In the previous article, Nucleic Acids 2, we looked at how cells maintain their identity and how they pass that identity on to the next cell or the next generation. During mitosis in somatic cells, DNA is first copied and then accurately divided into two, producing two daughter cells with essentially the same genetic information. In meiosis, which produces germ cells, the DNA is also copied, but homologous chromosomes undergo crossing-over and are rearranged into new combinations. This creates genetic diversity that can be passed on to the next generation. And in an even earlier article, we looked at another interesting question: If cells basically all have almost the same DNA, why do they end up looking and functioning so differently? The answer ultimately comes down to differences in which genes are selected, when they are used, and how much they are used.
So this time, I want to explore a question that naturally follows from all of that: How does a cell find the genes it needs and read them at the right time, despite having an enormous amount of genetic information? Neurons and heart muscle cells receive essentially the same genetic information. Yet in neurons, genes involved in nerve signaling and synapse formation are highly active, while genes related to muscle contraction are relatively suppressed because they are not particularly useful for what neurons need to do. In other words, cells can have the same genes but perform completely different functions depending on which genes they use. This is also why gene expression has attracted so much attention in the study of disease. Even identical twins, who share almost all of their DNA sequence, can develop different characteristics and health outcomes depending partly on differences in gene regulation and gene expression.
So, before we dive into gene expression itself, let's first straighten out a few terms and concepts related to the word “gene,” and then take a look at how the enormous space we call the genome is actually organized.
DNA, Genes, and the Genome
Although we often use terms such as DNA, genes, genome, and genetic material almost as if they were interchangeable, they actually mean slightly different things.
First, DNA is a real chemical and physical molecule. Its basic building blocks are nucleotides, which are made from a nitrogenous base, a sugar, and a phosphate group. DNA generally takes the form of a double helix. Inside the helix, opposing nitrogenous bases form base pairs through hydrogen bonds, while the sugars and phosphate groups connected to those bases form the outer backbone that supports and stabilizes the DNA structure. The information stored in DNA can be copied into RNA. In genes that contain instructions for making proteins, groups of three bases called codons are read like letters in a code, with each codon specifying a particular amino acid. Those amino acids are then linked together one after another to make a protein.
Storing the bases as paired bases in a double-stranded structure rather than as a single strand can, in simple terms, be seen as an evolutionary strategy for improving stability and accuracy. As we'll come back to later, one widely accepted hypothesis is that during the early stages of life's evolution, RNA rather than DNA may have played the major role in storing genetic information. RNA can exist as a single strand and fold back on itself into a remarkable variety of shapes and structures. Some RNAs can even catalyze chemical reactions, including self-cleavage reactions. DNA, on the other hand, has a relatively stable double-helical structure, making it better suited for safely storing information over long periods. So over the course of evolution, it is thought that RNA became increasingly specialized for transmitting, regulating, and carrying out information, while DNA became specialized for long-term information storage.
We often tend to confuse a gene with DNA itself. But a gene is not all of the DNA. A gene is a particular stretch of DNA that contains information used for a biological function. Some genes contain the information needed to make proteins, while others contain the information needed to make functional RNAs. These genes are scattered throughout the DNA. In other words, genes are only part of DNA, and they actually make up a surprisingly small portion of the entire genome. So DNA and genes should not be treated as the same thing.
And then there is the genome. The genome refers to all of the DNA carried by an organism. At the cellular level, it means the complete set of DNA contained in that cell. The genome includes not only the genes we just talked about, but also regulatory sequences that control gene expression, repetitive sequences, regions involved in maintaining chromosome structure, and many other regions whose functions are still not completely understood.
Finally, genetic material is the broadest of these concepts. It refers to the material that carries biological information from one generation to the next.
What Does DNA Look Like Inside a Cell?
So how does a cell find exactly the genes it needs and turn them on within such a vast genome? To answer this question, we first need to look at the physical state in which genes exist inside the nucleus. If you stretched out all the DNA contained in a single cell, it would be about two meters long. How could something that long possibly be squeezed(?) into such a tiny cell nucleus? Cells must have a remarkably clever and ingenious solution as usual.
Imagine wrapping thin strands of DNA tightly around histone proteins, like winding thread around a spool (or maybe a yo-yo?). These structures are called nucleosomes. Then, rather than leaving the nucleosomes loose, the cell folds and organizes them through several additional levels of packing, forming chromatin. The word chromatin may sound unfamiliar, but you have probably heard of chromosomes. Under normal conditions, chromatin is too loosely organized to be seen clearly as distinct structures under a microscope. However, as a cell prepares to divide, its DNA is replicated, producing sister chromatids. While these sister chromatids remain attached, the chromatin becomes extremely tightly condensed as cell division begins. At this stage, the chromosomes become visible under an optical microscope. Because the two sister chromatids remain attached to each other, a replicated chromosome looks like the familiar X shape. This highly condensed form of chromatin is what we call a chromosome. Chromatin and chromosomes refer to essentially the same material, but the terms describe different physical states. Chromatin generally refers to DNA associated with proteins in a relatively open and less condensed state, while a chromosome refers to that material in its highly condensed state during cell division.
But there is a problem. With so much DNA tightly folded and packed inside the nucleus, finding one particular gene buried somewhere in all that DNA is not exactly an easy task. But fortunately, cells have specialized proteins that help with this. They are called transcription factors. You can think of them as librarians who help you find the exact book you need in an enormous library.
Transcription Factors
Imagine that a cell needs to make a new protein in response to a particular signal from outside the cell. For example,if an inflammatory or stress signal is detected at the cell membrane, a chain of signaling events can unfold rapidly inside the cell, sometimes within minutes. As a result, a transcription factor such as NF-κB, which normally remains in an inhibited state in the cytoplasm, can become activated and move into the nucleus.
So what exactly is a transcription factor? First, transcription is the process of copying genetic information stored in DNA into RNA. The instructions for making proteins are stored in DNA, but DNA does not freely leave the nucleus. So the cell selects the genes it needs and copies the information from those regions into RNA. When the RNA contains instructions for making a protein, that RNA can eventually leave the nucleus and travel to a protein-making machine called the ribosome, where the information is translated into a protein.
This flow of information, “DNA → RNA → Protein” is known in modern molecular biology as the Central Dogma. Personally, I sometimes think the word “dogma” sounds a little strange here. I feel like calling it a “principle” would have been more intuitive.
Transcription factors act like switches that control whether transcription begins and how strongly it proceeds. When a transcription factor binds to the promoter region of a particular gene or to nearby regulatory regions, RNA polymerase and various helper proteins gather there to form a transcription initiation complex. Once the required gene is successfully transcribed into mRNA at the right time and in the right amount, the process moves on to protein synthesis. After all, transcription factors act almost like conductors, helping decide which genes should be turned on, when they should be turned on, and how strongly they should be used. They are key regulators connecting signals from outside the cell to gene expression.
Gene Regions vs. Non-Gene Regions
In the traditional view of the genome as a collection of blueprints for making proteins, DNA was broadly divided into regions containing genes and regions between genes. It was a very gene-centered way of looking at the genome. But as genome research has advanced, we have learned that many DNA regions that do not contain instructions for making proteins are not simply empty spaces filling the gaps between genes. Many of these regions perform important functions, including regulating gene expression and helping form and maintain the structure of chromosomes and chromatin.
With this in mind, it seems more appropriate to think of the genome not as a simple division between gene regions and non-gene regions, but as one enormous integrated system in which many different functional DNA elements work together.
Coding Genes
The regions of the human genome that contain protein-coding information make up only about 1–1.5% of the entire human genome. That's surprisingly little. There are only about 20,000 protein-coding genes in humans. Despite this relatively small number, we can produce an astonishingly large variety of proteins. One important reason is alternative splicing, along with the many modifications that can occur after a protein is translated, known as post-translational modifications. Even within a single gene, we find different functional regions, including exons, which contain the portions retained in the mature RNA, introns, which are removed during RNA processing, and UTRs (untranslated regions) located at the front and back of the transcript. Introns are often dismissed as meaningless regions simply because they do not contain information that is directly translated into protein. But they can actually play several roles, such as providing sequences involved in regulating splicing and serving as sources of some non-coding RNAs.
Non-Coding RNA Genes
The genome also contains genes that do not make proteins but instead produce RNAs that are essential for cell function. If DNA regions containing information for making proteins are called coding genes, then regions containing information for making functional RNAs such as mRNA, tRNA, rRNA, snRNA, miRNA, and lncRNA can be thought of as non-coding RNA (ncRNA) genes. The roles of these functional RNAs become easier to understand when we look at how proteins are actually made.
The Role of Functional RNA
To make a protein, RNA acts as an intermediary for genetic information because DNA itself remains physically inside the nucleus.
As we saw earlier, RNA polymerase, with help from transcription factors, finds the region containing the relevant protein-coding gene. It then locally opens the DNA double helix and uses one DNA strand as a template to make an RNA molecule. The new RNA is not simply an identical copy of the DNA strand. Instead, RNA polymerase builds an RNA strand using the complementary bases of the DNA template. That's why people often describe this process as “making a copy from a template.”
Among the RNAs produced through transcription, the ones used to carry protein-making instructions are called mRNA, or messenger RNA. After being processed and prepared for export, mRNA leaves the nucleus and travels to a nearby protein-making machine called the ribosome. There are countless ribosomes floating freely in the cytoplasm. The endoplasmic reticulum, or ER, can also have large numbers of ribosomes attached to its surface. Because of this rough-looking appearance, it is called the rough ER. By contrast, the ER without ribosomes attached is called literally the smooth ER.
Ribosomes read the mRNA and translate its codons into a sequence of amino acids, linking those amino acids together to make a protein. And tRNA acts like a delivery service, bringing the appropriate amino acids to the ribosome. As the amino acids are connected one after another, the protein is gradually assembled.
Then, who makes sure that the ribosome correctly reads the codons on mRNA and connects the amino acids delivered by tRNA in the proper order? One of the central players is rRNA. rRNA is a core component of the ribosome. It helps position mRNA and tRNA correctly and even plays a catalytic role in the actual formation of peptide bonds.
By the way, there is actually one more step that has to happen before all of this. The fresh, original RNA that has just been transcribed in the nucleus is called pre-mRNA. And before it can be used by the ribosome, it has to be processed and trimmed into mature mRNA. The introns inserted between the exons are removed, and the remaining exons are joined together. Depending on how the exons are combined, a single gene can give rise to several different proteins. This process is called alternative splicing. One of the key components of the spliceosome, the molecular machine that removes introns and joins exons, is snRNA.
So, as you can see, many different types of RNA—including mRNA, tRNA, rRNA, and snRNA—play important roles in protein synthesis and gene expression. And what happens if a cell produces too much mRNA and starts making more protein than it actually needs? Small RNAs such as miRNA can bind to mRNA like a little muzzle, reducing protein production or promoting the breakdown of the mRNA. There are also many different types of long non-coding RNA (lncRNA). And even today, new types of RNA and new functions for RNA continue to be discovered.
One point I really want to emphasize here is that when genetic information stored in DNA is actually read and used by a cell, RNA often acts as the intermediary. RNA can exist as a single strand and fold itself into many different shapes and structures. Life appears to have taken advantage of this flexibility by using RNA for information transfer, catalysis, and regulation, while leaving long-term genetic information storage mainly to DNA. RNA is a bit like a salted loach that simply cannot sit still: as a single strand, it naturally folds and changes into many different shapes, which makes it less ideal for safely storing information over very long periods. DNA, on the other hand, has a double-helical structure that is well suited to long-term information storage. And when DNA is damaged, the two strands are complementary to each other, so one strand can serve as a reference for accurately repairing the other. The same complementary relationship also makes accurate replication possible, because each original strand can serve as a template for the synthesis of a new strand during cell division. Given these advantages in stability and accuracy, it seems quite natural that humans and many other organisms evolved to use double-stranded DNA as their long-term information storage system.
Non-Coding RNA Genes and RNA Polymerases I, II, and III
As a side note, in eukaryotic cells, the RNA polymerases responsible for making RNA are broadly divided into three major types. Each type has a somewhat different job because different kinds of RNA are needed in different amounts, at different times, and under different forms of regulation.
RNA polymerase I mainly produces the major rRNAs, which are core components of ribosomes. Because cells need enormous numbers of ribosomes, polymerase I is specialized for producing large amounts of rRNA efficiently and rapidly. RNA polymerase II mainly produces mRNA, which requires relatively precise regulation. It also produces many different types of non-coding RNA. RNA polymerase III is best known for producing tRNA, and it also produces 5S rRNA.
Regulatory DNA
So far, we've looked at coding genes and non-coding RNA genes. But simply having a gene does not mean that the cell will automatically use it. Regulatory DNA, which determines when, where, and to what extent a gene is expressed, is also a crucial part of how the genome works. Just as a string of beads does not become a necklace until the beads are actually threaded together, having a gene is not enough. If the gene cannot be expressed at the right time and in the right place, it is of little use to the cell. Even in cell signaling pathways, the final question often comes down to whether a particular gene should be suppressed or upregulated. So let's take a look at some of the regulatory sequences involved in gene expression.
The promoter region is located near the transcription start site (TSS) of a gene and acts as its starting line for transcription. RNA polymerase and various transcription factors bind there to initiate transcription, so the promoter can be thought of as a kind of platform for starting transcription. An enhancer region can be located far away from the gene itself and still greatly increase its transcription. It is like a remote switch for gene expression. A silencer, on the other hand, helps suppress transcription, while an insulator can help prevent inappropriate interactions between different regulatory regions.
These regulatory elements can exist within genes as well as in the regions between genes. And they do not work simply because they happen to be close together along the DNA sequence. DNA is folded into a complex three-dimensional structure inside the nucleus. As a result, regulatory elements that look far apart when the DNA is stretched out can actually come into very close contact in three-dimensional space and work together to regulate a gene.
Structural DNA
There are also regions of DNA that are important for maintaining chromosome stability and ensuring that chromosomes are accurately separated during cell division. The centromere acts as a crucial structural region that helps chromosomes separate properly during cell division. A protein complex called the kinetochore forms at the centromere and connects the chromosome to spindle microtubules, helping move the chromosome to the correct location. Telomeres are structures that protect the ends of chromosomes. During DNA replication, chromosome ends are difficult to copy completely for several reasons, so telomeres act as an important buffer against the loss of DNA at chromosome ends. In certain cells, an enzyme called telomerase helps maintain telomere length and prevents excessive loss of DNA at chromosome ends. Telomeres also help prevent chromosome ends from being mistakenly joined together or becoming tangled with one another.
These regions are not directly involved in making proteins or functional RNAs, but they have a completely different—and essential—job: they help keep the genome stable so that cells can survive and function normally.
Repetitive DNA
Interestingly, more than half of the human genome consists of repetitive DNA sequences. A large portion of these repetitive sequences is related to transposable elements, which are often called “jumping genes.” As the name suggests, transposable elements can move to new locations in the genome, or in many cases make copies of themselves and insert those copies into other parts of the genome. These processes have been repeated over enormous periods of evolutionary time, contributing to the huge number of repetitive sequences found throughout the human genome.
But this movement is not simply a matter of structural change. Transposable elements can influence gene expression and chromatin states, and over much longer periods, they can also contribute to the evolution of the genome. How repetitive sequences can transform the standard DNA double helix into a variety of unusual DNA structures, and how those structural changes can in turn regulate gene expression, will be explored in detail in a later article.
As we've seen, the regions between genes—the intergenic regions—should no longer be thought of simply as empty spaces filling the gaps between genes. Many intergenic regions contain enhancers, various non-coding RNA genes, repetitive sequences, and elements involved in shaping the three-dimensional organization of the genome.
The fact that a DNA region does not directly make a protein does not mean that it has no function. In fact, many of these regions can be thought of as an important stage where the cell precisely controls which genes to use, when to use them, and how much to use.
Seeing the genome simply as “genes plus everything else” is a very narrow way of looking at it. It makes much more sense to think of the genome as one enormous integrated system in which many different functional elements are connected and work together. When we consider that protein-coding regions make up only about 1–1.5% of the human genome, leaving more than 98% outside those protein-coding regions, it is not surprising that there is enormous interest in this vast territory. It is easy to imagine that research into what functions may be hidden within these regions will remain an exciting area of science for many years to come.
We also know that humans are remarkably similar at the DNA sequence level while still carrying millions of genetic differences. That fact never stops being amazing. How can such a small amount of genetic variation, together with differences in gene regulation and environmental influences, contribute to such enormous diversity in our appearance, physiology, and susceptibility to disease? Once again, we come back to the same fundamental question: How can such similar DNA give rise to cells—and people—with such different forms and functions? And one of the most important keys to answering that question is gene expression and its regulation.
[Reference]
[1] Text Box 1 Human genetic variation and disease from “Whole genome sequencing in clinical practice”
https://link.springer.com/article/10.1186/s12920-024-01795-w/tables/1
