Molecular Genetics

Molecular genetics* is the field within genetics that deals with the molecular bases of inheritance. At the center of the field of study is the structure and function of the nucleic acids DNA and RNA.

Nucleotides in DNA

The study of modern genetics depends on an understanding of the physical and chemical characteristics of DNA. Some of the most fundamental properties of DNA emerge from the characteristics of its four basic building blocks, called nucleotides. Knowing the composition of nucleotides and the differences between the four nucleotides that make up DNA is central to understanding DNA’s role in living systems.



DNA is a nucleotide polymer, or polynucleotide. Each nucleotide contains three components:

  1. A five carbon sugar
  2. A phosphate molecule
  3. A nitrogen-containing base.

The sugar carbon atoms are numbered 1 to 5, with 1 being the point of attachment of the nitrogenous base, and 5 the point of attachment of the phosphate group. DNA polymers are built from individual nucleotides by linking the phosphate of one nucleotide to the #3 carbon of the neighboring nucleotide. The repeating pattern of phosphate, sugar, then phosphate again is commonly referred to as the backbone of the molecule.

The sugar in DNA is deoxyribose. Deoxyribose differs from ribose (found in RNA) in that the #2 carbon lacks a hydroxyl group (hence the prefix “Deoxy”). This missing hydroxyl group plays a role in the three-dimensional structure and chemical stability of DNA polymers.

Nucleotides in DNA contain four different nitrogenous bases: Thymine, Cytosine, Adenine, or Guanine. There are two groups of bases:
Pyrimidines: Cytosine and Thymine each have a single six-member ring.
Purines: Guanine and Adenine each have a double ring made up of a five-atom ring attached by one side to a six-atom ring.

The order of nucleotides along DNA polymers encode the genetic information carried by DNA. DNA polymers can be tens of millions of nucleotides long. At these lengths, the four letter nucleotide alphabet can encode nearly unlimited information.

Nucleosides are similar to nucleotides except they do not contain a phosphate group. Without this phosphate group, they are unable to form chains.

The illustration above introduces nucleotide and the terminology used to describe them.

Test your knowledge of Nucleotides

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Nucleotides in RNA

Ribonucleic acids, also called RNA, perform multiple important roles in living cells. RNA is needed for protein synthesis and DNA* replication. RNA containing molecules also contribute to the regulation of gene expression and function as enzymes.



Like DNA, RNA polymers are make up of chains of nucleotides*. These nucleotides have three parts: 1) a five carbon ribose sugar, 2) a phosphate molecule and 3) one of four nitrogenous bases: adenine, guanine, cytosine or uracil.

RNA nucleotides form polymers of alternating ribose and phosphate units linked by a phosphodiester bridge between the #3 and #5 carbons of neighboring ribose molecules.

RNA nucleotides differ from DNA nucleotides by the presence of a hydroxyl group linked to the #2 carbon of the sugar. The presence of this hydroxyl group allows RNA polymers to assume a more diverse number of shapes compared to DNA polymers. The extra hydroxyl group also makes RNA polymers less stable than DNA polymers.

The greater variety of shapes RNA polymers are able to form, is part of the reason RNA serves more functions than DNA.

Test your understanding of the concepts covered by answering the Nucleotides in RNA practice problems

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Complementary Nucleotide Bases

DNA* is the information molecule of the cell. DNA’s capacity to store and transmit heritable information depends on interactions between nucleotide bases and on the fact that some combinations of bases form stable links, while other combinations do not. Base pairs that form stable connections are called complementary bases.

Consistent pairings of complementary bases allow cells to make double-stranded DNA from a single strand template, create messenger RNA from DNA and synthesize proteins from individual amino acids by matching nucleotides bases on messenger RNA with their complementary bases on transfer RNA.




The polynucleotides chains that make up DNA and RNA form via covalent bond*s between sugar and phosphate subunits of neighboring nucleotides along a chain. In addition to the strong covalent bonds that hold polynucleotide chains together, bases along a polynucleotide chain can form hydrogen bonds with bases on other chains (or with bases elsewhere on the same chain, as with secondary structure in RNA).

The formation of stable hydrogen bonds depends on the distance between two strands, the size of the bases and geometry of each base. Stable pairings occur between guanine and cytosine and between adenine and thymine (or adenine and uracil in RNA). Three hydrogen bonds form between guanine and cytosine. Two hydrogen bonds form between adenine and thymine or adenine and uracil.

Complementary pairs always involve one purine and one pyrimidine base*. Pyrimidine-pyrimidine pairings do not occur because these relatively small molecules do not get close enough to form hydrogen bonds. Purine-purine links do not form because these bases are too large to fit in the space between the polynucleotide strands. Asymmetry in the structure of non-complimentary purine - pyrimidine pairs cause some crowding and prevent stable bonds from forming.

Take the concept quiz to test your understanding of complementary nucleotide bases.

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DNA Polymerase

DNA polymerases are the enzymes that replicate DNA in living cells. They do this by adding individual nucleotides to the 3-prime hydroxl group of a strand of DNA. The process uses a complementary, single strand of DNA as a template.

The energy required to drive the reaction comes from cutting high energy phosphate bonds on the nucleotide-triphosphate's used as the source of the nucleotides needed in the reaction.



The illustration above highlights important aspects of the reaction.

DNA polymerases can not create new strands of DNA. They only synthesis double stranded DNA from single stranded DNA. The starting point is a a stretch of single stranded DNA which is double stranded for at least part of its length. In the polymerase chain reaction the double stranded stretch is created by attaching short DNA primers. In living cells, RNA primers are used.

DNA polymerase uses the bases of the longer strand as a template. During strand elongation, two phosphates are cleaved from the incoming nucleotide triphosphate and the resulting nucleotide monophosphate is added to the DNA strand. This results in the:

  • Formation of a phosphodiester bond between the phosphate attached to the 5' carbon of the incoming nucleotide and the hydroxyl group on the trailing 3' carbon
  • Release of a pyrophosphate molecule
  • Extension of the DNA polymer by one nucleotide

Removing two phosphates from the incoming nucleotide and bonding the remaining phosphate to the oxygen on the 3' carbon of the existing strand maintains the repeating sugar-phosphate-sugar-phosphate pattern that makes up the backbone of each DNA polymer.

Orientation of the strand is important. Dependence on energy from the phosphates linked to the 5-prime carbon of the incoming nucleotides means that DNA polymerase can only extend DNA strands by adding nucleotides to the 3-prime end of a DNA strand.

Test your understanding of the concepts covered by this illustration with the DNA Polymerase concept questions.

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Semiconservative Replication

During DNA* replication, a double stranded DNA molecule separate, and each strand is used as a template for the synthesis of a new strand. This results in the formation of two identical copies of the original double stranded molecule. This is called semiconservative replication. This term captures the idea that each round of DNA replication produces hybrid molecules each of which contains one old strand and one newly synthesized strand.

The following illustration shows this process over two rounds of replication:



During each round of replication, the amount of DNA is doubled. The original strands remain intact and end up in different daughter strands.

The pattern of Semiconservative DNA replication was proposed in a 1953 paper by Watson and Crick. They did not call it semiconservative, but their description captures the idea that each of the two original strands are used as templates to make new double strands:

"…our model for deoxyribonucleic acid is, in effect, a pair of templates, each of which is complementary to the other. We imagine that prior to duplication the hydrogen bonds are broken, and the two chains unwind and separate. Each chain then acts as a template for the formation onto itself of a new companion chain, so that eventually we shall have two pairs of chains, where we only had one before. Moreover, the sequence of the pairs of bases will have been duplicated exactly."

Source Watson and Crick, 1953 (pdf)

Watson and Crick's paper proposed a mechanism, but provided no experimental evidence. The evidence for semiconservative replication was provided several years later by a 1958 paper by Matthew Meselson and Franklin W. Stahl (pdf).

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Replication Fork

During DNA* replication a DNA double helix must unwind and separate so that DNA polymerase enzymes can use each single strand as a template for the synthesis of a new double strand. Strand separation is catalyzed by a Helicase* enzyme. A number of helper proteins prevent the strands from coming back together before replication is complete. Partial separation of the double helix forms a replication fork*.



The primary enzymes responsible for DNA replication are DNA polymerases1. Understanding the activity and limitations of DNA polymerases help in making sense of why DNA replication occurs the way it does. Key points are:

  • Prokaryotic cells contain three different DNA polymerases. Each have slightly different activities. The two that are known to be required for DNA replication are DNA Polymerase III and DNA Polymerase I.
  • DNA polymerases need a template. They can not synthesize a strand of DNA without it. As a result, DNA replication is semiconservative and depends on the presence of the two single strands of DNA formed at the replication fork.
  • DNA polymerases can not create new polymers, they can only extend existing strands by adding new nucleotides* to one end. For replication to begin, another enzyme, Primase* ( a type of RNA polymerase), must create short priming sequences before DNA polymerases can begin their work. Unlike DNA polymerases, RNA polymerases can create new polymers by added based to a complimentary strand in the absence of an existing polymer. These short RNA polymer are called primers*. Once there is a short RNA primer, DNA polymerases can continue the elongation process.
  • The RNA primers need to be removed prior to the end of the replication process. DNA polymerase I, which has exonuclease activity, performs this task.
  • DNA polymerases can only add nucleotides to the 3'-hydroxyl end of a nucleotide polymer. Since the two strands of the original DNA helix are oriented in opposite directions. Double strand formation has to proceed in opposite directions on each of the two template strands at the replication fork.

In one direction DNA is replicated as one continuous strand. This is called the leading strand. Replication on the other strand occurs by the creation of many short segments. This is the lagging strand.

On both the leading and lagging strands, DNA replication starts with primase adding a short (3 to 10 base) RNA primer to the template strand. Once the primer is added, DNA polymerase III elongates the strand by added DNA nucleotides to the 3’-hydroxy end of the growing polymer.

These two steps are adequate to form long stretches of DNA on the leading strand.

On the lagging strand, the new strand's 3'-hydroxyl end points away from the replication fork. This forces the elongation process to occur in a discontinuous manner. As replication moves along the template strand, a series of shorter DNA polymers are formed. Each stretch is initiated with its own RNA primer. The shorter lengths of double stranded DNA formed along the lagging strand are called Okazaki fragments. When an Okazaki fragment extends to the point that it overlaps with the previous RNA primer, RNA nucleotides are removed and replaced by DNA. This requires DNA Polymerase I's exonuclease activity.

Once the RNA primer is completely by DNA the two DNA fragments are joined by a ligase enzyme.

1The process is quite complex and involves numerous enzymes and helper proteins. This discussion is limited to the major enzymes involved.

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Meselson–Stahl Experiment

In their second paper on the structure of DNA*, Watson and Crick (pdf) described how DNA's structure suggests a pattern for replication:

"…prior to duplication the hydrogen bonds are broken, and the two chains unwind and separate. Each chain then acts as a template for the formation onto itself of a new companion chain, so that eventually we shall have two pairs of chains, where we only had one before." - Watson and Crick, 1953

This is called semiconservative replication.

Today we know that this is the pattern used by living cells, but the experimental evidence in support of semiconservative replication was not published until 1958. In the 5 years between Watson and Crick's suggestion and the definitive experiment, semiconservative replication was controversial and other patterns were considered.



Three hypothesized patterns were proposed:

  • Semiconservative - The original double strand of DNA separates and each strand acts as a template for the synthesis of a complimentary strand.
  • Conservative replication - the original double strand of DNA remains intact and is used as a template to create a new double stranded molecule.
  • Dispersive replication - similar to conservative replication in that the original double strand is used as a template without being separated, but prior to cell division, the strands recombine such that each daughter cell gets a mix of new and old DNA. With each round of replication, the original DNA gets cut up and dispersed evenly between each copy.
Knowing what we know now about how DNA behaves, the fact that the dispersive pattern was a popular seems odd, but this pattern was favored by several well known, prominent scientists. These scientists did not like the semiconservative pattern. They thought the helical nature of the double stranded DNA molecule would make it difficult for the strands to be unwound, separated and copied in the way needed for semiconservative replication to be possible. The dispersive pattern of cutting the helix once every rotation eliminated the need for unwinding the helix. Support for the dispersive hypothesis remained strong until proof of semiconservative replication was provided by Meselson and stahl's 1958 paper (pdf).

The methods Meselson and Stahl developed allowed them to distinguish existing DNA from newly synthesized DNA and to track new and old DNA over several rounds of replication.

They accomplished this by labeling cells with different stable isotopes of nitrogen. First, a culture of bacterial cell were grown for several generations in a media containing only 15N ( a stable, heavy isotope of Nitrogen). After this period* of growth, all of the DNA in the cells contained 15N. These cells were then rinsed and put into a media containing only the more common, lighter isotope of nitrogen (14N). As the cells grew and divided in this fresh media, all newly synthesized DNA would contain only the lighter nitrogen isotope, while DNA from the original cells would still contain 15N. In this illustration above, 15N labeled DNA is shown in orange and 14N labeled in green.

The 15N and 14N labeled DNA was then tracked using high speed centrifugation and a density* gradient created with cesium chloride (CsCl).

During centrifugation in a CsCl gradient, DNA accumulates in bands along the gradient based on its density. Since 15N is more dense than 14N, 15N enriched DNA accumulates lower down in the centrifuge tube than the 14N DNA. DNA containing a mixture of 15N and 14N ends up in an intermediate position between the two extremes.

By spinning DNA extracted at different times during the experiment, Meselson and Stahl were able to see how new and old DNA interacted during each round of replication.

The beauty of this experiment was that it allowed them to distinguish between the three different hypothesized replication patterns. The key result occurs at the second generation when all three proposed replication patterns give different results in the CsCl gradient.

That Meselson and Stahl's experiment showed the pattern predicted by the semiconservative hypothesis provided the definitive experimental evidence in support of the process proposed by Watson and Crick.

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Restriction Enzymes

Restriction enzymes cut DNA* at specific sites based on the sequence of bases along the strand at the cut site. These enzymes were first identified and studied in strains of the bacteria E. Coli in the 1950’s and 60’s. The term restriction was used to describe them because their activity restricted the growth of viruses that infect E. coli.

Restriction enzymes are nucleases - enzymes that cut nucleic acid polymers (i.e. DNA and RNA). There are two types of nuclease: endonuclease and exonuclease. Endonucleases make cuts within a DNA polymer. Exonucleases remove individual nucleotides* from the end of a strand. Restriction enzymes are a type of endonuclease - they cut at specific sites in the middle of DNA strands.

The ability of these enzymes to cut DNA at specific sites provide bacteria with a type of immune system that cuts up and, therefore, deactivates foreign DNA such as that introduced by viruses. To be effective, the patterns recognized by each bacteria’s restriction enzymes do not recognize any sequence patterns found in that bacteria’s genome. The specificity of the activity of restriction enzymes has made them useful tools in many molecular biology procedures and techniques.



An important aspect of restriction recognition sites is that they are palindromic. This means the short recognition sequence reads the same way on both strands resulting in the enzyme cutting both strands of a double-stranded DNA molecule.

There are several ways to classify restriction enzymes. The most obvious way is in terms of the base pattern each enzyme recognizes. Many of the recognition sequences used in molecular biology are six bases long, but recognition sequence pattern and lengths vary from enzyme to enzyme. The most widely used enzymes require a perfect match to cut, but others allow for some variation.

Another useful classification system for restriction enzymes is the position of the cut. Many restriction enzymes do not cut in the center of their recognition sequence resulting in overhanging or ‘sticky ends’. Others cut at the mid-point of the recognition sequence leaving no overhang. These are referred to as blunt end cuts.

An example of an enzyme that leaves blunt ends is SmaI. SmaI recognizes the six base sequence: CCCGGG. When cut the end of the two strands are:

SmaI

Contrast this with the enzyme XmaI which recognizes the same six base pattern: CCCGGG, but cuts in a way that leaves overhangs on either side of the cut. The result of this type of cut is two new, matching strand ends:

XmaI

This cutting pattern leaves two matching ends with the four base 3`-CCGG… overhang.

The predictable way in which restriction enzymes cut DNA at specific locations makes them extremely useful for molecular biology. Knowing the recognition site of a particular restriction enzyme and the sequence of a DNA strand makes it possible to predict the number of cuts and the position of the cuts that that enzyme will make on that length of DNA. Cutting a set of strands with a panel of restriction enzymes and running them out on a gel is the basis for the comparative technique known as restriction mapping.

Cutting two sequences with a restriction enzyme and then gluing them back together with a ligase is a common and relatively easy way to make hybrid DNA sequences in the lab.

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