What is the DNA/protein charge ratio?

To study DNA-protein interaction, I want to do a DNA retardation test by mixing the protein with DNA and afterwarts loading it on an agarose gel to see if the DNA migrates slower. I've found some articles where they do this and they state that they mix the DNA with protein in an 1:8 charge ratio, but I can't really figure out how to interpret this

Could anyone tell me how to calculate a DNA:protein charge ratio? If 1:8 is stated, how should I interpret this?

This is really sounds like a guideline, a starting place, not necessarily your best protocol.

In practice its hard to be more specific as MS says. The pH of the buffer will affect the charge of the protein, and to a lesser extent the DNA.

Also the protein may have an ideal pH for binding which you could guess is in the range of 7.0-7.4, but might find that the strongest binding depends on unexpected pH and salt concentrations, or the addition of a small molecule.

The affinity of araC for instance is greatly increased by the presence of arabinose in sufficient concentration.

lac Repressor, in contrast releases itself from DNA in the presence of IPTG or lactose.

What is the DNA/protein charge ratio? - Biology

One common way of separating biological macro molecules is by taking advantage of the fact that many of these molecules either exist as ions in solution or can be modified to have ionic molecules associated with them and therefore will move in an electric field. There are a number of different types of electrophoresis, but all involve generating an electric field between to points and placing a matrix of some sort in-between through which the macromolecules must travel. The speed at which they pass through this matrix in the presence of the electric field is called their electrophoretic mobility. Electrophoretic mobility is typically measured in a relative sense. That is, one usually uses some sort of standard and determines the mobility of other molecules relative to that standard.

The theory of electrophoresis. The concept here is simple enough. Like centrifugation, the molecules feel a force pushing them in one direction. However, in this case, the force involved is due to the electric field acting on the charge of the molecule and is given by F = EQ where F is the force, E is the electric field and Q is the charge. Obviously the greater the charge on the molecule, the greater the force. Thus, for two molecules of the same size, the one with the larger charge will move faster in the electric field. Now, it is less obvious that molecules with a larger mass will move more slowly. This actually comes about because the frictional forces that slow a molecule traveling through solution down depend on the molecules size. The speed at which molecules go through a solution is determined by the point at which the forces driving it forward are just balanced by the frictional forces generated by the motion. The greater the mass of the molecule, the greater the size, in general, and therefore the friction the molecule will generate when traveling through the solution. It turns out that in fact the electrophoretic mobility of a molecule depends on its charge to mass ratio. Two different sized molecules with the same charge to mass ratio should run with the same mobility in a uniform electric field and a perfect world.

Types of electrophoresis. There are quite a number of types of electrophoresis commonly used. It is not possible to go through them all in any detail here, but a brief description of a few of the most common types follows:

SDS-PAGE. One of the most common means of analyzing proteins by electrophoresis is by using Sodium Dodecyl Sulfate - Polyacrylamide Gel Electrophoresis. SDS is a detergent which denatures proteins by binding to the hydrophobic regions and essentially coating the linear protein sequence with a set of SDS molecules. The SDS is negatively charged and thus becomes the dominant charge of the complex. The number of SDS molecules that bind is simply proportional to the size of the protein. Therefore the charge to mass ratio should not change with size. In solution (water), in principle all different sized proteins covered with SDS would run at about the same mobility. However, the proteins are not run through water. Instead they are run through an inert polymer, polyacrylamide. The density and pore size of this polymer can be varied by just how you make it (concentration of monomer and of cross-linking agent). Thus, the size of molecules that can pass through the matrix can be varied. This determines in what molecular weight range the gel will have the highest resolving power.

Native Gels. It is also possible to run protein gels without the SDS. These are called native gels in that one does not purposely denature the protein. Here, the native charge on the protein (divided by its mass) determines how fast the protein will travel and in what direction.

Electrofocusing Gels. Another variation of gel electrophoresis is to pour a gel that purposely has a pH gradient from one end to the other. As the protein travels through this pH gradient, its various ionizable groups with either pick up or lose protons. Eventually, it will find a pH where its charge is zero and it will get stuck (focused) at that point.

DNA Agarose Gels. A simple way of separating fairly large fragments of DNA from one another by size is to use an agarose gel. Agarose is another type of matrix used for many purposes (such as the support for the growth of bacteria on plates). DNA does not need a detergent, since it already has a large under of negative phosphate groups evenly spaced. Thus, as with SDS-PAGE, the charge to mass ratio is constant. Also like SDS-PAGE, the separation results from the matrix itself. The range of size sensitivity can be varied by changing the density of the agarose.

DNA denaturing polyacrylamide gels (often called sequencing gels). To look at smaller DNA molecules with much higher resolution, people generally denature the DNA via heat and run it through a thin polyacrylamide gel that is also kept near the denaturing temperature. These gels usually contain additional denaturing compounds such as Urea. Two pieces of DNA that differ in size by 1 base can be distinguished from each other this way.

Capillary electrophoresis. It has become popular to separate molecules electrophoretically by running them into and through a capillary tube. This is fast and accurate, but does not allow much sample to be loaded on the gel at once.

Detergents for Cell Lysis and Protein Extraction

Detergents are amphipathic molecules, meaning they contain both a nonpolar "tail" having aliphatic or aromatic character and a polar "head". Ionic character of the polar head group forms the basis for broad classification of detergents they may be ionic (charged, either anionic or cationic), nonionic (uncharged), or zwitterionic (having both positively and negatively charged groups but with a net charge of zero).

Detergents in solution

Like the components of biological membranes, detergents have hydrophobic-associating properties as a result of their nonpolar tail groups. Nevertheless, detergents are themselves water-soluble. Consequently, detergent molecules allow the dispersion (miscibility) of water-insoluble, hydrophobic compounds into aqueous media, including the extraction and solubilization of membrane proteins.

Detergents at low concentration in aqueous solution form a monolayer at the air–liquid interface. At higher concentrations, detergent monomers aggregate into structures called micelles. A micelle is a thermodynamically stable colloidal aggregate of detergent monomers wherein the nonpolar ends are sequestered inward, avoiding exposure to water, and the polar ends are oriented outward in contact with the water.

Idealized structure of a detergent micelle.

Both the number of detergent monomers per micelle (aggregation number) and the range of detergent concentration above which micelles form (called the critical micelle concentration, CMC) are properties specific to each particular detergent (see table). The critical micelle temperature (CMT) is the lowest temperature at which micelles can form. The CMT corresponds to what is known as the cloud point since detergent micelles form crystalline suspensions at temperatures below the CMT and are clear again at temperatures above the CMT.

Detergent properties are affected by experimental conditions such as concentration, temperature, buffer pH and ionic strength, and the presence of various additives. For example, the CMC of certain nonionic detergents decreases with increasing temperature, while the CMC of ionic detergents decreases with addition of counter ion as a result of reduced electrostatic repulsion among the charged head groups. In other cases, additives such as urea effectively disrupt water structure and cause a decrease in detergent CMC. Generally, dramatic increases in aggregation number occur with increasing ionic strength.

Detergents can be denaturing or non-denaturing with respect to protein structure. Denaturing detergents can be anionic such as sodium dodecyl sulfate (SDS) or cationic such as ethyl trimethyl ammonium bromide. These detergents totally disrupt membranes and denature proteins by breaking protein–protein interactions. Non-denaturing detergents can be divided into nonionic detergents such as Triton X-100, bile salts such as cholate, and zwitterionic detergents such as CHAPS.

Properties of common detergents.

Thermo Scientific Triton X-100Nonionic140647 (90K)0.24 (0.0155)64No
Thermo Scientific Triton X-114Nonionic537 ( – )0.21 (0.0113)23No
NP-40Nonionic149617 (90K)0.29 (0.0179)80No
Thermo Scientific Brij-35Nonionic401225 (49K)0.09 (0.0110)>100No
Thermo Scientific Brij-58Nonionic701120 (82K)0.08 (0.0086)>100No
Thermo Scientific Tween 20Nonionic1228 ( – )0.06 (0.0074)95No
Thermo Scientific Tween 80Nonionic601310 (76K)0.01 (0.0016)No
Octyl glucosideNonionic27292 (8K)23-24 (

‡Agg.# = Aggregation number, which is the number of molecules per micelle.

Purified detergent solutions

Although detergents are available from several commercial sources and used routinely in many research laboratories, the importance of detergent purity and stability is not widely appreciated. Detergents often contain trace impurities from their manufacture. Some of these impurities, especially peroxides that are found in most nonionic detergents, will destroy protein activity. In addition, several types of detergents oxidize readily when exposed to the air or UV light, causing them to lose their properties and potency as solubilizing agents. We offer several high purity, low peroxide–containing detergents that are packaged under nitrogen gas in clear glass ampules. These Thermo Scientific Surfact-Amps Detergent Solutions provide unsurpassed convenience, quality and consistency for all detergent applications. A sampler kit includes 10 different purified detergents (seven in the Surfact-Amps format and three in solid form).

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Structure of cell membranes

A major factor determining the behavior and interaction of molecules in biological samples is their hydrophilicity or hydrophobicity. Most proteins and other molecules with charged or polar functional groups are soluble (or miscible) in water because they participate in the highly ordered, hydrogen-bonded intermolecular structure of water. Some other proteins (or at least parts of proteins), as well as fats and lipids, lack polar or charged functional groups consequently, they are excluded from the ordered interaction of water with other polar molecules and tend to associate together in structures having minimal surface area contact with the polar environment. This association of nonpolar molecules in aqueous solutions is commonly called hydrophobic attraction, although it is more accurately understood as exclusion from the hydrophilic environment.

The formation and stability of biological membranes results in large measure from the hydrophobic attraction of phospholipids, which form bilayer sheets having hydrophobic lipid "tails" oriented within the sheet thickness and polar "head" groups oriented to the outer and inner aqueous environments. Membrane proteins completely span the membrane thickness or are embedded at one side of the membrane in accord with their structure of hydrophobic and hydrophilic amino acid side chains and other functional groups.

Membrane disruption, protein binding and solubilization

Generally, moderate concentrations of mild (i.e., nonionic) detergents compromise the integrity of cell membranes, thereby facilitating lysis of cells and extraction of soluble protein, often in native form. Using certain buffer conditions, various detergents effectively penetrate between the membrane bilayers at concentrations sufficient to form mixed micelles with isolated phospholipids and membrane proteins.

Detergent-based cell lysis. Both denaturing and non-denaturing cell lysis reagents may be used for protein extraction procedures.

Denaturing detergents such as SDS bind to both membrane (hydrophobic) and non-membrane (water-soluble, hydrophilic) proteins at concentrations below the CMC (i.e., as monomers). The reaction is equilibrium driven until saturated. Therefore, the free concentration of monomers determines the detergent concentration. SDS binding is cooperative (i.e., the binding of one molecule of SDS increases the probability that another molecule of SDS will bind to that protein) and alters most proteins into rigid rods whose length is proportional to molecular weight.

Non-denaturing detergents such as Triton X-100 have rigid and bulky nonpolar heads that do not penetrate into water-soluble proteins consequently, they generally do not disrupt native interactions and structures of water-soluble proteins and do not have cooperative binding properties. The main effect of non-denaturing detergents is to associate with hydrophobic parts of membrane proteins, thereby conferring miscibility to them.

At concentrations below the CMC, detergent monomers bind to water-soluble proteins. Above the CMC, binding of detergent to proteins competes with the self-association of detergent molecules into micelles. Consequently, there is effectively no increase in protein-bound detergent monomers with increasing detergent concentration beyond the CMC.

Detergent monomers solubilize membrane proteins by partitioning into the membrane bilayer. With increasing amounts of detergents, membranes undergo various stages of solubilization. The initial stage is lysis or rupture of the membrane. At detergent:membrane lipid molar ratios of 0.1:1 through 1:1, the lipid bilayer usually remains intact but selective extraction of some membrane proteins occurs. Increasing the ratio to 2:1, solubilization of the membrane occurs, resulting in mixed micelles. These include phospholipid–detergent micelles, detergent–protein micelles, and lipid–detergent–protein micelles. At a ratio of 10:1, all native membrane lipid:protein interactions are effectively exchanged for detergent:protein interactions.

The amount of detergent needed for optimal protein extraction depends on the CMC, aggregation number, temperature and nature of the membrane and the detergent. The solubilization buffer should contain sufficient detergent to provide greater than 1 micelle per membrane protein molecule to help ensure that individual protein molecules are isolated in separate micelles.

Detergents used for cell lysis. Major characteristics of denaturing and non-denaturing detergents used for protein extraction.


Column chromatography is one of the most common methods of protein purification. Like many of the techniques on this site, it is as much an art form as a science. Proteins vary hugely in their properties, and the different types of column chromatography allow you to exploit those differences. Most of these methods do not require the denaturing of proteins.

To be very general, a protein is passed through a column that is designed to trap or slow up the passing of proteins based on a particular property (such as size, charge, or composition).

There are three main steps to protein purification:

1. Capture. You need to get your protein into a concentrated form. If, for example, you are trying to isolate a protein you have synthesized in an E. coli cell, you could be looking at a protein to junk ratio of 1:1,000,000. For capture purification you need a high capacity method that is also fast. You need a speedy method because your crude solution is very likely to also contain proteases and these can quickly chew up your protein.

2. Intermediate. Intermediate purification requires both speed and good resolution.

3. Polishing. For the final step of purification you need a system that has both good resolution and speed. Capacity is usually irrelevant at this stage.

Some of the more common columns include:

IEX: Ion exchange chromatography. Good for capture, intermediate, and polish.

HIC: Hydrophobic interaction column. Good for intermediate purification.

AC: Affinity chromatography. Good for capture and intermediate purification.

GF: Gel filtration (size exclusion) chromatography. Good polishing step.

Let's look at these types of columns in more detail.

Ion exchange chromatography

Ion exchange chromatography is based on the charge of the protein you are trying to isolate. If your protein has a high positive charge, you'll want to pass it through a column with a negative charge. The negative charge on the column will bind the positively charged protein, and other proteins will pass through the column. You then use a procedure called "salting out" to release your positively charged protein from the negatively charged column. The column that does this is called a cation exchange column and often uses sulfonated residues. Likewise, you can bind a negatively charged protein to a positively charge column. The column that does this is called an anion exchange column and often uses quaternary ammonium residues.

Salting out will release, or elute, your protein from the column. This technique uses a high salt concentration solution. The salt solution will out-compete the protein in binding to the column. In other words, the column has a higher attraction for the charge of salts than for the charged protein, and it will release the protein in favor of binding the salts instead. Proteins with weaker ionic interactions will elute at a lower salt, so you will often want to elute with a salt gradient. Different proteins elute at different salt concentrations, so you will want to be sure you know well the properties of your protein best results.

Also be aware that changes in pH alter the charges in proteins. Be sure you know the isoelectric point of your protein (the isoelectric point is the pH at which the charge of a protein is zero) and make sure the pH of your system is adjusted and buffered accordingly.

The basic steps in using an ion exchange column are:

1. Prep the column. Pour your buffer over the column to make sure it has equilibrated to the required pH.

2. Load your protein solution. Some proteins in the solution don't bind and will elute during this loading phase.

3. Salt out. Increase the salt concentration to elute the bound proteins. It is best to use a salt gradient to gradually elute proteins with different ionic strengths. At the end bump the system with a very high salt concentration (2-3M) to make sure all proteins are off the column.

4. Remove salts. Use dialysis to remove the salts from your protein solution.

Temperature doesn't have a huge effect on column chemistry. However, it is better to work cold since proteins are more stable cold.

Hydrophobic interaction chromatography

Where ion exchange chromatography relies on the charges of proteins to isolate them, hydrophobic interaction chromatography uses the hydrophobic properties of some proteins. Hydrophobic groups on the protein bind to hydrophobic groups on the column. The more hydrophobic a protein is, the stronger it will bind to the column.

Load the proteins in the presence of a high concentration of ammonium sulfate (not ammonium persulfate). Ammonium sulfate is a chaotropic agent. It increases the chaos (entropy) in water, and thereby increases hydrophobic interactions (the more disordered the water, the stronger the hydrophobic interactions). Ammonium sulfate also stabilizes proteins. So as a result of using an HIC column you can expect your protein to be in its most stable form.

The hydrophobic column is packed with a phenyl agarose matrix. In the presence of high salt concentrations the phenyl groups on this matrix binds hydrophobic portions of proteins. You can control elution of different column-bound proteins by reducing the salt concentration or by adding solvents.

Affinity chromatography.

Affinity chromatography relies on the biological functions of a protein to bind it to a column. The most common type involves a ligand, a specific small biomolecule. This small molecule is immobilized and attached to a column matrix, such as cellulose or polyacrylamide. Your target protein is then passed through the column and bound to it by its ligand, while other proteins elute out. Elution of your target protein is usually done by passing through the column a solution that has in it a high concentration of free ligand. This is a very efficient purification method since it relies on the biological specificity of your target protein, such as the affinity of an enzyme for a substrate.

Gel filtration (size exclusion) chromatography d

Gel filtration, or size exclusion, chromatography separates proteins on the basis of their size. The column is packed with a matrix of fine porous beads.

It works somewhat like a sieve, but in reverse. The beads have in them very small holes. As the protein solution is poured on the column, small molecules enter the pores in the beads. Larger molecules are excluded from the holes, and pass quickly between the beads.

These larger molecules are eluted first. The smaller molecules have a longer path to travel, as they get stuck over and over again in the maze of pores running from bead to bead. These smaller molecules, therefore, take longer to make their way through the column and are eluted last.

PAGE (Polyacrylamide Gel Electrophoresis), is an analytical method used to separate components of a protein mixture based on their size. The technique is based upon the principle that a charged molecule will migrate in an electric field towards an electrode with opposite sign.The general electrophoresis techniques cannot be used to determine the molecular weight of biological molecules because the mobility of a substance in the gel depends on both charge and size. To overcome this, the biological samples needs to be treated so that they acquire uniform charge, then the electrophoretic mobility depends primarily on size. For this different protein molecules with different shapes and sizes, needs to be denatured(done with the aid of SDS) so that the proteins lost their secondary, tertiary or quaternary structure .The proteins being covered by SDS are negatively charged and when loaded onto a gel and placed in an electric field, it will migrate towards the anode (positively charged electrode) are separated by a molecular sieving effect based on size. After the visualization by a staining (protein-specific) technique, the size of a protein can be calculated by comparing its migration distance with that of a known molecular weight ladder(marker).

Packaging of DNA, Genome, chromosomal proteins, DNA in Prokaryotes & Eukaryotes

Prokaryotes are living organisms whose genetic material is not surrounded by a nuclear membrane, but found free in the cytoplasm such as bacteria, DNA molecules of mitochondria and chloroplasts (organelles of eukaryotic cells) are very similar to those of prokaryotes, Plasmids are found in the yeast cells (from eukaryotes).

DNA in Prokaryotes

DNA of Escherichia coli bacterium as an example for prokaryotes:

  1. DNA exists as a double helix with its ends joined to each other to form a circle.
  2. If DNA was stretched out in a straight line, it would be about 1.4 millimeters in length, whereas the cell itself is only about 2 micron in length.
  3. DNA is folded many times and occupied a nuclear area about 0.1 of the cell’s volume.
  4. DNA molecule is attached to the plasma membrane at one point or more.

Some bacteria contain one or several additional, much smaller and circular DNA molecules which are called “plasmids”, Plasmids are much smaller circular DNA molecules and not complexed with proteins.

Importance of plasmids: Plasmids are widely used in the field of genetic engineering, where the bacterial cell replicates any plasmid inside it during the replication of its main DNA and the scientists take advantage of this activity by introducing artificial plasmids into the bacterial cells, in order to obtain several copies of them.

Packaging of DNA

DNA in Eukaryotes

Eukaryotes are living organisms whose genetic material is surrounded by a nuclear membrane that separates it from the cytoplasm and DNA is organized into several chromosomes, The human’s somatic cell contains 46 chromosomes, Chromosomes appear in eukaryotic cells during the cell division.

Structure of chromosomes

Each chromosome contains a single DNA molecule, extending from one end of the chromosome to the other end, DNA molecule is coiled and folded many times and associated with various proteins, forming a “chromatin” which contains roughly equal amounts of DNA and proteins.

Chromatin is one molecule of DNA that is coiled and folded many times and associated with proteins, The chromosomal proteins are divided into Histone proteins and Non-histone proteins.

Histone proteins

Histones are a well-defined group of small structural proteins, where they have a high content of the basic amino acids “arginine” and “lysine” and present in great amounts in the chromatin of any cell.

At the normal pH of the cell, the amino acids “arginine” and “lysine” have a positively charged alkyl groups (R), So, they bind strongly to the negatively charged phosphate groups of DNA molecule, Histones proteins are responsible for shortening the DNA molecule tenfold by forming a string of nucleosomes.

Non-histone proteins

Non-histones are heterogeneous groups of structural and regulatory proteins with many functions found in the structure of chromatin and they present in a little amount, Non-histone proteins have many different functions because they contain :

  • Structural proteins enter in the structure of some definite parts of DNA molecule and play the main role in the spatial organization of DNA within the nucleus as they are responsible for shortening DNA about 100,000 times by forming the packed chromatin.
  • Regulatory proteins determine whether the DNA code will be used in making RNA, proteins and enzymes or not.
Packaging of DNA

If we imagine that DNA double helix of each chromosome was lined up and stretched out, it would be about 2 metres in length, The histones and other proteins are responsible for packing this long molecule into the cell’s nucleus that of 2 : 3 micron in diameter.

Steps of DNA packaging

Biochemical analysis and electron micrographs have shown that DNA is packed, as follows:

  1. DNA is wounded around clusters of histones, forming a string of particles that is called “nucleosomes”, this shortens the molecule about tenfold, but it must be packed about 100,000 times more tightly to fit into the nucleus.
  2. The string of nucleosomes is coiled to pack the nucleosomes together, However, even this is not sufficient to shorten the DNA molecule to the required length.
  3. The tightly coiled strings of nucleosomes are arranged in large loops that are held together by the structural non-histone proteins to form the chromatin, Chromatin is packed up as tightly as possible to be condensed into the chromosome.

Nucleosomes are a string of particles that found in the chromosomes and consist of DNA molecule wounded (wrapped) around clusters of histones to shorten the DNA molecule about tenfold.

When DNA is packed as chromatin, replication enzymes apparently can’t reach it, This packaging must be unwounded at least into a string of nucleosomes before DNA can serve as a template for DNA or RNA synthesis.

Structure of the genome

In 1977, researchers found methods that can be used for determining the sequence of nucleotides in DNA and RNA molecules, This provided the tools to describe precisely how genes are arranged within the cell’s DNA molecule, Genome is the total of all genes ( all DNA) found in the cell.

DNA contains genes that carry instructions to construct the :

  • The sequence of nucleotides that is responsible for making proteins.
  • The sequence of nucleotides that transcribes the ribosomal RNA (rRNA) which enters the building of ribosomes.
  • The sequence of nucleotides that transcribes the transfer RNA (tRNA) which carries the amino acids during protein synthesis.

The genes in prokaryotes are the genes that are responsible for the RNA and protein synthesis and represent most of the genome.

The genes in eukaryotes: less than 70% of the genome serve the function of RNA and protein synthesis and the rest of the genome is unaccounted ( has unknown function).

Repetitive DNA

Most genes are present in only one or few copies in the genome, such as:

Genes needed to synthesize the ribosomal RNA and histones that the cell needs in large amounts, where they are reasonable to suppose that having multiple copies of these genes to speed up the cell’s production of new ribosomes and histones, So, there are many -often hundreds- of copies of the genes in all the eukaryotic cells.

Some nucleotides sequences of DNA have been repeated many times, The role of most of this repetitive DNA is still unclear, For instance, in the fruit fly (Drosophila), the brief nucleotides sequence (A-G-A-A-G) is repeated about 100,000 times in the middle of one chromosome, This and many other repeated sequences are noncoding DNA.

Other noncoding DNA

The satellite DNA of some chromosomes is noncoding, Eukaryotic genomes contain a great deal of other noncoding DNA, where the geneticists observe that the amount of DNA in species’ genome bears little relationship to the complexity of the organism or the number of proteins that is produced by it, Little amount of DNA of the plants and animals actually codes for protein synthesis.

Example: The largest known genome belongs to the salamander, its cells contain about 30 times the amount of DNA found in the human cells, although they produce fewer proteins, this is due to the noncoding of a large amount of DNA.

Functions of noncoding DNA:

  1. Perhaps some of the noncoding DNA act on keeping the chromosomes structure.
  2. Some regions of DNA are references to the places at which the messenger RNA (mRNA) synthesis should start and these regions are important in the protein synthesis.

We can compare between prokaryotic DNA and eukaryotic DNA, as follows:

Prokaryotic DNA

It exists as a double helix, its ends are joined together, it is attached to the plasma membrane at one point or more and it is not organized in the form of chromosomes, It is found in the cytoplasm (not surrounded by the nuclear membrane), It is not complexed with proteins.

The most are coding, It starts from the attachment point with the plasma membrane, Plasmids present and not complexed with proteins, Most of them are responsible for making the RNA and proteins.

Eukaryotic DNA

Eukaryotic DNA exists as a double helix, its ends are free and it is organized in several chromosomes, It is found in the nucleus (surrounded by the nuclear membrane), It is complexed with histone and non-histone proteins.

Some are non-coding, It starts at any point along the DNA molecule, Plasmids present in the yeast only, Less than 70% serve the function of RNA and protein synthesis and the rest of the genome is unaccounted (has unknown function).

Amino Acids

Proteins are one of the most abundant organic molecules in living systems and have the most diverse range of functions of all macromolecules. Proteins may be structural, regulatory, contractile, or protective they may serve in transport, storage, or membranes or they may be toxins or enzymes. Each cell in a living system may contain thousands of proteins, each with a unique function. Their structures, like their functions, vary greatly. They are all, however, polymers of amino acids, arranged in a linear sequence.

Figure 1. Amino acids have a central asymmetric carbon to which an amino group, a carboxyl group, a hydrogen atom, and a side chain (R group) are attached.

Amino acids are the monomers that make up proteins. Each amino acid has the same fundamental structure, which consists of a central carbon atom, also known as the alpha (α) carbon, bonded to an amino group (NH2), a carboxyl group (COOH), and to a hydrogen atom. Every amino acid also has another atom or group of atoms bonded to the central atom known as the R group (Figure 1).

The name “amino acid” is derived from the fact that they contain both amino group and carboxyl-acid-group in their basic structure. As mentioned, there are 20 amino acids present in proteins. Ten of these are considered essential amino acids in humans because the human body cannot produce them and they are obtained from the diet.

For each amino acid, the R group (or side chain) is different (Figure 2).

Practice Question

Figure 2. There are 20 common amino acids commonly found in proteins, each with a different R group (variant group) that determines its chemical nature.

Which categories of amino acid would you expect to find on the surface of a soluble protein, and which would you expect to find in the interior? What distribution of amino acids would you expect to find in a protein embedded in a lipid bilayer?

The chemical nature of the side chain determines the nature of the amino acid (that is, whether it is acidic, basic, polar, or nonpolar). For example, the amino acid glycine has a hydrogen atom as the R group. Amino acids such as valine, methionine, and alanine are nonpolar or hydrophobic in nature, while amino acids such as serine, threonine, and cysteine are polar and have hydrophilic side chains. The side chains of lysine and arginine are positively charged, and therefore these amino acids are also known as basic amino acids. Proline has an R group that is linked to the amino group, forming a ring-like structure. Proline is an exception to the standard structure of an animo acid since its amino group is not separate from the side chain (Figure 2).

Amino acids are represented by a single upper case letter or a three-letter abbreviation. For example, valine is known by the letter V or the three-letter symbol val. Just as some fatty acids are essential to a diet, some amino acids are necessary as well. They are known as essential amino acids, and in humans they include isoleucine, leucine, and cysteine. Essential amino acids refer to those necessary for construction of proteins in the body, although not produced by the body which amino acids are essential varies from organism to organism.

Figure 3. Peptide bond formation is a dehydration synthesis reaction. The carboxyl group of one amino acid is linked to the amino group of the incoming amino acid. In the process, a molecule of water is released.

The sequence and the number of amino acids ultimately determine the protein’s shape, size, and function. Each amino acid is attached to another amino acid by a covalent bond, known as a peptide bond, which is formed by a dehydration reaction. The carboxyl group of one amino acid and the amino group of the incoming amino acid combine, releasing a molecule of water. The resulting bond is the peptide bond (Figure 3).

The products formed by such linkages are called peptides. As more amino acids join to this growing chain, the resulting chain is known as a polypeptide. Each polypeptide has a free amino group at one end. This end is called the N terminal, or the amino terminal, and the other end has a free carboxyl group, also known as the C or carboxyl terminal. While the terms polypeptide and protein are sometimes used interchangeably, a polypeptide is technically a polymer of amino acids, whereas the term protein is used for a polypeptide or polypeptides that have combined together, often have bound non-peptide prosthetic groups, have a distinct shape, and have a unique function. After protein synthesis (translation), most proteins are modified. These are known as post-translational modifications. They may undergo cleavage, phosphorylation, or may require the addition of other chemical groups. Only after these modifications is the protein completely functional.

The Evolutionary Significance of Cytochrome c

Cytochrome c is an important component of the electron transport chain, a part of cellular respiration, and it is normally found in the cellular organelle, the mitochondrion. This protein has a heme prosthetic group, and the central ion of the heme gets alternately reduced and oxidized during electron transfer. Because this essential protein’s role in producing cellular energy is crucial, it has changed very little over millions of years. Protein sequencing has shown that there is a considerable amount of cytochrome c amino acid sequence homology among different species in other words, evolutionary kinship can be assessed by measuring the similarities or differences among various species’ DNA or protein sequences.

Scientists have determined that human cytochrome c contains 104 amino acids. For each cytochrome c molecule from different organisms that has been sequenced to date, 37 of these amino acids appear in the same position in all samples of cytochrome c. This indicates that there may have been a common ancestor. On comparing the human and chimpanzee protein sequences, no sequence difference was found. When human and rhesus monkey sequences were compared, the single difference found was in one amino acid. In another comparison, human to yeast sequencing shows a difference in the 44th position.

Native Protein Electrophoresis

Proteins run on PAGE in the absence of SDS will separate on the basis of their charge to mass ratio. While native (nondenaturing) PAGE does not provide direct measurement of molecular weight, the technique can provide useful information such as protein charge or subunit composition. Native PAGE also has the potential for separating proteins of identical molecular weight which cannot be resolved with SDS - PAGE. In addition, proteins on native PAGE usually retain their activity. This allows enzymes to be detected by sensitive and specific activity stains and delicate proteins to be resolved and recovered in a biologically active form.

The interpretation of native gels is more complex than the interpretation of SDS - PAGE gels. Not only can differences in relative mobility reflect differences in charge, mass or both, but also, proteins may have a pI at or above the pH of the buffer, in which case they will not migrate or will "retro-phorese" backward into the upper buffer chamber.

The equation governing protein mobility in native gels is as follows:

  • Rf = relative mobility, normalized to the dye front or some other standard.
  • Yo = relative mobility of the protein in the absence of any sieving matrix.
  • KR = "retardation coefficient," the extent to which the gel matrix affects mobility.
  • T = % monomer of the gel matrix.

In the presence of SDS, all proteins have the same Yo, so that a simple relationship exists between Rf and KR at any given T. In other words, SDS treated proteins, having identical charge to mass ratio, migrate at the same speed in free solution under electric force. With such proteins, if you know the mechanical resistance exerted by the gel, you can determine the mobility. This mechanical resistance, KR, is directly related to molecular weight, so that a determination of KR allows calculation of molecular weight. In native gels, the situation is more complicated. Both Y0 and KR can vary between proteins. Y0 is related to the charge, while KR varies with the mass.

Ferguson Plots

Separation of protein mixtures and protein standards on gels of varying percentages allows the determination of both charge and mass of the sample proteins. The graphic analysis used is known as the Ferguson plot. On the Ferguson plot, the logarithm of Rf is graphed versus T for a range of T (the originators of this system covered 7 different percentage gels). By the above equation, the graph should have a slope of KR and a Y intercept (at T = 0) of Yo. Comparison with standards of known charge and size allows determination of the charge and molecular weight of the samples.

Native Gradient Gels

Another less laborious way of simplifying the interpretation of native PAGE gels is to run a gradient gel. Native gradient gels are poured in the same manner as gradient SDS PAGE gels. As proteins migrate through the increasing acrylamide concentration, into regions of ever smaller pore sizes, their mobility decreases. Eventually, each protein reaches its "pore-limit", at which point it slows to a minimum migration rate, which is constant for all proteins at their pore limit. The band pattern is stabilized at this point, so that gradient native gel PAGE approaches an equilibrium system, in that beyond a certain run length only minimal changes occur in the gel pattern.

Once proteins reach their pore limit, their relative positions are a direct reflection of their molecular weight. In a linear gradient, the logarithm of the molecular weight is proportional to log Rf over a wide range, although the curve is actually sigmoid in shape. This type of analysis is more subject to artifacts than the Ferguson plot, but is easier to carry out.

Finally, native gradient gels may be analyzed with activity stains, which simplify the pattern by only visualizing enzyme activities of interest. This can be useful when purifying an enzyme from a mixture of isozymes, or when studying the expression or activity of enzyme families. It also allows for the isolation of electrophoretically pure, active enzyme. Used in conjunction with gradient gels, activity stains can provide molecular weight information about otherwise uncharacterized enzymes. Of course, the technique is limited to enzymes for which chromogenic or chemiluminescent assays exist. Often an assay can be adapted from existing techniques with little effort. The critical requirement is that a colored compound be deposited in the gel in an insoluble form, either by the enzyme (positive stain) or in a reaction inhibited by the enzyme (negative stain).


Proteins are the most diverse biomolecules and play a significant role in various biological processes. Enzymes are the most important functional proteins that catalyse biological reactions. Proteins are sensitive to heat and pH changes. They often undergo irreversible changes in structure, called protein denaturation, due to extreme change in temperature or pH. Amino acids are the monomers of complex proteins.

Peptide bonds and polypeptides

Carboxyl group of one amino acid reacts with amino group of another amino acid to form a dipeptide linked through a peptide bond. When 100 or more amino acids link through peptide bonds, the resulting chain of amino acids is called polypeptide. One or more polypeptide chains constitute a protein.

Structure of proteins

Proteins have four levels of structural organization including primary, secondary, tertiary and quaternary structures. Chain of amino acids joined by peptide bond forming a polypeptide is known as the primary structure of protein. The polypeptide chains may further bend or fold to form the secondary structure. There are two types of secondary structures including α-helix and β-sheets. The α-helix is a chain of amino acids that coil into a helix shape and stabilized by the formation hydrogen bonds between C=O and NH groups of various amino acids. The β-sheets are formed when the polypeptide chains arrange side by side and held together by hydrogen bonds between C=O and NH groups.

The side chain R groups of amino acids in polypeptide chain interact to through various interatomic forces like hydrogen bonds, hydrophobic bonds, disulphide bonds or van der Waals forces. This level of organization is known is tertiary structure. Two or more polypeptide chains may interact through hydrogen bonds, covalent bonds and/or hydrophobic interactions to form the quaternary structure. This is the most stable structure of proteins and gives a final 3-dimensional shape to the proteins.

What is the DNA/protein charge ratio? - Biology

The human genome encodes over 20,000 genes each of the 23 pairs of human chromosomes encodes thousands of genes. The DNA in the nucleus is precisely wound, folded, and compacted into chromosomes so that it will fit into the nucleus. It is also organized so that specific segments can be accessed as needed by a specific cell type.

The first level of organization, or packing, is the winding of DNA strands around histone proteins. Histones package and order DNA into structural units called nucleosome complexes, which can control the access of proteins to the DNA regions (Figure 1a). Under the electron microscope, this winding of DNA around histone proteins to form nucleosomes looks like small beads on a string (Figure 1b). These beads (histone proteins) can move along the string (DNA) and change the structure of the molecule.

Figure 1. DNA is folded around histone proteins to create (a) nucleosome complexes. These nucleosomes control the access of proteins to the underlying DNA. When viewed through an electron microscope (b), the nucleosomes look like beads on a string. (credit “micrograph”: modification of work by Chris Woodcock)

If DNA encoding a specific gene is to be transcribed into RNA, the nucleosomes surrounding that region of DNA can slide down the DNA to open that specific chromosomal region and allow for the transcriptional machinery (RNA polymerase) to initiate transcription (Figure 2). Nucleosomes can move to open the chromosome structure to expose a segment of DNA, but do so in a very controlled manner.

Practice Question

Figure 2. Nucleosomes can slide along DNA. When nucleosomes are spaced closely together (top), transcription factors cannot bind and gene expression is turned off. When the nucleosomes are spaced far apart (bottom), the DNA is exposed. Transcription factors can bind, allowing gene expression to occur. Modifications to the histones and DNA affect nucleosome spacing.

In females, one of the two X chromosomes is inactivated during embryonic development because of epigenetic changes to the chromatin. What impact do you think these changes would have on nucleosome packing?

How closely the histone proteins associate with the DNA is regulated by signals found on both the histone proteins and on the DNA. These signals are functional groups added to histone proteins or to DNA and determine whether a chromosomal region should be open or closed (Figure 3 depicts modifications to histone proteins and DNA). These tags are not permanent, but may be added or removed as needed. Some chemical groups (phosphate, methyl, or acetyl groups) are attached to specific amino acids in histone “tails” at the N-terminus of the protein. These groups do not alter the DNA base sequence, but they do alter how tightly wound the DNA is around the histone proteins. DNA is a negatively charged molecule and unmodified histones are positively charged therefore, changes in the charge of the histone will change how tightly wound the DNA molecule will be. By adding chemical modifications like acetyl groups, the charge becomes less positive, and the binding of DNA to the histones is relaxed. Altering the location of nucleosomes and the tightness of histone binding opens some regions of chromatin to transcription and closes others.

The DNA molecule itself can also be modified by methylation. DNA methylation occurs within very specific regions called CpG islands. These are stretches with a high frequency of cytosine and guanine dinucleotide DNA pairs (CG) found in the promoter regions of genes. The cytosine member of the CG pair can be methylated (a methyl group is added). Methylated genes are usually silenced, although methylation may have other regulatory effects. In some cases, genes that are silenced during the development of the gametes of one parent are transmitted in their silenced condition to the offspring. Such genes are said to be imprinted. Parental diet or other environmental conditions may also affect the methylation patterns of genes, which in turn modifies gene expression. Changes in chromatin organization interact with DNA methylation. DNA methyltransferases appear to be attracted to chromatin regions with specific histone modifications. Highly methylated (hypermethylated) DNA regions with deacetylated histones are tightly coiled and transcriptionally inactive.

Figure 3. Histone proteins and DNA nucleotides can be modified chemically. Modifications affect nucleosome spacing and gene expression. (credit: modification of work by NIH)

Epigenetic changes are not permanent, although they often persist through multiple rounds of cell division and may even cross generational lines. Chromatin remodeling alters the chromosomal structure (open or closed) as needed. If a gene is to be transcribed, the histone proteins and DNA in the chromosomal region encoding that gene are modified in a way that opens the promoter region to allow RNA polymerase and other proteins, called transcription factors , to bind and initiate transcription. If a gene is to remain turned off, or silenced, the histone proteins and DNA have different modifications that signal a closed chromosomal configuration. In this closed configuration, the RNA polymerase and transcription factors do not have access to the DNA and transcription cannot occur (Figure 3).

View this video that describes how epigenetic regulation controls gene expression.

In Summary: Eukaryotic Epigenetic Gene Regulation

In eukaryotic cells, the first stage of gene expression control occurs at the epigenetic level. Epigenetic mechanisms control access to the chromosomal region to allow genes to be turned on or off. These mechanisms control how DNA is packed into the nucleus by regulating how tightly the DNA is wound around histone proteins. The addition or removal of chemical modifications (or flags) to histone proteins or DNA signals to the cell to open or close a chromosomal region. Therefore, eukaryotic cells can control whether a gene is expressed by controlling accessibility to transcription factors and the binding of RNA polymerase to initiate transcription.

Watch the video: Η αλληλουχία των βάσεων στο DNA καθορίζει την δομή των πρωτεϊνών (January 2022).