Thursday, April 25, 2013

Biomolecules of the Cell - Nucleic Acids (Part 2)

In the previous post, we have seen the basics of nucleic acids and the native form of DNA i.e.; B-DNA in details.
There are various other forms which DNA can assume. In this post, I will discuss some of the other forms of DNA (i.e.; A-DNA and Z-DNA) and another type of nucleic acid - RNA.
Starting the post with the A-form of DNA.

A-form of DNA: 
The following are certain characteristics of A-DNA:
i. This form of DNA exists when the relative humidity is around 75%.  
ii. The A-DNA is also right-handed helix like B-DNA.
iii. A-DNA is shorter and wider than B-DNA. 
iv. The diameter of A-DNA is 23Å as against 20Å in B-DNA. 
v. There are 11 base pairs per turn (more than B-DNA which has 10 base pairs per turn). 
vi. The rise per turn is 28Å
vii. Thus, if we calculate, rise per base pair (28Å/11base pairs), we get 2.6Å
viii. The base pairs are not perpendicular to the helical axis instead they are tilted 20˚ w.r.t. helical axis. 
ix. Just like B-DNA, A-DNA also has major groove and minor groove; however, the former is very deep and latter is very shallow as can be seen in the adjacent diagram.

Does A-DNA have any biological importance? Yes. A-DNA is observed in some biological context as follows. 
A ̴ 3 base pair segment of A-DNA is present at the active site of DNA polymerase.
Also, gram positive bacteria undergoing sporulation contain a high proportion (20%) of small acid soluble-spore proteins (SASPs). Some of these SASPs induce B-DNA to assume the A-form of DNA (atleast in vitro).
Another instance where A-form of DNA is present is during RNA-DNA hybrids. RNA-DNA hybrids do occur in the cell at different junctions for example, during replication (where replication is initiated by RNA primer) and also during transcription (where RNA is made on DNA template). These RNA-DNA hybrids cannot take up the B-form of DNA (because of the 2' oxygen of RNA) and hence, they resemble A-form of DNA.

Z-form of DNA:
Another form of DNA is Z-DNA and is called so because of its zigzag pattern of the phosphate backbone. The following are some of the characteristics:
i. The main characteristic is that Z-DNA is a left-handed double helix.
ii. The formation of Z-DNA is base sequence dependent (composition dependent). Only alternating purine and pyrimidine polymers can form Z-DNA. Thus, this zigzag forms the repeating unit of Z-DNA which is a dinucleotide. 
iii. The right handed DNA can be transited to left handed DNA (Z-DNA) in solutions that include high ionic strength, hydrophobic solvents, presence of certain trivalent cations or covalent modification with bulky alkylating agents.
iv. It contains approximately 12 base pairs per turn of the helix. 
v. Rise per turn of the helix is 44Å.
vi. The helix rises by 2.7Å per base pair. 
vii. The base pairs are perpendicular of the helix (just like that of B-DNA).
viii. Regarding the grooves in Z-DNA, it possesses only a minor groove. There is no discernible major groove and the minor groove is extremely deep and narrow.

The following table will give you an idea of major structural differences between three major forms of DNA (B-DNA, A-DNA and Z-DNA).

Ribonucleic Acid (RNA):
Coming to another type of nucleic acid which is RNA. As we have seen the major difference between RNA and DNA in previous post, here I will discuss about its types and structure. 
Types of RNA:
There are various kinds of RNAs. Here, the RNAs have been classified into two major groups as coding RNAs and non-coding RNAs. 
Coding RNA - mRNA
a. Coding RNAs:
mRNA (messenger RNA) is coding RNA that is involved in the process of translation in the cell. It is called messenger as it carries information from DNA to the ribosome which is the site of protein synthesis. The coding sequence of mRNA determines the amino acid sequence in the protein that is produced.

b. Non-coding RNAs:
Most of the RNAs do not code for any proteins and these types of RNA fall under another group as non-coding RNAs (ncRNAs). These so called non-coding RNAs can be encoded by their own genes. The most prominent examples are rRNA (ribosomal RNA) and tRNA (transfer RNA), both of which are involved in the process of translation.
Some other examples of noncoding RNAs are those involved in gene regulation and RNA processing. There are also some non coding RNAs that are able to catalyse chemical reactions such as cutting and ligating other RNA molecules and catalysis of peptide bond formation in ribosome. Such catalytic RNAs are called ribozymes

Structure of RNA:
The structure of RNA is described as follows:
Primary Structure of RNA:
Primary structure of RNA is somewhat similar to that of DNA; the only difference being the sugar (ribose) component which has an additional hydroxyl group at 2' carbon and thymine is replaced with uracil (see figure on the left).
Note: The hydroxyl group on the 2' carbon atom of ribose make it more labile as compared to DNA and it provides a chemically reactive group. Because of this lability, RNA is cleaved into mononucleotides by alkaline solution and DNA is not.
RNA is a polynucleotide chain that can be double stranded or single stranded, linear or cicular. 

Secondary Structure of RNA
There are various types of RNA exhibiting different conformations. The simplest secondary structures in single stranded RNAs are formed by pairing of complementary bases. Two major forms of secondary structures are hairpins and stem loops.
‘Hairpins’ are formed by pairing within approximately 5-10 nucleotides of each other whereas in stem loop, there is pairing of bases that are separated by approximately 50 to several hundreds of nucleotides.

Tertiary Structure of RNA:
These simple folds (hairpin and stem-loops) can cooperate to form more complicated tertiary structures, and one of such structure is 'pseudoknot' as can be seen in the adjacent figure.
tRNA is a well-defined example of tertiary structure of RNA. tRNA is a type of RNA that is involved in translation process of protein synthesis. They have a L-shaped 3D structure that allows them to fit into the P and A-sites of the ribosome. The figure depicted as tRNA above under non-coding RNAs is the three-dimensional structure of tRNA.

Here, we complete the nucleic acids topic. Any comments or doubts are welcome.!

Friday, April 19, 2013

Biomolecules of the Cell - Nucleic Acids (Part 1)

Nucleic acids are considered to be the building blocks of all the living organisms. The building blocks of nucleic acids are nucleotides. DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) are the nucleic acids. They can be described as the polymers of nucleotides linked through phosphodiester bonds. Don't worry, before going ahead and learning more about nucleic acids, we will go through the basics. Keeping in mind the basics, lets will start with nucleotides.

Bases, Nucleosides and Nucleotides:
Nucleotide has a distinctive structure and is composed of the following components which are bound together covalently:
a. Base (contains nitrogen) – This can be either a pyrimidine or a purine (explained below)
b. Sugar (5-carbon or pentose) – This can be either ribose or deoxyribose
c. A phosphate group
When a base and sugar is present (no phosphate), then it is called a nucleoside as can be seen in the adjacent figure.
When all the three components (base, sugar and phosphate) are bonded together, then this is known as nucleotide. Nucleotides can also exist in activated forms containing either two phosphates (diphosphate) or three phosphates (triphosphate).
When the sugar in the nucleotide is ribose, then the nucleotide is called ribonucleotide and when the sugar is deoxyribose, then nucleotide becomes deoxynucleotide.
Let us have a look at the structure of a nucleotide and make the concepts all the more clear. In this adjacent figure, the structure of deoxyadenosine monophosphate will depict all the three components as sugar, base (here, deoxyribose) and phosphate. In comparison, the diagram on the right has an extra hydroxyl group (-OH) on 2’ carbon atom of ribose sugar, making it ribonucleotide (instead of deoxyribonucleotide).  Also, in this diagram, note the 5’ and 3’ carbon atoms. If we understand these 5' and 3' carbon atoms, this will aid in understanding the polarity of the nucleic acids. The 5’ carbon atom is attached to the phosphate group while 3’ carbon atom is attached to a hydroxyl (-OH) group. 

There are five bases known as Adenine (A), Guanine (G), Cytosine (C), Uracil (U) and Thymine (T). The point to remember is that Uracil is not present in DNA but present in RNA while Thymine is not present in RNA, but present in DNA. Here is the table showing all the five bases with their structure, abbreviations and their nucleoside and nucleotide forms.
Another way to categorize nucleotide bases is as ‘purines’ and ‘pyrimidines’. Purines include A and G (which are double-ring members) while pyrimidines include the remaining T, C and U (which are single-ring members). A point to remember is that in double-stranded nucleic acids, the pairing is always between a purine and a pyrimidine.

The polynucleotides are polymeric compounds consisting of 15 or more nucleotide monomers covalently bonded together in a chain. As we have seen above, the carbon atom in the sugar is numbered 1’ to 5’. The hydroxyl (–OH) group on the 3’ carbon of one nucleotide can react with the phosphate attached to the 5’ carbon of another (adjacent) nucleotide to from a dinucleotide held together by phosphate ester bonds. These bonds are also called phosphodiester bonds. As the chain contributes more and more nucleotides to itself; it begins to grow and hence, becomes a polynucleotide.
A short segment in the adjacent figure will make it all the more clear. Remember that DNA is read from 5’ end to the 3’ end. If we read this DNA segment, then the sequence goes like this – adenine (A), cytosine (C), guanine (G) and thymine (T).

DNA and RNA are the main polynucleotides. First, we will have a look at DNA.

DNA (Deoxyribonucleic Acid):
DNA is the molecule that encodes the blueprint of an organism meaning the DNA contains all the information required to build up and maintain an organism. Let me ask you one question, by whom was DNA first discovered? You might be thinking, Watson and Crick..!! Let me make it clear to you, the answer here is NO. The DNA was first discovered in 1868 by Swiss-physician Friedrich Miescher. He isolated a compound from the nuclei of white blood cells. This compound was neither a protein, nor a carbohydrate, nor a lipid but a unique type of biomolecule. Miescher named it ‘nuclein’ as he had isolated it from the nuclei of the cell. Today, this molecule is called DNA.

Erwin Chargaff was a biochemist who did an extensive research on chemical analysis of the base composition of DNA and stated that the base composition of DNA varied from organism to organism but was independent of age, sex, nutritional status or any other environmental factors. He came up with certain relationships which were called the 'Chargaff’s rule' and they are as follows:
a. The amount of adenine is always equal to the amount of thymine (A=T).
b. The amount of guanine is always equal to the amount of cytosine (G=C).
c. The amount of A+G is 50% of the total amount of bases in the molecule.
d. The amount of T+C is 50% of the total amount of bases in the molecule.

 Then, what was the contribution of Watson and Crick. So, here lies the answer - in 1953, James D. Watson and Francis Crick described the molecular structure and shape of DNA.
When the counterion is Na+ and the relative humidity is 92%, then DNA fibers assume what is known as “B conformation”. I would like to jot some points/features of B form of DNA from my HOD, Dr. Avinash Upadhyay’s book “Molecular Biology” because they are so beautifully explained in the book that I still remember them even after years. 
Watson and Crick described the shape of DNA as ‘double helix’ and following are the features of B-DNA:
i. Double-stranded helix: DNA consists of two polynucleotide strands coiled around each other to form a double stranded helix.
ii. Plectonomic coil: The two strands are coiled around each other in such a way that they cannot be separated without unwinding the helix (plectonomic coil).
iii. Antiparallel: The two strands are antiparallel to one another meaning they run in opposite directions. Both the strands have one 5' phosphate terminus and one 3’ hydroxyl (-OH) terminus. Antiparallel strands mean that the 5’ terminus of one strand is adjacent to 3’ adjacent of another strand and vice-versa (as can be seen in the adjacent figure).
iv. Right handed helix: The helix is a right-handed helix.
v. Diameter: The diameter of the helix is 20Å.
vi. Sugar phosphate backbone - Hydrophilic: The sugar-phosphate backbones of both the strands are hydrophilic in nature and follow the helical path and are towards the outer edge of the molecule where they will be able to interact with the aqueous environment.
vii. Bases - Hydrophobic: The bases are hydrophobic in nature and they are placed at the interior of the helix.
viii. Planar base pairs: The planes of the bases are perpendicular to helical axis. Each base is hydrogen bonded to a base on the opposite strand to from a planar base pair. Two types of purine-pyrimidine base pairs can occur as A..T and C...G. There are two hydrogen bonds between A and T and three hydrogen bonds between C and G. This is the reason why the helix has a constant diameter of 20Å.
xi. Complementary base pairing: This specificity of the base pairing is referred to as ‘complementary base pairing’.
x. Thickness: The bases have a Vander Waal’s thickness of 3.4Å.
xi. Base pairs per turn: There are 10 base pairs per turn of the helix and hence, the helix rises by 34Å per turn.
xii. Rotation per base pair: If there are 10 base pairs per turn, then each base pair rotates by 36˚ (as one rotation is 360˚ and divided by 10 will give 36˚) and hence, a 36˚ turn per base pair is present.
xiii. Major and minor grooves: The angle of the glycosidic linkages lead to the formation of two external helical grooves. One of the groove is deep and wide and is called the major groove. Another groove is shallow and narrow. This is the minor groove.

This was all about B-form of DNA. There are some other forms of DNA as well, which we will discuss in the next post along with RNA.

Tuesday, April 16, 2013

Biomolecules of the Cell - Carbohydrates (Part 2)

In the previous post, we have completed the introduction of carbohydrates and the characteristics of monosaccharides. In this post, we will discuss about characteristics of disaccharides, oligosaccharides and some polysaccharides. 
Let us first understand the disaccharides.

A. Disaccharides:

Disaccharides are formed by the condensation reaction (or dehydration reaction) of two monosaccharide units and water is the by-product of this reaction (as can be seen in the adjacent figure). The covalent linkage that is formed between two monosaccharides is called O-glycosidic bond and it represents the formation of an hemiacetal from an aldehyde and an alcohol. Similarly, a hemiketal is formed from a ketone and an alcohol.
In the adjacent figure (right side), is the disaccharide, maltose, the linkage is α(1-4) linkage. We can see the hemiacetal and acetal ends in the figure.

Types of Disaccharides (Sugars):
a. Reducing and Non-reducing Sugars:
The disaccharides are classified as reducing disaccharide (or sugar) and non-reducing disaccharide (or sugar). The disaccharides that have hemiacetals, are grouped under reducing sugar. Hemiacetals contain a free aldehyde group which can be oxidized into carboxylic acids (or diverse products). Thus, these types of sugars are reducing in nature, hence, they are called reducing sugars.
Another type is non-reducing sugars (disaccharides). Here, the sugar or the disaccharide in an acetal or ketal which cannot be oxidized readily and neither monosaccharide has a free hemiacetal unit. This is so, because both of its anomeric carbon atoms are involved in glycosidic linkage.

In the figure, the glucose on the right is designated as the reducing end of the disaccharide molecule as it can participate in a reduction reaction. In contrast, the glucose on the left represents the non-reducing end as the C-1 carbon atom is the part of the  α(1-4) linkage and it cannot form the open chain. Thus, as maltose contains one reducing end, it is called, reducing sugar. 
b. Properties of Disaccharides: 
The glycosidic bond can be formed between the hydroxyl groups on its component monosaccharide. So, even if both monosaccharides (forming a disaccharide) are the same (e.g., glucose), different bond combinations which is regiochemistry and stereochemistry (like alpha- or beta-) result in disaccharides that are isomers of each other. These are diastereoisomers with different chemical and physical properties.
For example, both maltose and cellobiose are the disaccharides of glucose monomers. However, maltose is the disaccharide with α(1-4) linkage between C1 hydroxyl of one glucose and C4 hydroxyl of another glucose. The configuration is 'α' because the O at the anomeric carbon atom points down from the ring. On the other hand, cellobiose, is the disaccharide with β(1-4) linkage but here, the configuration is β as O points up from the ring. (This β glycosidic linkage is generally depicted by a zig-zag line; however, one glucose molecule is actually flipped over relative to the other).

c. Nomenclature:
As per the standard conventions, the disaccharide is named such that first listing is that of non-reducing monosaccharide on the left followed by glycosidic linkage between the two monosaccharides and then the monosaccharide on the right. For example, with this nomenclature, maltose can be described as Glc(α1-4)Glc (where Glc stands for glucose).
Here is the table of some common reducing and non-reducing disaccharides with their glycosidic linkages.

Reducing Disaccharides
Unit 1
Unit 2
Either α(1-2)
-3), α(1-4) or

Non-reducing Disaccharides

B. Oligosaccharides:
Most of the oligosaccharides are not found as isolated molecules. Instead, they may be attached to other biomolecules like proteins or lipids, generally referred to as glycoconjugates. For example, the blood group serotypes (A, B, AB and O) are the result of various oligosaccharides involved in cellular recognition. The lipids on the surface of the erythrocytes are conjugated with various oligosaccharides.

C. Polysaccharides:
Polysaccharides are long chains of monosaccharides joined together by glycosidic bonds (linkages). As mentioned in previous post, polysaccharides maybe branched or unbranched. When all the monosaccharides in a polysaccharide are of the same type, the polysaccharide is called a homopolysaccharide or homoglycan, but when more than one type of monosaccharide is present, then they are called heteropolysaccharides or heteroglycans.
Here, we are going to see about some polysaccharides:
a. Cellulose: It is the most abundant (structural) polysaccharide on the earth. It is a straight chain homopolymer consisting of thousands of glucose moieties attached together by β(1-4) glycosidic linkage present in plants. It is a polymer of cellobiose units (repeats of Glcβ(1-4)Glc) as can be seen in the figure. Humans and many other animals lack the enzyme cellulose which is required to hydrolyze β-glycosidic linkages.
Many hydroxyl groups on the glucose molecules from one chain form hydrogen bonds with the oxygen atoms on the same or neighboring chain thereby holding the chains firmly together side-by side forming microfibrils giving high tensile strength.

b. Chitin: Chitin is to animal kingdom what cellulose is to plant kingdom. It is another abundant linear polysaccharide that forms the structural components of many invertebrates exoskeletons of insects and crustaceans. It is a polymer of units of N-acetyl glucosamine (abbreviated as NAG or GlcNAc) which is linked by β(1-4) glycosidic bond. The only difference between the structure of glucose and N-acetyl-glucosamine is the replacement of the C-2 hydroxyl group with that of an acetylated amino group. This allows for increased hydrogen bonding between adjacent polymers giving chitin more strength than cellulose.

c. Starch: Starch is the homopolymer in which the glucose units are linked via alpha linkages. It is made up of amylose (15-20%) and amylopectin (80-85%). Amylose is a linear polysaccharide linked by α(1-4) linkages while amylopectin is a branched polysaccharide connected by α(1-4) and α(1-6) linkages. The linear linkage is α(1-4) while the branched linkage is α(1-6) between glucose residues which greatly increases the number of free ends in the homopolymeric molecule. The branch points occur in chain after every 20-30 residues. Being the alpha linkages, these can easily be hydrolyzed by alpha amylase which cleaves α(1-4) glycosidic bonds.

d. Glycogen: What starch is to plants, glycogen is to animals and human. The structure is similar to amylopectin meaning the linear, glucose molecules are linked together by α(1-4) glycosidic bond and the branches are linked to these linear chains branching off from α(1-6) glycosidic bond between first glucose of new branch and a glucose on the stem branch.
There are certain differences between starch (amylopectin, more specifically) and glycogen. One of them is that of branching. In glycogen, the branching occurs more frequently  i.e.; branch point is after every 6-10 residues.
The glucose units can be added or removed only from the non-reducing ends of amylopectin and glycogen. The more branch points, the more ends are available for glucose retrieval and storage. Another difference is between the macromolecular structures of both. Amylopectin contains one free glucose at the reducing end of the 'tree branch' whereas glycogen lacks a free reducing end. This is because the glucose residue at the center of the glycogen 'spiral' is covalently linked to a protein called glycogenin (see adjacent figure).

e. Heparin: Heparin is a polysaccharide that is heteropolysaccharide with anti-clotting properties. It has medicinal value for surgery and is used to treat thrombosis. Heparin is found in arterial walls where it facilitates interactions between antithrombin (an inhibitor of blood coagulation) and thrombin (a clot-forming protein).