Biology

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Genetic Code and Its Mutation

DNA is the genetic material that carries genetic information in a cell and from generation to generation. At this stage, an attempt will be made to determine in what manner the genetic information exists in DNA molecule? Are they written in coded language on a DNA molecule? If they occur in the language of codes what is the nature of genetic code?

The translation of proteins follows the triplet rule; a sequence of three mRNA base (a codon) designates one of the 20 different kinds of amino acids used in protein synthesis. Genetic code is the sequence relationship between nucleotide in genes (or mRNA) and the amino acids in the proteins they encode.

There are 64 possible triplets, and 61 of them are used to represent amino acids. The remaining three triplet codons are termination signals for polypeptide chains. Since there are only 20 amino acids involved in protein synthesis, most of them are encoded by more than one triplet. Two things make this multiple (degenerate) coding possible. First, there is more than one tRNA for most amino acids.

Each tRNA has a different anticodon. Second, this pairing is highly specific for the first two portions on the codon, permitting Watson and Crick base pairs (A – U and G – C) to be formed. But at the third position there is a great deal of flexibility as to which base pairs are acceptable. Most part of the genetic code is universal, being the same in prokaryotes and eukaryotes.

The order of base pairs along DNA molecule controls the kind and order of amino acids found in the proteins of an organism. This specific order of base pairs is called genetic code, the blue print establishing the kinds of proteins to be synthesized which makes and organism unique.

Marshall Nirenberg, Severo Ochoa (enzyme polynucleotide phosphorylase called Ochoa’s enzyme), Hargobind Khorana, Francis Crick and many others have contributed signifiantly to decipher the genetic code. The order in which bases are arranged in mRNA decides the order in which amino acids are arranged in proteins. Finally a checker board for genetic code was prepared (table 5.1). The salient features of genetic code are as follows:

1. The genetic codon is a triplet code and 61 codons code for amino acids and 3 codons do not code for any amino acid and function as stop codon (Termination).

2. The genetic code is universal. It means that all known living systems use nucleic acids and the same three base codons (triplet codon) direct the synthesis of protein from amino acids. For example, the mRNA (UUU) codon codes for phenylalanine in all cells of all organisms. Some exceptions are reported in prokaryotic, mitochondrial and chloroplast genomes. However similarities are more common than differences.

3. A non-overlapping codon means that the same letter is not used for two different codons. For instance, the nucleotide sequence GUU GUC represents only two codons.

4. It is comma less, which means that the message would be read directly from one end to the other i.e., no punctuation are needed between two codes.

5. A degenerate code means that more than one triplet codon could code for a specific amino acid. For example, codons GUU, GUC, GUA and GUG code for valine.

6. Non-ambiguous code means that one codon will code for one amino acid.

7. The code is always read in a fixed direction i.e. from 5′ → 3′ direction called polarity.

8. AUG has dual functions. It acts as a initiator codon and also codes for the amino acid methionine.

9. UAA, UAG and UGA) codons are designated as termination (stop) codons and also are known as “non sense” codons.

Mutation and genetic code

Comparative studies of mutations (sudden change in a gene) and corresponding alteration in amino acid sequence of specific protein have confirmed the validity of the genetic code. The relationship between genes and DNA are best understood by mutation studies.

The simplest type of mutation at the molecular level is a change in nucleotide that substitutes one base for another. Such changes are known as base substitutions which may occur spontaneously or due to the action of mutagens. A well studied example is sickle cell anaemia in humans which results from a point mutation of an allele of β-haemoglobin gene (βHb).

A haemoglobin molecule consists of four polypeptide chains of two types, two α chains and two β-chains. Each chain has a heme group on its surface. The heme groups are involved in the binding of oxygen. The human blood disease, sickle cell anaemia is due to abnormal haemoglobin. This abnormality in haemoglobin is due to a single base substitution at the sixth codon of the beta globin gene from GAG to GTG in β – chain of haemoglobin. It results in a change of amino acid glutamic acid to valine at the 6^th position of the β – chain.

This is the classical example of point mutation that results in the change of amino acid residue glutamic acid to valine (Fig. 5.10). The mutant haemoglobin undergoes polymerisation under oxygen tension causing the change in the shape of the RBC from biconcave to a sickle shaped structure. The effect of point mutation can be understood by the following example.

ABC DEF GHI JKL

If we insert a letter O between DEF and GHI the arrangement would be

ABC DEF OGH IJK L

If we insert OQ at the same place the arrangement would be

ABC DEF OQG HIJ KL

The above information shows that insertion or deletion of one or two bases, changes the reading frame from the point of insertions or deletions. Such mutations are referred to as frame shift insertion or deletion mutations. This forms the genetic basis of proof that codon is a triplet and is read in a continuous manner.

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Process Of Transcription and Its Unit Gene

Francis Crick proposed the Central dogma of protein synthesis in molecular biology states that genetic information flows as follows:

The process of copying genetic information from one strand of DNA into RNA is termed transcription. This process takes place in presence of DNA dependent RNA polymerase. In some retroviruses that contain RNA as the genetic material (e.g, HIV), the flow of information is reversed. RNA synthesizes DNA by reverse transcription, then transcribed into mRNA by transcription and then into proteins by translation.

For a cell to operate, its genes must be expressed. This means that the gene products, whether proteins or RNA molecules must be made. The RNA that carries genetic information encoding a protein from genes into the cell is known as messenger RNA (mRNA). For a gene to be transcribed, the DNA which is a double helix must be pulled apart temporarily, and RNA is synthesized by RNA polymerase.

This enzyme binds to DNA at the start of a gene and opens the double helix. Finally, RNA molecule is synthesized. The nucleotide sequence in the RNA is complementary to the DNA template strand from which it is synthesized.

Both the strands of DNA are not copied during transcription for two reasons. 1. If both the strands act as a template, they would code for RNA with diffrent sequences. This in turn would code for proteins with different amino acid sequences.

This would result in one segment of DNA coding for two different proteins, hence complicate the genetic information transfer machinery. 2. If two RNA molecules were produced simultaneously, double stranded RNA complementary to each other would be formed. This would prevent RNA from being translated
into proteins.

Transcription unit and gene

A transcriptional unit in DNA is defined by three regions, a promoter, the structural gene and a terminator. The promoter is located towards the 5′ end of the coding strand. It is a DNA sequence that provides binding site for RNA polymerase. The presence of promoter in a transcription unit, defies the template and coding strands.

The terminator region located towards the 3′ end of the coding strand contains a DNA sequence that causes the RNA polymerase to stop transcribing. In eukaryotes the promoter has AT rich regions called
TATA box (Goldberg-Hogness box) and in prokaryotes this region is called Pribnow box. Besides promoter, eukaryotes also require an enhancer.

The two strands of the DNA in the structural gene of a transcription unit have opposite polarity. DNA dependent RNA polymerase catalyses the polymerization in only one direction, the strand that has the polarity 3′ → 5′ acts as a template, and is called the template strand.

The other strand which has the polarity 5′ → 3′ has a sequence same as RNA (except thymine instead of uracil) and is displaced during transcription. This strand is called coding strand (Fig. 5.7).

The structural gene may be monocistronic (eukaryotes) or polycistronic (prokaryotes). In eukaryotes, each mRNA carries only a single gene and encodes information for only a single protein and is called monocistronic mRNA. In prokaryotes, clusters of related genes, known as operon, oftn found next to each other on the chromosome are transcribed together to give a single mRNA and hence are polycistronic.

Before starting transcription, RNA polymerase binds to the promoter, a recognition sequence in front of the gene. Bacterial (prokaryotic) RNA polymerase consists of two major components, the core enzyme and the sigma subunit. The core enzyme (2α, β, β¹ and ω) is responsible for RNA synthesis whereas a sigma subunit is responsible for recognition of the promoter. Promoter sequences vary in different organisms.

RNA polymerase opens up the DNA to form the transcription bubble. The core enzyme moves ahead, manufacturing RNA leaving the sigma subunit behind at the promoter region. The end of a gene is marked by a terminator sequence that forms a hair pin structure in the RNA. The sub-class of terminators require a recognition protein, known as rho (ρ), to function.

Process of transcription

In prokaryotes, there are three major types of RNAs: mRNA, tRNA, and rRNA. All three RNAs are needed to synthesize a protein in a cell. The mRNA provides the template, tRNA brings amino acids and reads the genetic code, and rRNAs play structural and catalytic role during translation. There is a single DNA-dependent RNA polymerase that catalyses transcription of all types of RNA.

It binds to the promoter and initiates transcription (Initiation). The polymerases binding sites are called promoters. It uses nucleoside triphosphate as substrate and polymerases in a template depended fashion following the rule of complementarity. After the initiation of transcription, the polymerase continues to elongate the RNA, adding one nucleotide after another to the growing RNA chain.

Only a short stretch of RNA remains bound to the enzyme, when the polymerase reaches a terminator at the end of a gene, the nascent RNA falls off so also the RNA polymerase. The question is, how the RNA polymerases are able to catalyse the three steps initiation, elongation and termination? The RNA polymerase is only capable of catalyzing the process of elongation.

The RNA polymerase associates transiently with initiation factor sigma (σ) and termination factor rho (σ) to initiate and terminate the transcription, respectively. Association of RNA with these factors instructs the RNA polymerase either to initiate or terminate the process of transcription (Fig. 5.8).

In bacteria, since the mRNA does not require any processing to become active and also since transcription and translation take place simultaneously in the same compartment (since there is no separation of cytosol and nucleus in bacteria), many times the translation can begin much before the mRNA is fully transcribed. This is because the genetic material is not separated from other cell organelles by a nuclear membrane
consequently; transcription and translation can be coupled in bacteria.

In Eukaryotes, there are at least three RNA polymerases in the nucleus (in addition to RNA polymerase found in the organelles). There is a clear division of labour. The RNA polymerase I transcribes rRNAs (28S, 18S and 5.8S), whereas the RNA polymerase III is responsible for transcription of tRNA, 5S rRNA and snRNA.

The RNA polymerase II transcribes precursor of mRNA, the hnRNA (heterogenous nuclear RNA). In eukaryotes, the monocistronic structural genes have interrupted coding sequences known as exons (expressed sequences) and noncoding sequences called introns (intervening sequences). The introns are removed by a process called splicing.

hnRNA undergoes additional processing called capping and tailing. In capping an unusual nucleotide, methyl guanosine triphosphate is added at the 5′ end, whereas adenylate residues (200-300) (Poly A) are added at the 3′ end in tailing (Fig. 5.9). Thereafter, this processed hnRNA, now called mRNA is transported out of the nucleus for translation.

The split gene feature of eukaryotic genes is almost entirely absent in prokaryotes. Originally each exon may have coded for a single polypeptide chain with a specific function. Since exon arrangement and intron removal are flexible, the exon coding for these polypeptide subunits act as domains combining in various ways to form new genes.

Single genes can produce different functional proteins by arranging their exons in several different ways through alternate splicing patterns, a mechanism known to play an important role in generating both protein and functional diversity in animals. Introns would have arosen before or after the evolution of eukaryotic gene.

If introns arose late how did they enter eukaryotic gene? Introns are mobile DNA sequences that can splice themselves out of, as well as into, specific ‘target sites’ acting like mobile transposon-like elements (that mediate transfer of genes between organisms – Horizontal Gene Transfer HGT). HGT occurs between lineages of prokaryotic cells, or from prokaryotic to eukaryotic cells and between eukaryotic cells. HGT is now hypothesized to have played a major role in the evolution of life on earth.

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Packaging Of DNA Helix

The distance between two consecutive base pairs is 0.34nm (0.34 × 10^-9 m) of the DNA double helix in a typical mammalian cell. When the total number of base pairs is multiplied with the distance between two consecutive base pairs (6.6 × 10⁹ × 0.34 × 10^-9 m/bp), the length of DNA double helix is approximately 2.2 m. (The total length of the double helical DNA = total number of base pairs × distance between two consecutive base pairs).

If the length of E. coli DNA is 1.36 mm, the number of base pairs in E. coli is 4 × 10⁶bp (1.36 × 10³ m/0.34 × 10^-9). The length of the DNA double helix is far greater than the dimension of a typical mammalian nucleus (approximately 10^-6 m). How is such a long DNA polymer packaged in a cell?

Chromosomes are carriers of genes which are responsible for various characters from generation to generation. Du Praw (1965) proposed a single stranded model (unineme), as a long coiled molecule which is associated with histone proteins in eukaryotes. Plants and animals have more DNA than bacteria and must fold this DNA to fit into the cell nucleus.

In prokaryotes such as E. coli though they do not have defined nucleus, the DNA is not scattered throughout the cell. DNA (being negatively charged) is held with some proteins (that have positive charges) in a region called the nucleoid. The DNA as a nucleoid is organized into large loops held by protein. DNA of prokaryotes is almost circular and lacks chromatin organization, hence termed genophore.

In eukaryotes, this organization is much more complex. Chromatin is formed by a series of repeating units called nucleosomes. Kornberg proposed a model for the nucleosome, in which 2 molecules of the four histone proteins H2A, H2B, H3 and H4 are organized to form a unit of eight molecules called histone octamere. The negatively charged DNA is wrapped around the positively charged histone octamere to form a structure called nucleosome.

A typical nucleosome contains 200 bp of DNA helix. The histone octameres are in close contact and DNA is coiled on the outside of nucleosome. Neighbouring nucleosomes are connected by linker DNA (H1) that is exposed to enzymes. The DNA makes two complete turns around the histone octameres and the two turns are sealed of by an H1 molecule. Chromatin lacking H1 has a beads-on-a-string appearance in which DNA
enters and leaves the nucleosomes at random places. H1 of one nucleosome can interact with H1 of the neighbouring nucleosomes resulting in the further folding of the fire.

The chromatin fier in interphase nuclei and mitotic chromosomes have a diameter that vary between 200-300 nm and represents inactive chromatin. 30 nm fire arises from the folding of nucleosome, chains into a solenoid structure having six nucleosomes per turn. This structure is stabilized by interaction between different H1 molecules. DNA is a solenoid and packed about 40 folds.

The hierarchical nature of chromosome structure is illustrated in (Fig. 5.3). Additional set of proteins are required for packing of chromatin at higher level and are referred to as non-histone chromosomal proteins (NHC). In a typical nucleus, some regions of chromatin are loosely packed (lightly stained) and are referred to as euchromatin. The chromatin that is tightly packed (stained darkly) is called heterochromatin. Euchromatin is transcriptionally active and heterochromatin is transcriptionally inactive.

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Properties Of Genetic Material

The experiment by Hershey and Chase clearly indicates that it is DNA that acts as a genetic material. However, in some viruses like Tobacco mosaic virus (TMV), bacteriophage θB, RNA acts as the genetic material. A molecule that can act as a genetic material should have the following properties:

Self Replication:

It should be able to replicate. According to the rule of base pairing and complementarity, both nucleic acids (DNA and RNA) have the ability to direct duplications. Proteins fail to fulfill this criteria.

Stability:

It should be stable structurally and chemically. The genetic material should be stable enough not to change with different stages of life cycle, age or with change in physiology of the organism. Stability as one of property of genetic material was clearly evident in Griffi’s transforming principle. Heat which killed the bacteria did not destroy some of the properties of genetic material.

In DNA the two strands being complementary, if separated (denatured) by heating can come together (renaturation) when appropriate condition is provided. Further 2′ OH group present at every nucleotide in RNA is a reactive group that makes RNA liable and easily degradable.

RNA is also known to be catalytic and reactive. Hence, DNA is chemically more stable and chemically less reactive when compared to RNA. Presence of thymine instead of uracil in DNA confers additional stability to DNA.

Information storage:

It should be able to express itself in the form of ‘Mendelian characters’. RNA can directly code for protein synthesis and can easily express the characters. DNA, however depends on RNA for synthesis of proteins. Both DNA and RNA can act as a genetic material, but DNA being more stable stores the genetic information and RNA transfers the genetic information.

Variation through mutation:

It should be able to mutate. Both DNA and RNA are able to mutate. RNA being unstable, mutates at a faster rate. Thus viruses having RNA genome with shorter life span can mutate and evolve faster. The above discussion indicates that both RNA and DNA can function as a genetic material. DNA is more stable, and is preferred for storage of genetic information.

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Rna World and The Origins Of Life

A typical cell contains about ten times as much RNA as DNA. The high RNA content is mainly due to the variety of roles played by RNA in the cell. Fraenkel-Conrat and Singer (1957) first demonstrated that RNA is the genetic material in RNA containing viruses like TMV (Tobacco Mosaic Virus) and they separated RNA from the protein of TMV viruses.

Three molecular biologists in the early 1980’s (Leslie Orgel, Francis Brick and Carl Woese) independently proposed the ‘RNA world’ as the first stage in the evolution of life, a stage when RNA catalysed all molecules necessary for survival and replication. The term ‘RNA world’ first used by Walter Gilbert in 1986, hypothesizes RNA as the first genetic material on earth.

There is now enough evidence to suggest that essential life processes (such as metabolism, translation, splicing etc.,) evolved around RNA. RNA has the ability to act as both genetic material and catalyst. There are several biochemical reactions in living systems that are catalysed by RNA. This catalytic RNA is known as ribozyme. But, RNA being a catalyst was reactive and hence unstable.

This led to evolution of a more stable form of DNA, with certain chemical modifications. Since DNA is a double stranded molecule having complementary strand, it has resisted changes by evolving a process of repair. Some RNA molecules function as gene regulators by binding to DNA and affect gene expression. Some viruses use RNA as the genetic material.

Andrew Fire and Craig Mellow (recipients of Nobel Prize in 2006) were of the opinion that RNA is an active ingredient in the chemistry of life. The types of RNA and their role have been discussed in class XI.