Chapter 3: The Central Dogma of Molecular Biology
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James Crick (cofounder of DNA’s secondary structure) proposed that DNA is an informational storage molecule capable of replicating itself. Further, he proposed that the information that was transmitted had to be “read” by a manufacturing body within the cell which put amino acids together in a specific sequence ultimately synthesizing a protein. This became known as the central dogma of molecular biology.
The central dogma of molecular biology suggests that DNA serves as a template for the direct synthesis of a messenger RNA (mRNA) molecule, in a process known as transcription. Secondly, mRNA is “read” at a ribosome by transfer RNAs (tRNAs) , which work together to assemble a specific chain of amino acids, which collectively assemble to generate a protein. This process is known as translation.
DNA is directly responsible for creating all of the intermediate players of transcription and translation. DNA’s day-to-day function is the production of RNA molecules. Messenger RNA (mRNA) is directly generated by a specific segment of DNA. That segment of DNA is known as a gene. The mRNA travels to a ribosome, which is made up of protein and another type of RNA, ribosomal RNA (rRNA). At the ribosome, the mRNA serves as a code for the synthesis of protein by linking specific amino acids in an exact sequence. The overall collection of an amino acid chain is a protein.
Deoxyribonucleic Acid (DNA) is a nucleic acid that is a polymer, or a molecule that made up of a linking chain of repeating molecules. Repeating molecules are known as monomers. DNA’s, monomers are known as deoxyribonucleotides (or D-nucleotides). A D-nucleotide is made up of three components: a phosphate group, the sugar deoxyribose, and one of four nitrogenous bases.
Nitrogenous bases are the only molecules that differ in deoxyribonucleotides: adenine (A), thymine (T), cytosine (C), and guanine (G). Thymine and cytosine are each composed of a single carbon ring skeleton, and are known as pyrimidines; whereas, adenine and guanine are composed of two carbon ring skeletons connected together (one six-sided and the other five-sided), and are known as purines. DNA is double stranded, and the complementary strands connect, adenine (A) bonds with a thymine (T), and cytosine (C) bonds with guanine (G).
Ribonucleic acids (RNAs) are single stranded nucleic acid polymers made up of the monomers, ribonucleotides (R-nucleotides) (Fig. 4). R-nucleotides are nearly identical to D-nucleotides with two exceptions. First, ribonucleotides are made of the sugar ribose, in which a hydrogen (H) atom takes the place of the hydroxide (OH) at the 2’ carbon of deoxyribose. Second, R-nucleotides differ in their suite of nitrogenous bases. Three R-nucleotides have the same nitrogenous bases as D-nucleotides: cytosine, guanine and adenine. While the fourth R-nucleotide is composed of the nitrogenous base: uracil. Uracil is very similar to thymine, except there is a hydrogen atom at the 3’ location of uracil, while thymine has a has a methyl (CH3) group.
Nucleic acids are polymers of nucleotides, connected via phosphodiester linkages.
In nucleic acids, nucleotides are connected to another via a phosphodiester linkage. The phosphodiester linkage is a covalent bond that occurs when the phosphate (PO4H) of one nucleotide connects directly to the 3’ carbon (C) of a sugar of an adjacent nucleotide. During this chemical reaction, the hydroxide (OH) of the phosphate group is removed and the phosphorous (P) combines with with the sugar of the adjacent nucleotide by removing the sole hydrogen (H) of the 3’ carbon (C) of the sugar, deoxyribose in the case of D-nucleotides or ribose in the case of R-nucleotides. In addition, the hydrogen (H) from the deoxyribose and the hydroxide (OH) from the phosphate combine to form water (H2O) as a byproduct, making this a condensation reaction.
Complementary base pairing
The reason A only binds with T and C only binds with G is due to the structural makeup of the nitrogenous bases. On the interior edge of an adenine-containing D-nucleotide is an exposed nitrogen (N) and hydrogen (H), while on the interior edge of a thymine D-nucleotide is an exposed oxygen (O) and hydrogen (H). Nitrogen and oxygen atoms are highly electronegative. This means that when these atoms are covalently bonded in a molecule, they tend to hold onto the electrons more than the atom(s) they are bonded with. Effectively this makes oxygen and nitrogen partially negative (δ-). In contrast, hydrogen has a very low electronegativity due to its relative inability to hold onto its electrons giving it a partially positive (δ+) charge. Hydrogen bonds in biology occur when different molecules have exposed nitrogen (N) or oxygen (O) atoms in close contact with exposed H atoms. Since these charges are only partial, hydrogen bonds are relatively weak compared with covalent bonds.
Deoxyribonucleic acid (DNA) is a molecule that encodes the genetic instructions used in the development and functioning of all known living organisms and many viruses. DNA molecules are double-stranded helices, consisting of two long biopolymers made of simpler units called nucleotides—each nucleotide has one of the following nitrogenous bases (guanine, adenine, thymine, and cytosine), as well as a backbone made of alternating sugars (deoxyribose) and phosphate groups, with the bases (G, A, T, C) attached to the sugars.
The two strands of DNA run in opposite directions to each other and are therefore anti-parallel, one backbone being 3′ (three prime) and the other 5′ (five prime). This refers to the direction the 3rd and 5th carbon on the sugar molecule is facing. Attached to each sugar is one of four types of molecules called nitrogenous bases). It is the sequence of these four nitrogenous bases along the backbone that encodes genetic information. This information is read using the genetic code, which specifies the sequence of the amino acids within proteins. The code is read by copying stretches of DNA into the related nucleic acid RNA in a process called transcription.
The double-helix model of DNA structure was first published in the journal Nature by James D. Watson and Francis Crick in 1953, based upon the crucial X-ray diffraction image of DNA labeled as "Photo 51", from Rosalind Franklin in 1952. The realization that the structure of DNA is that of a double-helix elucidated the mechanism of base pairing by which genetic information is stored and copied in living organisms and is widely considered one of the most important scientific discoveries of the 20th century. Crick, Wilkins, and Watson each received one third of the 1962 Nobel Prize in Physiology or Medicine for their contributions to the discovery. (Franklin, whose breakthrough X-ray diffraction data was used to formulate the DNA structure, died in 1958, and thus was ineligible to be nominated for a Nobel Prize.)
Ribonucleic acid (RNA) is a ubiquitous family of large biological molecules that perform multiple vital roles in the coding, decoding, regulation, and expression of genes. Together with DNA, RNA comprises the nucleic acids, which, along with proteins, constitute the three major macromolecules essential for all known forms of life. Like DNA, RNA is assembled as a chain of nucleotides, but is usually single-stranded. Cellular organisms use messenger RNA (mRNA) to convey genetic information (often notated using the letters G, A, U, and C for the nucleotides guanine, adenine, uracil and cytosine) that directs synthesis of specific proteins.
Some RNA molecules play an active role within cells by catalyzing biological reactions, controlling gene expression, or sensing and communicating responses to cellular signals. One of these active processes is protein synthesis, a universal function whereby mRNA molecules direct the assembly of proteins on ribosomes. This process uses transfer RNA (tRNA) molecules to deliver amino acids to the ribosome, where ribosomal RNA (rRNA) links amino acids together to form proteins.
Amino Acids and Peptide Bonds
Amino acids are the physical building blocks of all living things. The primary purpose of DNA is to produce a message (mRNA) which codes for the linking of amino acids in a specific pattern, producing a protein. Amino acids are composed of an amino group (NH) connected to a central carbon which is also connected to a carboxylic acid group (COO-). These three parts of an amino acid are identical from one to another. Also connected to the central carbon is an R-group. There are 20 different R-groups, which interact with water and each other to form a specific 3-dimensional structure that determines the function of a protein.
Proteins are polymers of amino acids chained together via peptide bonds. A peptide bond is a condensation reaction, the oxygen ion (O-) of the carboxylic acid from one amino acid is removed and combines with two hydrogen atoms (H+) from an amino group of an adjacent amino acid to produce water (H2O). A peptide bond forms between two amino acids when the carbon of the carboxyl group that lost an oxygen during the condensation reaction combines with an adjacent nitrogen (N) of another amino acid that lost the two hydrogen atoms, bonding two adjacent amino acids.
Levels of Protein Structure
There are four levels of protein structure.
The three dimensional shape of a protein determines its function, and the shape of proteins are ultimately dependent upon the sequence of amino acids coded for by DNA. The unique amino acid sequence is considered a protein’s primary structure.
When amino acids are grouped into polypeptide chains, neighboring amino acids begin to interact via hydrogen bonding. Oxygen has very high electronegativity, while hydrogen has very low electronegativity. This differential in electronegativity results in hydrogen bonding between neighboring amino acids. The oxygen of the carboxylic acid group of the polypeptide backbone have a tendency to form a hydrogen bond with the hydrogen of a neighboring (but not adjacent) amino group of a different amino acid. These interactions can happen very regularly and result in one of two shapes common in proteins: an α-helix or a β-pleated sheet.
While the secondary structure of proteins is determined by the interactions between amino groups and carboxylic acid groups of neighboring (but not adjacent) amino acids, tertiary structure is defined by how the R-groups of neighboring amino acids interact. These interactions result in very specific folding patterns eventually helping to stabilize a specific 3-dimensional structure of the polypeptide. Several types of interactions occur between neighboring R-groups to create the physical 3-dimensional structure of a polypeptide.
While the hydrogen bonding determines the secondary structure of proteins, hydrogen bonding can also occur between the R-groups of a polypeptide chain. The 20 R-groups of amino acids are either polar or non-polar. Polar R-groups have oxygen or nitrogen atoms which characteristically have high electronegativity due to their high affinity for electrons. These polar R-groups tend to bond with hydrogen atoms of neighboring non-polar R-groups or the amino hydrogen of the peptide backbone. Likewise, non-polar R-groups have a lack of affinity for electrons and can form hydrogen bonds with neighboring polar R-groups or the oxygen of the carboxylic acid group (or the nitrogen of the amino group) of the peptide backbone. While hydrogen bonding is relatively weak, the overarching abundance of these interactions forms very stable polypeptide structures.
Hydrophobic and hydrophilic interactions
All proteins are surrounded by water within the cell. Polar R-groups are hydrophilic and bend to turn towards the water; whereas non-polar R-groups are hydrophobic and turn away from water. Hydrophobic R-groups tend to amass in the internal section of the protein forming globular masses.
While hydrogen bonding is facilitated by the interactions of partial charges, certain R-groups have full charges and are involved in ionic bonding. Ionic bonding happens between completely positive R-groups with neighboring R-groups that are completely negative.
The overall structure of a fully-functional protein is known as the quaternary structure. Most proteins are composed of several polypeptides. A polypeptide is composed of either a series of α-helices with tertiary level interactions, or a series of β-pleated sheet with tertiary level interactions.
While proteins serve several functions in the cell, proteins known as enzymes, are specifically designed to speed up (or catalyze) certain chemical reactions necessary for adequate cellular functioning. Enzymes can actually accelerate reactions up to thousands of times compared with chemicals in solution. How is this possible?
Proteins are very specific three-dimensional molecules. Enzymes’ three-dimensional structure perfectly aligns specific molecules (substrates) in a way that encourages chemical bonding to occur between them, efficiently synthesizing specific molecules (products) necessary for cellular function. In solution, the substrates would eventually become products. The unique shape of enzymes allows them to play the role of matchmaker, bringing together substrates much faster than normal, generating specific products. The image to the right represents how the enzyme sucrase catalyzes the hydrolysis of sucrose into glucose and fructose.
Every unique combination of substrates requires their own specific enzyme. Amazingly, enzymes don’t react during the bonding of substrates. They are capable of maintaining their molecular structure and therefore are capable of repeatedly combining reactants to make the desired products without losing functionality.
Benzoquinone is an antimicrobial molecule produced by plants in order to prevent bacterial infections when their internal tissues are exposed. When the cells of exposed tissues come into contact with oxygen, an enzyme known as catecholase catalyzes benzoquinone from catechol and oxygen.
In this lab, you will measure the effect of the enzyme, catecholase, under varying conditions. Catecholase binds catechol and oxygen creating benzoquinone. Benzoquinone turns fruit brown. You will measure the effect of catecholase by determining the degree of brownness in a variety of experiments.
The rate of enzyme efficiency can be affected by a number of factors. In this lab, you will experimentally alter the availability of reactants, pH, and temperature.
Enzyme efficiency relative to availability of reactants
You will examine the effect of the availability of substrates, by comparing the relative change in brownness by comparing two apples with different surface areas: one sliced and one grated.
Enzyme efficiency relative to pH
Enzymes are pH specific, meaning they work most efficiently with an optimal range of pH values. Above or below an enzyme’s optimal range can cause the enzyme to denature (altering their 3-D structure), and therefore lose their functionality. You will compare the relative change in brownness by comparing two apple slices immersed in two different pH solutions: neutral (H2O) and acidic (lemon juice).
Enzyme efficiency relative to temperature
Enzymes also have a specific optimal range of temperatures. Similar to pH, temperatures above or below an enzyme’s optimal range will cause the protein to denature, impeding their functionality.
- ½ an apple
- 3 - Petri dishes
- Lemon juice
- Plate warmer
- pH indicator
- 3 - 600ml beakers
- 3 - 100ml beakers
- In a large cup, warm 150ml of water to 40˚C. While that is warming, in a second cup create a 150ml ice bath. Fill a third large cup with 150ml of room temperature water.
- Cut your apple into six slices and assemble the following treatments:
- Room temperature incubator. Place apple slice in dry, small cup
- Hot incubator. Place apple slice in dry, small cup.
- Cold incubator. Place apple slice in dry, small cup.
- Lemon juice. Place apple slice on a plante. Flip apple every 5 minutes to moisten.
- Water. Place apple slice on a plate. Flip apple every 5 minutes to moisten.
- Grated apple. Place grated apple on a plate.
- Check your apples every 5 minutes and determine their level of brownness relative to the scale in The Biology Lab Primer.