DNA is the genetic material in most of the eukaryocytes. In order for the cells to replicate, DNA must also be divided between the parental cell and the progeny. Replication of DNA is a semi-conservative process in which the daughter's DNA retains the originality of the parental strands. There are 3 billion double-stranded DNAs in the human genome that need to be replicated. Replications of DNA is an active process demanding a lot of energy, enzymatic processes, and mediation by the different biomolecules. In order to preserve the similarity between the parental cells and the daughter cells, the parental DNA should deliver the exact copy of karyotype tot heir daughter cells. Meselson and Franklin Stahl have conducted a trial on E-coli to understand DNA replication. E-coli was given a preference as the human DNA resembles with the Ecoil. In the year 1958, they have explained the process of replication of the DNA process as follows.
DNA -replication ( semi-conservative )
Prerequisites of DNA replication
The replication of DNA is a semiconservative process as each parental DNA of a double helix is an original template from which 2 new daughter strands erupt as shown in the image. The newly developing pair is called the complementary strand. DNA replication is mediated by many enzymes, but the DNA polymerases play a significant role. Polymerases need an original template and a starter called primer. During the DNA replication, one new strand - the leading strand will be formed. The leading strand in a template becomes 2 small pieces called daughter cells or the lagging strands. At the same time, in addition to the polymerases, other enzymes such as DNA helicase, DNA primase, DNA ligase, and topoisomerase also assists the process of DNA replication. I t is called a semi-conservative because the replication process produces 2-daughter molecules in which each newly formed double helix receiving one new daughter strand and one old parental strand( Half- saved from the original and the raining half is added as new daughter strand). In order for a parental DNA to replicate the exact copy , there must be an error-free transmission of DNA into the daughters. During the course, one of the key processes involved here is the DNA polymerase reaction.
DNA polymerase reaction
DNA Polymerase reaction
1. The DNA replication begins at specific locations on the DNA, called origins of replication. The group of enzymes work in pairs to generate 2 identical DNA strands from an original parent template. During the process, DNA polymerase "reads" the genetic code existing on the parental DNA strands to build 2 new strands exactly as that of parental DNA. The formula for the replication is, Deoxynucleoside triphosphate + DNA ⇌diphosphate + DNAn+1. DNA polymerases will slowly add one by one of the nucleotides to the growing DNA chain. DNA polymerase must be highly efficient because they have to catalyze the polymerisation of many nucleotides in a quick time. One of the examples is, E-coli having only 4.6 × 106 base pairs compared to the diploid content of human which is 6.6 × 109 bp. The process of polymerization hardly takes 38 minutes and the rate of polymerization is 2000 base pairs per second. Another enzyme, deoxyribonucleoside triphosphate act as substrates that provide the energy needed for the polymerisation reaction. In addition to the enzymes, a tool called replication fork (see image on top) helps in separating the lengthy parental DNA strand. DNA ligase is another enzyme assists in the replication of DNA. The DNA-dependent DNA polymerases catalyze polymerization only in one direction that is 5’ to 3’. This creates some additional complications at the replicating fork. Consequently, on one strand (the template with polarity 3' to 5, the replication is continuous, while on the other (the template with polarity 5' to 3' ), it is discontinuous. The fragments synthesized in a disorderly fashion are later joined by the enzyme DNA ligase.
The process of copying genetic information from one strand of the DNA into RNA is termed as transcription. It is the initial step in gene expression where a segment of template DNA is copied into mRNA (messenger RNA) with the help of the enzyme RNA polymerase. During the process, a sequence of DNA scanned by an RNA polymerase produces a complementary and antiparallel RNA strand called a Primary transcript.
Steps of transcription
1. RNA polymerase, together with one or more general transcription factors, binds to promoter DNA.
2. RNA polymerase creates a transcription bubble, which separates the two strands of the DNA helix. This is done by breaking the hydrogen bonds between complementary DNA nucleotides.
3. RNA polymerase adds RNA nucleotides (which are complementary to the nucleotides of one DNA strand).
4. RNA sugar-phosphate backbone forms with assistance from RNA polymerase to form an RNA strand.
5. Hydrogen bonds of the RNA–DNA helix break, freeing the newly synthesized RNA strand.
6. If the cell has a nucleus, the RNA may be further processed. This may include polyadenylation, capping, and slicing.
7. The RNA may remain in the nucleus or exit to the cytoplasm through the nuclear pore complex.
A transcription unit is a sequence of nucleotides in DNA that codes alongside the single RNA molecule in a way that requires information in the sequence. A transcription unit normally has a promoter, an RNA-coding sequence, and the terminator. There is a narrow principle that transcription can occur only in 5’→ 3′ direction and such strand of DNA is called template strand/ master strand.
Mechanism of Transcription:
1. Activation of Ribo-nucleotides
During this process, Enzyme phosphorylase along with the energy, phosphorylated Ribonucleotides are activated. The activated Ribonucleotides are adenosine triphosphate (ATP), guanosine triphosphate (GTP), uridine triphosphate (UTP) and cytidine triphosphate (CTP) and the process is called phosphorylation. Which appears prior to the Transcription and the nucleotides are activated through phosphorylation.
2. Formation of DNA Template
In order to form a template, 3 major regions are required, namely, initiation site, a promoter region, coding region, and a terminator region. As the name indicates, the initiation site launches the transcription. Promoter region has an RNA polymerase recognition site and RNA polymerase binding site. In a coding region, the chain opening occurs. Chain opening needs unwindases, gyrases, and single-stranded binding proteins. In a terminator region, the two strands of DNA uncoil progressively from the site of polymerase binding. One of the two strands of DNA (3’—» 5′) functions as a template for transcription of RNA. It is called master, template or antisense strand.
3. DNA strand formation
The Ribonucleoside triphosphates come closer to lie just opposite the nitrogenous bases of the DNA template (Antisense strand). This results in the formation of complementary pairs, I,e they form complementary pairs, U goes opposite to A, A goes opposite to T, С goes opposite to G, and G goes opposite to C. A pyrophosphate is released from each ribonucleoside triphosphate to form Ribonucleotides. The pyrophosphate is hydrolyzed with the help of enzyme pyrophosphatase. It releases energy.
With the help of RNA polymerase, the adjacent ribonucleotides are held tightly against the DNA template to form an RNA chain.
3.Separation of RNA
Separation is enhanced by the factor called ATPase activity. This helps in the release of the completed RNA chain from the previous phase of chain formation. The released RNA is called primary transcript which will further get processed to form a functional RNA.
4.Duplex Formation:Once the primary transcript is released, the 2 strands of DNA form linkages amongst complementary base pairs. At this stage, Gyrases, unwindases and SSB proteins are released, consequently, there is double-helical DNA formation.