NEET Biology Notes Molecular Basis of Inheritance DNA Replication
DNA replication takes place during S-phase of interphase in cell cycle. DNA replication is semiconservative process.
In this process
- The two strands of the parent DNA molecule separate.
- Complementary strands are synthesised using old strands as template.
- Each progeny strand has one old and one new strand.
- It is a semiconservative process, i.e. out of two strands, present in one molecule, one is the old strand and other is the new one.
Meselson and Stahl (1958) provided the evidence in support of semiconservative replication of DNA. They grew E. coli on 15N (i.e. a heavy isotope of 14N). He observed that 50% of new DNA duplexes extracted from the culture possess 14N strands (normal) while another 50% have both 15N and 14N strands; This shows that one parent strand is conserved in daughter while second is freshly prepared.
Requirement for DNA Replication
- DNA template.
- A primer (usually RNA).
- Deoxyribonucleotide triphosphates (i.e. d-ATP, d-GTP, d-TTP, d-CTP).
- DNA unwinding proteins (helicase and gyrase).
- RNA polymerase to synthesise the RNA primer.
- Polynucleotide ligase (a joining enzyme).
- DNA polymerase (a DNA synthesising enzyme).
- DNA replication is bidirectional. A replicon is the segment of DNA, which is capable to perform DNA replication and independent of other segments of DNA. Each replicon has an origin of replication, at which DNA replication starts and a terminus, at which replication stops.
The two strands of DNA double helix unwind with the help of DNA unwinding proteins (e.g. DNA gyrase and DNA helicase). Unwinding produces two Y-shaped forks at origin, one fork is located at each end of the origin. When replication begins, these forks become replication forks. ‘
The unwinding of the strands imposes by the action of a superhelix relaxing protein. Single Strand Binding (SSB) Proteins stabilise the unwound parental DNA. Initiation of DNA synthesis requires an RNA primer. RNA primer is synthesised on DNA template by an enzyme primase.
The free 3’—OH of primer RNA provides the initiation point for DNA polymerase, which requires a free 3’—OH of a pre-existing polynucleotide for the initiation of DNA replication. DNA replication is semiconservative, semidiscontinuous, which occurs always in 5’ —> 3′ direction. Replication of 3′ —> 5′ strand of a DNA molecule proceeds continuously therefore, the 3’—>5′ strand of DNA molecule is known as leading strand. The replication of 5’ —>3′ strand of the DNA molecule is discontinuous and is known as lagging strand. Replication of lagging strand generates small nucleotide fragments in 5′ 3′ direction called Okazaki fragments.
DNA Polymerases of Eukaryotes
In eukaryotes, DNA polymerase-a replicates the leading strand, while DNA polymerase-8 synthesises the lagging strand. DNA polymerase p and E are nuclear DNA repair enzymes, whereas DNA polymerase-y is found in mitochondria and chloroplasts.
Some Proteins (enzymes) Involved in DNA.
It is the flow of information fromDNA to mRNA (transcription) and then decoding the information present in mRNA in the formation of polypeptide chain or protein (translation). The concept of central dogma was advanced by Crick in 1958 that proposes unidirectional flow of information from DNA to RNA and then to protein.
Commoner (1968) propounded a circular flow of information (from DNA —> RNA —> Protein RNA —>( DNA) Temin (1970) and Baltimore (1970) reported that double stranded RNA of Rous Sarcoma Virus (RSV) operates a central dogma reverse (inverse flow of information).
RNA of these viruses first synthesises DNA through reverse transcription or teminism.
The process of copying genetic information from one strand of the DNA into RNA is termed as transcription. The principle of complementarity governs the process of transcription, (except of adenine that pairs with uracil instead of thymine). However, in the process of replication the total DNA of an organism gets duplicated, while in transcription only a segment of DNA and only one of the strands of DNA is copied into RNA.
In bacteria, RNA polymerase binds to the promoter sequence just after upstream of the start site of transcription. The enzme moves down the DNA template and synthesises an RNA molecule. RNA synthesis stops, once the enzyme has recognised a terminator sequence.
In E coli, the enzyme RNA polymerase consists of five different polypeptide chains a, a’ p, p’ and a.
The holoenzyme (with five, units) has a molecular weight of 450000 Da. The enzyme without cr (sigma) subunit is called core enzyme.
- Promoter is the specific sequence of 20-200 bases, where the RNA polymerase binds to DNA. In a region from 5-10 bases to the left of the first base copied into mRNA is the right end of a sequence called the pribnow box.
- Pribnow box is a sequence that orients RNA polymerase so that synthesis proceeds from left to right.
- The pribnow box sequence TATAATG is an example of a consensus sequence (à pattern of bases from which actual sequences are observed) in prokaryotes.
- In eukaryotes, the corresponding consensus sequence is TATA AAA or TATA box or Hogness box.
Steps of Transcription
RNA polymerase binds to a specific sequence of bases on the DNA of the gene to be expressed called promotor and initiates transcription by associating transiently with initiation factor (o).
The DNA begins to unwind and the strands begin to separate.
The RNA polymerase begins to move along one strand of the exposed DNA (the sense strand), linking ribonucleotides together in order specified by the sequence of bases on the DNA.
Transcription continues unit the RNA polymerase reaches a ‘stop’ message on the DNA (a terminator).
Here, RNA polymerase associates transiently with the termination factor (p) to terminate the transcription.
The unzipped DNA closes back up, the enzymes drop off and the messenger RNA is released into the nucleus .
In eukaryotes, there arft two additional complexities
- There are at least three RNA polymerases in the nucleus (in addition to the RNA polymerase found in the organelles). There is a clear cut division of labour. The RNA polymerase-I transcribes rRNAs (28S, 18S, and 5.8S), whereas the RNA
polymerase-III is responsible «for transcription of fRNA, 5&rRNA, and snRNAs (small nuclear RNAs). The RNA polymerase-II transcribes precursor of mRNA, the heterogeneous nuclear RNA (hnRNA).
- The second complexity is that the primary transcripts contain both the exons and the introns. Hence, it is subjected to a process called splicing, where the introns are removed and exons are joined in a defined order. hnRNA undergoes additional processing called as capping and tailing.
In capping an unusual nucleotide (methyl guanosine triphosphate) is added to the 5′-end of hnKNA. In tailing, adenylate residues (200-300) are added at 3′-end in a template independent manner.
- It is the fully processed hnKNA, now called mRNA, that is transported out of the nucleus for translation.