The process of DNA replication at biochemical level
DNA or deoxyribonucleic acid is a biopolymer that encodes all the structures needed for the organism’s lifespan and development. In a cell it is located in the nucleus, mitochondria and chloroplasts. DNA consists of four types of deoxyribonucleotides, the sequence of which is used to store the data.
The essential characteristic of DNA molecule that allows it to pass the stored information to the next generations of cells or organisms is the way of self-reproduction known as replication. The molecule consists of two complementary chains that are held together with the help of hydrogen bonds between the complementary bases. In the course of the replication these chains are separated, and each of them is used as a template or parental chain to create new daughter chains. Hence, the process is semiconservative that means that each of the new cells receives one copy of parental and one copy of daughter strain.
The replication of the DNA molecule in the nucleus is a complicated process that requires the participation of the great amount of enzymes and non-enzymatic proteins. Its initiation starts with the separation of two strands that starts in strictly determined sequences of nucleotides called origin sites. A number of these sites depends on the size of the replicated chromosome. For instance, bacterial chromosome, being relatively small, contains only one replication origin, while human chromosomes might contain up to thousands of such sites that significantly increases the speed of the replication.
The specific sequence of the replication origins is recognized by helicase – the enzyme that ruins the hydrogen bonds between two strains of DNA, therefore, separates them. The free sites of the chains form the replication bubble. Unwinding the molecule in such bubbles increases the twisting of the spiral beyond the bubbles. To reduce the strain and prevent the chains from breaking enzymes that are called topoisomerases create single or even double-strand breaks, rotate the molecule and rejoin the strands. Single-strand binding proteins bind to the unwound chains to prevent the renaturation, stabilize the chains and prepare them to serve as a template for the creation of new strands.
The size of the replication bubble increases in both directions and two replication forks are formed. The process of the DNA synthesis is initiated by DNA dependent RNA polymerase called primase that creates a short sequence of RNA called a primer. This sequence is needed for the next enzyme, DNA dependent DNA polymerase that starts attaching nucleotides that are complementary to the template strand of DNA. Because of the enzyme’s structure, the nucleotides are added only to the free 3’ end of the previous nucleotide; therefore, the direction of the new strand elongation is 5’→3’. Since two parental chains are antiparallel, the directions of the chains elongation are antiparallel too. That is why there is only one stand that can be synthesized continuously, starting with one primer – the leading strand. The replication of another strand - the lagging strand - starts later, and as the replication fork moves further the short fragments of DNA called Okazaki fragments are synthesized. That process requires the constant synthesis of new primers.
The replication complex moves along the DNA strand while DNA polymerase I starts replacing RNA primers with DNA nucleotides. Afterwards, the fragments are joined in a single strand with the help of DNA ligase in ATP-dependent reaction. The proofreading and correction of the nucleotide sequence are also done by DNA polymerase.
The role of mRNA in transcription and translation
DNA contains the information that encodes all the possible proteins in a cell. Still for the direct synthesis of proteins it is more reasonable to use the molecules that contain the data needed to synthesize only one particular protein, since it facilitates its transportation and regulation. That is why RNA or ribonucleic acid molecules are used as messengers to transfer the information from DNA to ribosomes responsible for the process of protein synthesis called translation.
Messenger RNA molecules are created by DNA dependent RNA polymerase II or transcriptase in a process called transcription. The enzyme breaks the hydrogen bonds of two DNA strands, separates them and uses the sense DNA strand to create a complementary chain that consists of RNA nucleotides. The transcription always starts at specific nucleotide sequences called promoters that are recognized by transcription factors – the proteins that bind to the DNA strand and promote or block the binding of the enzyme. The typical promoter contains the following sequences: TATA box in eukaryotic cells or Pribnow box in prokaryotic ones, the initiator element and Downstream Promoter Element. The work of the enzyme is also regulated by enhancers and silencers – the sequences that might be located even in thousands of nucleotides from the start of transcription. The end of the sequence known as terminator is a sign for enzyme to stop transcription.
The obtained transcript is still not ready for a further translation, since it contains introns – the non-coding segments of DNA that play the regulatory role. That is why pre-mRNA undergoes modification called processing that includes the addition of 5’-cap, formation of poly-A tail and splicing or removing the intron sequences by spliceosome – the complex containing proteins and small nuclear RNA. After that mRNA is transferred from nucleus to cytoplasm.
The synthesis of proteins or translation takes place on ribosomes – the organelles that consist of ribosomal RNA and proteins. The sequence of RNA nucleotides in converted into amino acids sequence with the help of transfer RNA. The initiation of translation occurs when two subunits of ribosome bind to mRNA and a specific initiator tRNA. After that the ribosome elongates the chain of amino acids adding them one by one with the help of tRNA. At the end of mRNA sequence a stop codon is located. It consists of three nucleotides that bind to a release factor protein and terminate the movement of the ribosome. Afterwards, its subunits separate and release the ready amino acid chain to form the structure needed for the protein functioning.
The poisonous effect of the death cup mushroom
The main toxin of the death cup mushroom is alpha-Amanitin that is responsible for the deadly effect of the mushroom. It is a cyclic peptide that consists of eight amino acids. In cells of a human organism it immediately binds to the largest subunit of RNA Polymerase II that is called RPB1 (Nguyen et al., 1996). The toxin interacts with the bridge α-helix, the cleft and the funnel regions of the enzyme that catalyzes the transcription of mRNA, small nucleus RNA and microRNA and promotes the degradation of the subunit (Gong, Nedialkov & Burton, 2004). The result is a block of translocation and the inhibition of the transcription. That leads to system failure in kidneys and liver, therefore, cause the death of the organism.
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