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Structure of DNA – Watson and Crick model Proof for semi conservative method of DNA replication; Models of DNA replication; steps involved in DNA replication. RNA types - mRNA, tRNA, rRNA – Protein synthesis
The salient features of double helix structure of DNA are:
The DNA molecule consists of two polynucleotide chains wound around each other in a right-handed double helix.
The two strands of a DNA molecule are oriented anti-parallel to each other i.e. the 5’ end of one strand is located with the 3’ end of the other strand at the same end of a DNA molecule.
Each polydeoxyribonucleotide strand is composed of many deoxyribonucleotides joined together by phosphodiester linkage between their sugar and phosphate residues and the sugar phosphate backbones are on the outsides of the double helix with the nitrogen bases oriented toward the central axis.
The half steps of one strand extend to meet half steps of the other strand and the base pairs are called complementary base pairs. The adenine present in one stand of a DNA molecule is linked by two hydrogen bonds with the thymine located opposite to it in the second strand, and vice-versa. Similarly, guanine located in one strand forms three hydrogen bonds with the cytosine present opposite to it in the second strand, and vice-versa. The pairing of one purine and one pyrimidine maintains the constant width of the DNA double helix.
The bases are connected by hydrogen bonds. Although the hydrogen bonds are weaker, the fact that so many of them occur along the length of DNA double helix provides a high degree of stability and rigidity to the molecule.
The diameter of this helix is 200A, while its pitch (the length of helix required to complete one turn) is 340A. In each DNA strand, the bases occur at a regular interval of 3.40A so that about 10 base pairs are present in one pitch of a DNA double helix.
The helix has two external grooves, a deep wide one, called major groove and a shallow narrow one, called minor groove. Both these groves are large enough to allow protein molecules to come in contact with the bases.
This DNA structure offers a ready explanation of how a molecule could form perfect copies of itself. During replication, the two strands of a DNA molecule unwind and the unpaired bases in the single-stranded regions of the two strands by hydrogen bonds with their complementary bases present in the cytoplasm as free nucleotides. These nucleotides become joined by phospho-diester linkages generating complementary strands of the old ones with the help of appropriate enzymes.
The DNA molecule satisfies the requirement of genetic material in the following ways:
It can replicate itself accurately during cell growth and division.
Its structure is sufficiently stable so that heritable charges i.e., mutations can occur only very rarely.
It has a potential to carry all kinds of necessary biological information.
It transmits all the biological information to the daughter cells.
Thus, the essential functions of DNA are the storage and transmission of genetic information and the expression of this information in the form of synthesis of cellular proteins.
Types of DNA:
The double helix described by Watson and Crick has right-handed helical coiling and is called B-DNA.
It is a biologically important form of DNA that is commonly and naturally found in most living systems.
This double helical structure of DNA exists in other alternate forms such as A-form, C-form etc. which differ in features such as the number of nucleotide base pairs per turn of the helix.
The B form contains ~ 10 (range 10.0 – 10.6) base pairs per turn.
The B-DNA is the most stable form and it can change to another form depending upon the humidity and salt concentration of the sample.
The A- form is also a right-handed helix, but it has 11 base pairs per turn.
The C-form of DNA has 9.3 base pairs per turn, while the D-form of DNA, which is rare form, has 8 base pairs per turn.
Another form of DNA, in which the helix is left-handed, called Z-DNA was discovered by Rich.
In Z-DNA sugar and phosphate linkages follow a zigzag pattern. Z-DNA plays a role in the regulation of the gene activity.
Denaturation: The hydrogen bonds between the DNA strands break on heating the DNA to high temperature (nearly 100oC). The process of separation of DNA strands is known as denaturation.
Renaturation: Reunion of the separated or denatured DNA strands on cooling is called renaturation or annealing. The optimum temperature for renaturation is 20 – 25oC.
DNA replication: The process by which a DNA molecule makes its identical copies is called DNA replication.
Modes of DNA Replication: There are three possible modes of DNA replication.
Dispersive: In dispersive mode of replication, the old DNA molecule would break into several pieces, each fragment would replicate and the old and new segments would recombine randomly to yield the progeny DNA molecule. Each progeny molecule would have both old and new segments along its length.
Conservative: According to conservative scheme, the two newly synthesized strands following the replication of a DNA molecule would associate to form one double helix, while the other two old strands would remain together as one double helix.
Semi conservative: In this model of DNA replication, each newly synthesized strand of DNA would remain associated with old strand against which it was synthesized. Thus, each progeny DNA molecule would consist of one old and one newly synthesized strand.
Evidence for Semi conservative replication:
The experiment conducted by Matthew Meselson and Franklin Stahl in 1958 on E. coli provided a conclusive proof that replication of DNA is by semi conservative model.
Meselson and Stahl labelled DNA of E. coli bacteria with heavy nitrogen i.e. 15N by growing them on a medium containing 15N for many generations to replace the normal nitrogen (14N).
The density of normal and heavy nitrogen differs. The 14N is lighter (1.710 g/cm3) than 15N (1.724 g/cm3).
It is possible to detect such minute differences in density through density gradient centrifugation.
Distinct bands are formed in the centrifuge tube for different density DNA. DNA extracts of E. coli with 15N gave a characteristic heavy band at one end of a tube that had been centrifuged at a high speed in an ultra-centrifuge.
These labelled cells were then grown on a normal unlabelled media containing 14N for one generation.
DNA was again extracted and processed and it was found to consist of a hybrid DNA containing both 14N and 15N at the same time.
This indicated that the DNA had not replicated in two separate labelled and unlabelled forms.
The next generation of growth on unlabelled DNA was found to be in amounts equal to the partially labelled hybrid DNA.
Additional generation of growth on unlabelled media gave a relative increase in the amount of unlabelled DNA.
After two generations half the DNA was with intermediate density and half with light bands which further confirm semi conservative mode of DNA replication.
After third generation, ¾ DNA was found with 14N and ¼ with hybrid nitrogen (14N+15N). When the hybrid DNA was denatured by heating upto 100oC it was found to produce two separate single strands and in the ultracentrifuge density gradient, it was observed to form two separate bands; one band containing 15N and the other 14N.
Thus, it was concluded that DNA replication was by semi-conservative mode. The major objection put forth for the semi-conservative replication was that the DNA molecule must unwind a number of times (1/10 of the total number of nucleotides) which cannot be accomplished without breaking with in a short span of time say two minutes.
Cairns (1968) provided evidence for this in his experiments with radio-active labelled E. coli chromosomes.
The E. coli chromosome is a double stranded circular chromosome. It was shown that the two circular component strands separate during replication with each strand duplicating individually producing a θ shaped structures during replication. This indicates that unwinding and replication proceed simultaneously.
Method of semi-conservative replication:
DNA replication was found to begin at various initiation points, called origin of replication, and proceed bi-directionally.
Two enzymes, DNA gyrase and DNA helicase induce unwinding of complementary strands of DNA.
Single-strand DNA binding (SSB) proteins bind to the single-stranded DNA, stabilizing it and preventing it from reannealing.
An enzyme, primase, initiates replication by synthesizing the primer. DNA polymerases synthesize the complementary strand by progressively adding deoxyribonucleotides.
The DNA replication always proceeds in 5’ à 3’ direction. During replication one strand of DNA can replicate continuously and the other strand discontinuously or in pieces.
The continuously replicating strand is known as leading strand and the discontinuously replicating strand is known as lagging strand.
The replication of lagging strand generates small polynucleotide fragments called ‘Okazaki fragments’ which are later joined together by the enzyme, DNA ligase.
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