, as well as in regulation of gene expression.
Benefits of the Human Genome Project
The sequencing of the human genome holds benefits for many fields, including molecular medicine and human evolution.
- Knowing the human DNA sequence can help us understand many human diseases. For example, it is helping researchers identify mutations linked to different forms of cancer. It is also yielding insights into the genetic basis of cystic fibrosis, liver diseases, blood-clotting disorders, and Alzheimer's disease, among others.
- The human DNA sequence can also help researchers tailor medications to individual genotypes. This is called personalised medicine, and it has led to an entirely new field called pharmacogenomics. Pharmacogenomics, also called pharmacogenetics, is the study of how our genes affect the way we respond to drugs.
- The analysis of similarities between DNA sequences from different organisms is opening new avenues in the study of evolution. For example, analyses are expected to shed light on many questions about the similarities and differences between humans and our closest relatives the nonhuman primates.
Ethical, Legal, and Social Issues of the Human Genome Project
From its launch in 1990, the HGP proactively established and funded a separate committee to oversee potential ethical, legal, and social issues associated with the project. A major concern was the possible use of the knowledge generated by the project to discriminate against people. One issue was the fear that employers and health insurance companies would refuse to hire or insure people based on their genetic makeup, for instance, if they had genes that increased their risk of getting certain diseases. In response, in 1996, the U.S. passed the Health Insurance Portability and Accountability Act (HIPAA). It protects against unauthorised, nonconsensual release of individually identifiable health information to any entity not actively engaged in providing healthcare to a patient. This was followed in 2008 by the Genetic Information Nondiscrimination Act (GINA), which specifically prohibits genetic discrimination by health insurance companies and workplaces.
Transcription factors that prevent gene transcription in response to an external stimulus by binding to operator DNA sequences.
Transcription factors that increase gene transcription in response to an external stimulus by binding to promoter elements.
Small environmental or cellular molecules that regulate transcription by binding to repressors or activators to modulate their DNA binding.
A regulatory DNA sequence upstream of a coding region that recruits RNA polymerase and transcription factors to initiate transcription.
A regulatory DNA sequence within or near an operon to which a repressor protein binds to physically block transcription.
The specific DNA nucleotide at which transcription begins, designated as the +1 site.
A repressible, negatively regulated bacterial operon encoding five enzymes for tryptophan synthesis, which is shut down when tryptophan is plentiful.
Proteins (such as repressors) that actively bind to DNA sites to prevent or silence gene expression.
The active multi-subunit bacterial RNA polymerase complex (α₂ββ') responsible for elongation but lacking the sigma (σ) factor needed for initiation specificity.
The complete, functional bacterial RNA polymerase complex consisting of the core enzyme and the sigma (σ) initiation factor.
A prokaryotic transcription termination mechanism where the rho protein tracks the transcript and collides with a stalled RNA polymerase to release the transcript.
A prokaryotic transcription termination mechanism where C-G rich transcript regions fold into a stable hairpin that stalls the polymerase on an A-T rich region, releasing the transcript.
The RNA secondary structure formed by C-G base pairing that physically stalls the polymerase during Rho-independent transcription termination.
Structural RNA molecules (snRNAs) localised in the nucleus that function in splicing pre-mRNAs and regulating transcription factors.
A conserved eukaryotic promoter consensus sequence (GGCCAATCT) located around -80 that increases transcription efficiency.
Upstream promoter consensus sequences (GGCG) in active eukaryotic genes that bind transcription factors to increase initiation efficiency.
Upstream eukaryotic promoter elements (ATTTGCAT) that bind cellular factors to enhance transcription initiation.
A chromatin-remodelling protein complex (Facilitates Chromatin Transcription) that temporarily pulls histones away from the DNA template ahead of RNA polymerase.
A post-transcriptional eukaryotic mechanism where different combinations of exons are joined and introns excised from a pre-mRNA to yield multiple unique protein products from a single gene.
An international collaborative research project (1990-2003) (HGP) that sequenced all 3 billion base pairs and mapped all genes in the human genome.
The study of how an individual's genetic makeup influences their physiological response to therapeutic drugs.
