3

THE MOLECULAR BASIS OF GENETICS

Professor Alfred Cuschieri

Deartment of Anatomy

University of Malta

Msida MSD06

OBJECTIVES:

By the end of this session the student should be able to:

* Outline the molecular structure of DNA and RNA

* Explain the significance of specific base pairing

* Define the main properties of DNA

* Work out the sequence on m-RNA from that on the coding and transcribed strands of DNA

* Define transcription and translation and state where they occur

* Distinguish between single- stranded and double-stranded nucleic acids

* Explain the meaning of denaturation and re-annealing of DNA

NUCLEOTIDES

The molecules of Deoxyribose Nucleic Acid (DNA) and Ribose Nucleic Acid (RNA) are common to all living organisms and form the basis of life. Both molecules consist of long chains of nucleotides.

A nucleotide consists of three main components (Fig 3.1):

a) a pentose sugar consisting of a five-sided ring containing 5 carbon atoms, numbered clockwise from 1 to 5. This sugar is ribose in RNA or deoxyribose in DNA,

b) a phosphate group (PO₄^--) attached to C5 of the pentose ring.

c) a nitrogenous base attached to C1 of the pentose ring. The bases present in DNA are Adenine (A), Guanine (G), Cytosine (C), and Thymine (T). In RNA Uracil (U) replaces Thymine.

A nucleoside consists of a ribose sugar with an attached base. Addition of a phosphate group to the nucleoside forms a nucleotide. Thus there are five nucleotides that form the building blocks of DNA and RNA - Adenine, guanine, cytosine, thymine and uracil nucleotides.

A hydroxyl group (OH), situated at C3 of the pentose ring, is the reactive site where one nucleotide interacts with the phosphate group of another nucleotide to form phosphodiester bond (fig. 3.2).

Di-nucleotides and tri-nucleotides consist of two and three nucleotides respectively. Poly-nucleotides, such as DNA and RNA consists of long chains of nucleotides.

A polynucleotide chain has a distinct polarity. It has a PO₄ group attached to the C5 at one end - this is termed the 5’ end; and an -OH group attached to C3 at the other end - this is termed the 3’ end. New nucleotides may be added to a nucleotide chain only at the 3’ end. Thus, increase in length of a nucleotide chain is always unidirectional and occurs only in the 5’ to 3’ direction

The bases present in DNA and RNA are divided into two groups (Fig3.3).

1. Adenine and guanine are purines and consist of two nitrogenous rings. They differ in the position of the amino group (NH₂).

2. Cytosine, thymine and uracil are pyrimidines and consist of one nitrogenous ring. They differ in the nature and position of the side groups that are NH₂ and CH₃ respectively.

Specific base pairing occurs between nucleotides on opposite strands of DNA (Fig. 3.4). Thymine on one strand is always linked to adenine on the other strand by two hydrogen bonds. Cytosine on one strand is always linked to Guanine on the other strand by three hydrogen bonds. Specific base pairing is one of the most important features of DNA. It is crucial in the processes of DNA replication and transcription, which are two of the main functions of this molecule.

DNA

The DNA molecule consists of two very long nucleotide chains (or strands) arranged in the form of a double spiral with the following properties:

* the two strands are antiparallel i.e. the 5' end of one strand lies next to the 3' end of the complementary strand.

* the two strands are twisted in the form of a double spiral

* the bases on one strand are linked to corresponding bases on the other strand by hydrogen bonds

RNA

RNA is similar to DNA in that it consists of a polynucleotide chain, but differs from it in the following respects: (a) it consists of only one strand; (b) the pentose sugar is ribose; (c) uracil replaces thymine.

DIMENSIONS OF DNA

The double helical molecule of DNA has a diameter of 2 nanometers (nm). One spiral turn covers a length of 3.4 nm and contains 10 base pairs. (Fig. 3.5)

Note: 1 nm = 10^-9m. (In old terminology, molecular dimensions were measured in Ǻngstrom Units (Ǻ) where 1 nm=10Ǻ.)

PROPERTIES OF DNA

The unique structure of the DNA molecule explains its main properties and functions as the basis of life:

1. DNA REPLICATES ITSELF

DNA replication is the process by which a DNA molecule makes an exact copy or replica of itself (Fig. 3.6). The two strands forming the DNA molecule separate, and each strand then serves as a template for synthesis of a complementary chain of nucleotides. This function is possible because of specific base pairing. Adenine on one strand always pairs with Thymine on the other (A - T), and Cytosine on one strand always pairs with Guanine on the other (C - G). Specific base pairing ensures that the sequence of bases is preserved on both daughter DNA molecules. DNA replication is the basis of propagation of life.

2. DNA CARRIES THE GENETIC MESSAGE.

The sequence of bases on a stretch of a DNA forming a gene carries a coded message for a protein molecule. The complete DNA sequence of an organism is its genome, which contains all the genes coding for all the proteins present in that organism. The number of sequences contained in the genome varies according to the organism. The genome of a virus contains only a few thousand base pairs, forming the genes that code for a few proteins. More complex organisms have larger genomes and in man the genome contains 3 billion (3x10⁹) base pairs. The size of the genome, however, is not proportional to the complexity of the organism. The sizes of the genomes of some organisms are shown in Table 3.1

Table 3.1. Sizes of genomes in a various organisms
Organism	Number of base pairs
SV40 virus	5,226
Escherichia coli, a bacterium	4.1 x10⁶
Salmonella typhimurium, a bacterium	1.1x10⁷
Saccharomyces cerevisiae, a yeast	1.75x10⁷
Zea mays (maize)	6.6x10⁹
Lilium longiflorum (lily)	3x10¹¹
Cyanorhabditis elegans, a worm	9x10⁷
Drosophila melanogoster, the fruit fly	1.75 x 10⁸
Mus musculus, the mouse	2.2 x 10⁹
Homo sapiens, Man	3 x 10⁹

3. DNA CAN REPAIR ITSELF

When some bases on one strand of DNA are damaged or lost, the complementary strand serves as a template for replacement of the damaged nucleotides ensuring that the exact sequence is preserved. This would be impossible with a single-stranded molecule. When the two strands of DNA become separated, the complementary strands can find each other even in a complex mixture of molecules. They can join up once again by formation of hydrogen bonds. This is possible because of the specific base pairing.

4. DNA IS UNIVERSAL.

In all living organisms, whether bacteria, plants, animals or man, the structure of DNA is basically the same. Only the sequence of bases differs and consequently the genetic message. The resulting proteins and their specific functions are also different. However, because the basic structure of DNA is the same, DNA from one organism can be grafted on to the DNA of another organism. This is the basis of genetic engineering. DNA is the genetic material of prokaryotic and eukaryotic organisms but some viruses have RNA and others single-stranded DNA as their genetic material. However, the principles of replication and transcription remain the same.

The Genetic code

The genetic message is read in units of three bases, called codons. Each codon (triplet of bases) represents an amino acid. A chain of amino acids forms a specific polypeptide or protein, which is responsible for a particular function in a cell. Only one of the strands of DNA carries the genetic message. This is known as the template or transcribed strand because it forms the template from which the message is transcribed. The other strand is not transcribed and is known as the complementary or coding strand.

The template strand is transcribed to messenger RNA (m-RNA), which then itself acts as a template for the assembly of a polypeptide chain (Fig. 3.7) The template strand is read in the 3' to 5' direction while the m-RNA is synthesised in the 5' to 3' direction. The coding strand of DNA is never transcribed. Its sequence, however, happens to be the same as that on m-RNA (with the exception that T on DNA is replaced by U on RNA). This is why it is termed the "coding" strand. This nomenclature may be rather confusing and one should spend some time to ensure that the concept of complimentary sequences is understood.

TRANSCRIPTION AND TRANSLATION

This section explains how the coded message in DNA results in the synthesis of a specific protein. The process involves two main steps, namely transcription and translation. (Fig. 3.7)

Transcription

Transcription is the process in which the genetic message on the transcribed strand of DNA is copied on to m-RNA. The sequence of bases on DNA acts as a template for the synthesis of corresponding bases on m-RNA. Transcription proceeds in the 5’ to 3’ direction i.e. nucleotides are added at the 3’ end of the growing m-RNA. Note, however, that the coded message on DNA is read in the 3' to 5' direction. The enzyme RNA polymerase is necessary for this process, which occurs in the nucleus. The m-RNA moves out of the nucleus into the cytoplasm.

TRANSLATION

Translation is the process in which the m-RNA attaches itself to the ribosomes and acts as a template for the synthesis of a specific protein. The sequence of bases on m-RNA are read in triplets of bases termed codons, which determine the sequence of amino acids forming the protein.

THREE TYPES OF RNA

The processes of transcription and translation involve three different types of RNA as intermediaries, each having different functions: messenger RNA (m-RNA), ribosomal RNA (r-RNA) and transfer RNA (t-RNA).

m-RNA

m-RNA is formed in the nucleus by transcription of a segment of DNA corresponding to a particular gene. The sequence of bases on m-RNA is complementary to that on the template strand of DNA and not a direct copy of it. m-RNA (representing a particular gene) detaches itself from the DNA and moves out from the nucleus into the cytoplasm. In the cytoplasm it attaches itself to a ribosome where it acts as a template for the synthesis of a protein (Fig. 3.8).

r-RNA

r-RNA is the main component of ribosomes, which also contain a number of associated proteins. Ribosomes are the sites of protein synthesis. There are four different r-RNAs. They all consist of single stranded RNA molecules coiled upon themselves.

Ribosomes consist of two sub-units, one large sub-unit and one small sub-unit (Fig. 3.8). The m-RNA inserts itself between the large and the small sub-units of the ribosomes. The ribosomes provide the synthetic machinery for the assembly of amino acids to form a protein using m-RNA as a template. Here each codon (triplet of bases) on m-RNA serves as a template for a corresponding amino acid.

t-RNA

t-RNA is a special type of RNA which binds to a specific amino acid and transfers it to the growing polypeptide chain (Fig 3.9). It consists of a single strand of RNA folded to form three loops. It has, at the 3’ end of the RNA, the attachment site for a specific amino acid. On the second loop there is a triplet of bases, which are complementary to the codon corresponding to the amino acid, called an anticodon. The anticodon on t-RNA recognizes a codon on m-RNA and transfers the appropriate amino acid to the growing polypeptide chain in the ribosome. The t-RNA must first be activated by combining with the amino acid and with GTP to provide energy for the chemical reaction.

TRANSLATION: PROTEIN SYNTHESIS

The complex process of translation leading to protein synthesis is summarized below and illustrated graphically in Fig. 3.10:

1. m-RNA inserts itself in between the large and the small sub-units of the ribosomes starting at the 5’ end; translation proceeds in 5’ to 3’ direction. In this example three codons are shown (CAU - AGU - UAU)

2. The codons are exposed, one at a time at the "A" (aminoacyl) site of the ribosome; here the codon CAU occupies the site - this codon codes for the amino acid histidine (his).

3. A t-RNA having an anticodon (GUC) at one end and the amino acid histidine at the other, attaches to the exposed codon (CAU) and transfers the corresponding amino-acid

4. The t-RNA detaches itself and m-RNA moves to expose the next codon (AGU). The t-RNA having the corresponding anticodon (UCA) and the amino acid (serine) attaches to the codon and transfers its amino acid to the growing polypeptide chain. GTP (a substance similar to ATP) provides energy for this process.

5. Another t-RNA is shown ready for the next codon and the cycle is repeated.

***********************

SINGLE-STRANDED AND DOUBLE- STRANDED NUCLEIC ACIDS

Biochemical analysis can be used to estimate the relative quantities of bases in nucleic acids. The proportions of bases (adenine, guanine, thymine and cytosine) vary in different specimens of DNA. However, in all double- stranded DNA, because of the specific base pairing (A - T and C - G) the proportion of thymine is equal to that of adenine and the proportion of cytosine is equal to that of guanine. In contrast, in single-stranded polynucleotides, whether RNA or single-stranded DNA, the proportion of the base is not equal. This is an important distinguishing feature between single- stranded and double- stranded nucleic acids. The presence of uracil and the absence of thymine distinguish RNA from DNA.

DENATURATION AND RENATURATION OF DNA

When a solution of DNA is heated slowly to about 100⁰C the H-bonds, which bind the two strands of DNA are broken and single-stranded DNA molecules are formed. This is called denaturation or melting of DNA. If this solution is then cooled rapidly the DNA remains denatured or single-stranded. However, if the solution is reheated and cooled gradually, double stranded DNA will again be formed. Through specific base pairing the single strands can identify their complementary strand and H-bonds are reformed. This is called renaturation or annealing of DNA.

PROBLEMS

Problem 1.

If the sequence of bases on the template strand of DNA is 3'-A G C A T T C G A G A T -5', what is the sequence on the coding strand of DNA?

Solution

The sequence of bases on the coding strand of DNA is worked out using specific base pairing. The two strands are antiparallel; therefore the complementary strand is in the 5' to 3' direction.

template strand: 3'-A G C A T T C G A G A T -5'

coding strand: 5'-T C G T A A G C T C T A -3'

Problem 2.

On analysis a sample of DNA is found to contain 30% adenine. What is its percentage content of the other three bases?

Solution

Because of specific base pairing an equal amount (30%) must be thymine. Therefore A-T forms 60% of the bases. The remaining 40% must be formed by C and G which must be present in equal amounts of 20% each.

Adenine 30% - Thymine 30%; Guanine 20% - Cytosine 20%.