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Chapter 2: RNA and DNA Revealed: New Roles, New Rules
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For many years, when scientists thought about heredity, DNA was the first thing to come to mind. It's true that DNA is the basic ingredient of our genes and, as such, it often steals the limelight from RNA, the other form of genetic material inside our cells.
But, while they are both types of genetic material, RNA and DNA are rather different. The chemical units of RNA are like those of DNA, except that RNA has the nucleotide uracil (U) instead of thymine (T). Unlike double-stranded DNA, RNA usually comes as only a single strand. And the nucleotides in RNA contain ribose sugar molecules in place of deoxyribose.
RNA is quite flexible—unlike DNA, which is a rigid, spiral-staircase molecule that is very stable. RNA can twist itself into a variety of complicated, three-dimensional shapes. RNA is also unstable in that cells constantly break it down and must continually make it fresh, while DNA is not broken down often. RNA's instability lets cells change their patterns of protein synthesis very quickly in response to what's going on around them.
Many textbooks still portray RNA as a passive molecule, simply a "middle step" in the cell's gene-reading activities. But that view is no longer accurate. Each year, researchers unlock new secrets about RNA. These discoveries reveal that it is truly a remarkable molecule and a multi-talented actor in heredity.
Today, many scientists believe that RNA evolved on the Earth long before DNA did. Researchers hypothesize—obviously, no one was around to write this down—that RNA was a major participant in the chemical reactions that ultimately spawned the first signs of life on the planet.
RNA World
At least two basic requirements exist for making a cell: the ability to hook molecules together and break them apart, and the ability to replicate, or copy itself, from existing information.
RONALD BREAKER
RNA probably helped to form the first cell. The first organic molecules, meaning molecules containing carbon, most likely arose out of random collisions of gases in the Earth's primitive atmosphere, energy from the Sun, and heat from naturally occurring radioactivity. Some scientists think that in this primitive world, RNA was a critical molecule because of its ability to lead a double life: to store information and to conduct chemical reactions. In other words, in this world, RNA served the functions of both DNA and proteins.
What does any of this have to do with human health? Plenty, it turns out.
Today's researchers are harnessing some of RNA's flexibility and power. For example, through a strategy he calls directed evolution, molecular engineer Ronald R. Breaker of Yale University is developing ways to create entirely new forms of RNA and DNA that both work as enzymes.
Recently, Breaker and others have also uncovered a hidden world of RNAs that play a major role in controlling gene activity, a job once thought to be performed exclusively by proteins. These RNAs, which the scientists named riboswitches, are found in a wide variety of bacteria and other organisms.
This discovery has led Breaker to speculate that new kinds of antibiotic medicines could be developed to target bacterial riboswitches.
Molecular Editor
Scientists are learning of another way to customize proteins: by RNA editing. Although DNA sequences spell out instructions for producing RNA and proteins, these instructions aren't always followed precisely. Editing a gene's mRNA, even by a single chemical letter, can radically change the resulting protein's function. Nature likely evolved the RNA editing function as a way to get more proteins out of the same number of genes. For example, researchers have found that the mRNAs for certain proteins important for the proper functioning of the nervous system are particularly prone to editing. It may be that RNA editing gives certain brain cells the capacity to react quickly to a changing environment.
Which molecules serve as the editor and how does this happen? Brenda Bass of the University of Utah School of Medicine in Salt Lake City studies one particular class of editors called adenosine deaminases. These enzymes "retype" RNA letters at various places within an mRNA transcript.
They do their job by searching for characteristic RNA shapes. Telltale twists and bends in folded RNA molecules signal these enzymes to change the RNA sequence, which in turn changes the protein that gets made.
Bass' experiments show that RNA editing occurs in a variety of organisms, including people. Another interesting aspect of editing is that certain disease-causing microorganisms, such as some forms of parasites, use RNA editing to gain a survival edge when living in a human host. Understanding the details of this process is an important area of medical research.
Small But Powerful
Recently, molecules called microRNAs have been found in organisms as diverse as plants, worms, and people. The molecules are truly "micro," consisting of only a few dozen nucleotides, compared to typical human mRNAs that are a few thousand nucleotides long.
What's particularly interesting about microRNAs is that many of them arise from DNA that used to be considered merely filler material (see Getting the Message).
How do these small but important RNA molecules do their work? They start out much bigger but get trimmed by cellular enzymes, including one aptly named Dicer. Like tiny pieces of Velcro®, microRNAs stick to certain mRNA molecules and stop them from passing on their protein-making instructions.
First discovered in a roundworm model system (see Living Laboratories), some microRNAs help determine the organism's body plan. In their absence, very bad things can happen. For example, worms engineered to lack a microRNA called let-7 develop so abnormally that they often rupture and practically break in half as the worm grows.
AMY PASQUINELLI
Perhaps it is not surprising that since microRNAs help specify the timing of an organism's developmental plan, the appearance of the microRNAs themselves is carefully timed inside a developing organism. Biologists, including Amy Pasquinelli of the University of California, San Diego, are currently figuring out how microRNAs are made and cut to size, as well as how they are produced at the proper time during development.
MicroRNA molecules also have been linked to cancer. For example, Gregory Hannon of the Cold Spring Harbor Laboratory on Long Island, New York, found that certain microRNAs are associated with the severity of the blood cancer B-cell lymphoma in mice.
Since the discovery of microRNAs in the first years of the 21st century, scientists have identified hundreds of them that likely exist as part of a large family with similar nucleotide sequences. New roles for these molecules are still being found.
RNA Interference (RNAi)
Healthy Interference
RNA controls genes in a way that was only discovered recently: a process called RNA interference, or RNAi. Although scientists identified RNAi less than 10 years ago, they now know that organisms have been using this trick for millions of years.
Researchers believe that RNAi arose as a way to reduce the production of a gene's encoded protein for purposes of fine-tuning growth or self-defense. When viruses infect cells, for example, they command their host to produce specialized RNAs that allow the virus to survive and make copies of itself. Researchers believe that RNAi eliminates unwanted viral RNA, and some speculate that it may even play a role in human immunity.
Oddly enough, scientists discovered RNAi from a failed experiment! Researchers investigating genes involved in plant growth noticed something strange: When they tried to turn petunia flowers purple by adding an extra "purple" gene, the flowers bloomed white instead.
This result fascinated researchers, who could not understand how adding genetic material could somehow get rid of an inherited trait. The mystery remained unsolved until, a few years later, two geneticists studying development saw a similar thing happening in lab animals.
The researchers, Andrew Z. Fire, then of the Carnegie Institution of Washington in Baltimore and now at Stanford University, and Craig Mello of the University of Massachusetts Medical School in Worcester, were trying to block the expression of genes that affect cell growth and tissue formation in roundworms, using a molecular tool called antisense RNA.
To their surprise, Mello and Fire found that their antisense RNA tool wasn't doing much at all. Rather, they determined, a doublestranded contaminant produced during the synthesis of the single-stranded antisense RNA interfered with gene expression. Mello and Fire named the process RNAi, and in 2006 were awarded the Nobel Prize in physiology or medicine for their discovery.
Further experiments revealed that the doublestranded RNA gets chopped up inside the cell into much smaller pieces that stick to mRNA and block its action, much like the microRNA pieces of Velcro discussed above (see drawing).
Today, scientists are taking a cue from nature and using RNAi to explore biology. They have learned, for example, that the process is not limited to worms and plants, but operates in humans too.
Medical researchers are currently testing new types of RNAi-based drugs for treating conditions such as macular degeneration, the leading cause of blindness, and various infections, including those caused by HIV and herpes virus.
Dynamic DNA
A good part of who we are is "written in our genes," inherited from Mom and Dad. Many traits, like red or brown hair, body shape, and even some personality quirks, are passed on from parent to offspring.
But genes are not the whole story. Where we live, how much we exercise, what we eat: These and many other environmental factors can all affect how our genes get expressed.
You know that changes in DNA and RNA can produce changes in proteins. But additional control happens at the level of DNA, even though these changes do not alter DNA directly. Inherited factors that do not change the DNA sequence of nucleotides are called epigenetic changes, and they too help make each of us unique.
Epigenetic means, literally, "upon" or "over" genetics. It describes a type of chemical reaction that can alter the physical properties of DNA without changing its sequence. These changes make genes either more or less likely to be expressed (see drawing).
Currently, scientists are following an intriguing course of discovery to identify epigenetic factors that, along with diet and other environmental influences, affect who we are and what type of illnesses we might get.
Secret Code
DNA is spooled up compactly inside cells in an arrangement called chromatin. This packaging is critical for DNA to do its work. Chromatin consists of long strings of DNA spooled around a compact assembly of proteins called histones.
One of the key functions of chromatin is to control access to genes, since not all genes are turned on at the same time. Improper expression of growth-promoting genes, for example, can lead to cancer, birth defects, or other health concerns.
Many years after the structure of DNA was determined, researchers used a powerful device known as an electron microscope to take pictures of chromatin fibers. Upon viewing chromatin up close, the researchers described it as "beads on a string," an image still used today. The beads were the histone balls, and the string was DNA wrapped around the histones and connecting one bead to the next.
Decades of study eventually revealed that histones have special chemical tags that act like switches to control access to the DNA. Flipping these switches, called epigenetic markings, unwinds the spooled DNA so the genes can be transcribed.
The observation that a cell's gene-reading machinery tracks epigenetic markings led C. David Allis, who was then at the University of Virginia Health Sciences Center in Charlottesville and now works at the Rockefeller University in New York City, to coin a new phrase, the "histone code." He and others believe that the histone code plays a major role in determining which proteins get made in a cell.
Flaws in the histone code have been associated with several types of cancer, and researchers are actively pursuing the development of medicines to correct such errors.
Genetics and You: The Genetics of Anticipation
Occasionally, unusual factors influence whether or not a child will be born with a genetic disease.
An example is the molecular error that causes Fragile X syndrome, a rare condition associated with mental retardation. The mutation leading to a fragile X chromosome is not a typical DNA typing mistake, in which nucleotides are switched around or dropped, or one of them is switched for another nucleotide. Instead, it is a kind of stutter by the DNA polymerase enzyme that copies DNA. This stutter creates a string of repeats of a DNA sequence that is composed of just three nucleotides, CGG.
Some people have only one repeat of the CGG nucleotide triplet. Thus, they have two copies of the repeat in a gene, and the extra sequence reads CGGCGG. Others have more than a thousand copies of the repeat. These people are the most severely affected.
The number of triplet repeats seems to increase as the chromosome is passed down through several generations. Thus, the grandsons of a man with a fragile X chromosome, who is not himself affected, have a 40 percent risk of retardation if they inherit the repeat-containing chromosome. The risk for great-grandsons is even higher: 50 percent.
Intrigued by the evidence that triplet repeats can cause genetic disease, scientists have searched for other examples of disorders associated with the DNA expansions. To date, more than a dozen such disorders have been found, and all of them affect the nervous system.
Analysis of the rare families in which such diseases are common has revealed that expansion of the triplet repeats is linked to something called genetic anticipation, when a disease's symptoms appear earlier and more severely in each successive generation.
Battle of the Sexes
A process called imprinting, which occurs naturally in our cells, provides another example of how epigenetics affects gene activity.
With most genes, the two copies work exactly the same way. For some mammalian genes, however, only the mother's or the father's copy is switched on regardless of the child's gender. This is because the genes are chemically marked, or imprinted, during the process that generates eggs and sperm.
As a result, the embryo that emerges from the joining of egg and sperm can tell whether a gene copy came from Mom or Dad, so it knows which copy of the gene to shut off.
One example of an imprinted gene is insulin-like growth factor 2 (Igf2), a gene that helps a mammalian fetus grow. In this case, only the father's copy of Igf2 is expressed, and the mother's copy remains silent (is not expressed) throughout the life of the offspring.
Scientists have discovered that this selective silencing of Igf2 and many other imprinted genes occurs in all placental mammals (all except the platypus, echidna, and marsupials) examined so far, but not in birds.
Why would nature tolerate a process that puts an organism at risk because only one of two copies of a gene is working? The likely reason, many researchers believe, is that mothers and fathers have competing interests, and the battle- field is DNA!
The scenario goes like this: It is in a father's interest for his embryos to get bigger faster, because that will improve his offspring's chances of survival after birth. The better an individual's chance of surviving infancy, the better its chance of becoming an adult, mating, and passing its genes on to the next generation.
Of course mothers want strong babies, but unlike fathers, mothers provide physical resources to embryos during pregnancy. Over her lifetime, a female is likely to be pregnant several times, so she needs to divide her resources among a number of embryos in different pregnancies.
Researchers have discovered over 200 imprinted genes in mammals since the first one was identified in 1991. We now know that imprinting controls some of the genes that have an important role in regulating embryonic and fetal growth and allocating maternal resources. Not surprisingly, mutations in these genes cause serious growth disorders.
Marisa Bartolomei of the University of Pennsylvania School of Medicine in Philadelphia is trying to figure out how Igf2 and other genes become imprinted and stay silent throughout the life of an individual. She has already identified sequences within genes that are essential for imprinting. Bartolomei and other researchers have shown that these sequences, called insulators, serve as "landing sites" for a protein that keeps the imprinted gene from being transcribed.
Starting at the End
When we think of DNA, we think of genes. However, some DNA sequences are different: They don't encode RNAs or proteins. Introns, described in Chapter 1, are in this category.
HESED PADILLA-NASH AND THOMAS RIED
Another example is telomeres—the ends of chromosomes. There are no genes in telomeres, but they serve an essential function. Like shoelaces without their tips, chromosomes without telomeres unravel and fray. And without telomeres, chromosomes stick to each other and cause cells to undergo harmful changes like dividing abnormally.
Researchers know a good deal about telomeres, dating back to experiments performed in the 1970s by Elizabeth Blackburn, a basic researcher who was curious about some of the fundamental events that take place within cells.
At the time, Blackburn, now at the University of California, San Francisco, was working with Joseph Gall at Yale University. For her experimental system, she chose a single-celled, pond-dwelling organism named Tetrahymena. These tiny, pear-shaped creatures are covered with hairlike cilia that they use to propel themselves through the water as they devour bacteria and fungi.
Tetrahymena was a good organism for Blackburn's experiments because it has a large number of chromosomes—which means it has a lot of telomeres!
CAROL GREIDER
Her research was also perfectly timed, because methods for sequencing DNA were just being developed. Blackburn found that Tetrahymena's telomeres had an unusual nucleotide sequence: TTGGGG, repeated about 50 times per telomere.
Since then, scientists have discovered that the telomeres of almost all organisms have repeated sequences of DNA with lots of Ts and Gs. In human and mouse telomeres, for example, the repeated sequence is TTAGGG.
The number of telomere repeats varies enormously, not just from organism to organism but in different cells of the same organism and even within a single cell over time. Blackburn reasoned that the repeat number might vary if cells had an enzyme that added copies of the repeated sequence to the telomeres of some but not all chromosomes.
With her then-graduate student Carol Greider, now at Johns Hopkins University, Blackburn hunted for the enzyme. The team found it and Greider named it telomerase.
The telomerase enzyme, it turns out, consisted of a protein and an RNA component, which the enzyme uses as a template for copying the repeated DNA sequence.
What is the natural function of telomerase? As cells divide again and again, their telomeres get shorter.Most normal cells stop dividing when their telomeres wear down to a certain point, and eventually the cells die. Telomerase can counteract the shortening. By adding DNA to telomeres, telomerase rebuilds the telomere and resets the cell's molecular clock.
The discovery of telomerase triggered new ideas and literally thousands of new studies. Many researchers thought that the enzyme might play important roles in cancer and aging. Researchers were hoping to find ways to turn telomerase on so that cells would continue to divide (to grow extra cells for burn patients, for example), or off so that cells would stop dividing (to stop cancer, for instance).
So far, they have been unsuccessful. Although it is clear that telomerase and cellular aging are related, researchers do not know whether telomerase plays a role in the normal cellular aging process or in diseases like cancer.
Recently, however, Blackburn and a team of other scientists discovered that chronic stress and the perception that life is stressful affect telomere length and telomerase activity in the cells of healthy women. Blackburn and her coworkers are currently conducting a long-term, follow-up study to confirm these intriguing results.
The Other Human Genome
Before you think everything's been said about DNA, there's one little thing we didn't mention: Some of the DNA in every cell is quite different from the DNA that we've been talking about up to this point. This special DNA isn't in chromosomes— it isn't even inside the cell's nucleus where all the chromosomes are!
So where is this special DNA? It's inside mitochondria, the organelles in our cells that produce the energy-rich molecule adenosine triphosphate, or ATP. Mendel knew nothing of mitochondria, since they weren't discovered until late in the 19th century. And it wasn't until the 1960s that researchers discovered the mitochondrial genome, which is circular like the genomes of bacteria.
In human cells, mitochondrial DNA makes up less than 1 percent of the total DNA in each of our cells. The mitochondrial genome is very small—containing only about three dozen genes. These encode a few of the proteins that are in the mitochondrion, plus a set of ribosomal RNAs used for synthesizing proteins for the organelle.
Mitochondria need many more proteins though, and most of these are encoded by genes in the nucleus. Thus, the energy-producing capabilities of human mitochondria—a vital part of any cell's everyday health—depend on coordinated teamwork among hundreds of genes in two cellular neighborhoods: the nucleus and the mitochondrion.
Mitochondrial DNA gets transcribed and the RNA is translated by enzymes that are very different from those that perform this job for genes in our chromosomes. Mitochondrial enzymes look and act much more like those from bacteria, which is not surprising because mitochondria are thought to have descended from free-living bacteria that were engulfed by another cell over a billion years ago.
Scientists have linked mitochondrial DNA defects with a wide range of age-related diseases including neurodegenerative disorders, some forms of heart disease, diabetes, and various cancers. It is still unclear, though, whether damaged mitochondria are a symptom or a cause of these health conditions.
Scientists have studied mitochondrial DNA for another reason: to understand the history of the human race. Unlike our chromosomal DNA, which we inherit from both parents, we get all of our mitochondrial DNA from our mothers.
Thus, it is possible to deduce who our maternal ancestors were by tracking the inheritance of mutations in mitochondrial DNA. For reasons that are still not well understood, mutations accumulate in mitochondrial DNA very slowly compared to chromosomal DNA. So, it's possible to trace your maternal ancestry way back beyond any relatives you may know by name—all the way back to "African Eve," the ancestor of us all!
The Tools of Genetics: Recombinant DNA and Cloning
In the early 1970s, scientists discovered that they could change an organism's genetic traits by putting genetic material from another organism into its cells. This discovery, which caused quite a stir, paved the way for many extraordinary accomplishments in medical research that have occurred over the past 35 years.
ROSLIN INSTITUTE, EDINBURGH
How do scientists move genes from one organism to another? The cutting and pasting gets done with chemical scissors: enzymes, to be specific. Take insulin, for example. Let's say a scientist wants to make large quantities of this protein to treat diabetes. She decides to transfer the human gene for insulin into a bacterium, Escherichia coli, or E. coli, which is commonly used for genetic research (see Living Laboratories). That's because E. coli reproduces really fast, so after one bacterium gets the human insulin gene, it doesn't take much time to grow millions of bacteria that contain the gene.
The first step is to cut the insulin gene out of a copied, or "cloned," version of the human DNA using a special bacterial enzyme from bacteria called a restriction endonuclease. (The normal role of these enzymes in bacteria is to chew up the DNA of viruses and other invaders.) Each restriction enzyme recognizes and cuts at a different nucleotide sequence, so it's possible to be very precise about DNA cutting by selecting one of several hundred of these enzymes that cuts at the desired sequence. Most restriction endonucleases make slightly staggered incisions, resulting in "sticky ends," out of which one strand protrudes.
The next step in this example is to splice, or paste, the human insulin gene into a circle of bacterial DNA called a plasmid. Attaching the cut ends together is done with a different enzyme (obtained from a virus), called DNA ligase. The sticky ends join back together kind of like jigsaw puzzle pieces. The result: a cut-and-pasted mixture of human and bacterial DNA.
The last step is putting the new, recombinant DNA back into E. coli and letting the bacteria reproduce in a petri dish. Now, the scientist has a great tool: a version of E. coli that produces lots of human insulin that can be used for treating people with diabetes.
So, what is cloning? Strictly speaking, it's making many copies of a gene—in the example above, E. coli is doing the cloning. However, the term cloning is more generally used to refer to the entire process of isolating and manipulating a gene. Dolly the cloned sheep contained the identical genetic material of another sheep. Thus, researchers refer to Dolly as a clone.
Got It?
Besides the sequence of nucleotides in genes, what are some other changes to DNA and RNA that can affect our health and who we are?
Can you imagine treatments— other than vaccines and current medicines—crafted from genetic information and new molecular tools?
How is cloning a gene different from cloning an animal or a person? How do researchers use gene cloning to study health and disease?
Do you have any recurring illnesses in your extended family?