Biological Functions of DNA 2024-2025 PDF

Republic of Iraq Ministry of Higher Education and Scientific Research University of Babylon College of Pharmacy Department of Laboratory and Clinical Sciences Biochemistry I For...

Republic of Iraq Ministry of Higher Education and Scientific Research University of Babylon College of Pharmacy Department of Laboratory and Clinical Sciences Biochemistry I For 3rd Year Pharmacy Students/ Semester I-2024/2025 Biological functions of DNA By Lec. Dr. Zeyad H. Nafaee 1 University of Babylon /College of Pharmacy Structure of DNA 2024-2025/ Semester I/ 3rd year  A diagrammatic representation of the Watson and Crick model of the double-helical structure of the B form of DNA. The horizontal arrow indicates  Width of the double helix (20 Å), and the vertical  arrow indicates the distance spanned by one complete turn of the double helix (34 Å).  One turn of B-DNA includes ten base pairs (bp), so the rise is 3.4 Å per bp.  Central axis of the double helix is indicated by the vertical rod.  Short arrows designate the polarity of the antiparallel strands.  Major and minor grooves are depicted.  (A, adenine; C, cytosine; G, guanine; T, thymine; P, phosphate; S, sugar [deoxyribose].) 2 University of Babylon /College of Pharmacy Biological functions of DNA 2024-2025/ Semester I/ 3rd year 3 University of Babylon /College of Pharmacy Biological functions of DNA 2024-2025/ Semester I/ 3rd year  DNA replication is the biological process of producing two identical DNA from one original DNA molecule by DNA polymerase. DNA replication occurs in almost living organisms, it is essential part of biological inheritance. This is essential for cell division during growth and repair of damaged tissues, while it ensures that each of the new cells receives its own copy of the DNA.  DNA Transcription is the biological process of copying a segment of DNA into RNA. Some segments of DNA are transcribed into RNA molecules called messenger RNA (mRNA) that can encode proteins, During transcription, a DNA sequence is read by an RNA polymerase, which produces a complementary, antiparallel RNA strand called a primary transcript.  DNA Translation is the biological process in living cells in which proteins are produced using RNA molecules as templates. The generated protein is a sequence of amino acids. In translation, messenger RNA (mRNA) is decoded in a ribosome, outside the nucleus, to produce a specific amino acid chain, or polypeptide. The polypeptide later folds into an active protein and performs its functions in the cell. 4 University of Babylon /College of Pharmacy DNA Replication 2024-2025/ Semester I/ 3rd year  DNA replication is the process by which the genome's DNA is copied in cells.  Before a cell divides, DNA must first copy (or replicate) its entire genome so that each resulting daughter cell ends up with its own complete genome.  Four major stages for replication: initiation, unwinding, primer synthesis, and elongation. 5 University of Babylon /College of Pharmacy DNA Replication 2024-2025/ Semester I/ 3rd year 6 University of Babylon /College of Pharmacy DNA Replication 2024-2025/ Semester I/ 3rd year  Genetic information stored in the nucleotide sequence of DNA serves two purposes. (i) It is the source of information for the synthesis of all protein molecules of the cell and organism, (ii)and it provides the information inherited by daughter cells or offspring.  Both of these functions require that the DNA molecule serve as a template  In the first case for the transcription of the information into RNA and in the second case for the replication of the information into daughter DNA molecules.  Complementarity of the Watson and Crick double-stranded model of DNA strongly suggests that replication of the DNA molecule occurs in a semiconservative manner.  Each strand of the double- stranded parental DNA molecule separates from its complement during replication, each serves as a template on which a new complementary strand is synthesized.  Two newly formed double stranded daughter DNA molecules, each containing one strand (but complementary rather than identical) from the parent double-stranded DNA molecule, are then sorted between the two daughter cells.  Each daughter cell contains DNA molecules with information identical to that which the parent possessed; yet in each daughter cell the DNA molecule of the parent cell has been only semiconserved. 7 University of Babylon /College of Pharmacy DNA Replication 2024-2025/ Semester I/ 3rd year  Double-stranded structure of DNA and the template function of each old strand (dark shading) on which a new (light shading) complementary strand is synthesized. 8 University of Babylon /College of Pharmacy PCR 2024-2025/ Semester I/ 3rd year  Polymerase chain reaction (PCR) is a laboratory technique for rapidly producing (amplifying) millions to billions of copies of a specific segment of DNA, which can then be studied in greater detail.  PCR involves using short synthetic DNA fragments called primers to select a segment of the genome to be amplified, and then multiple rounds of DNA synthesis to amplify that segment.  The reaction consists of cycles, each of which can be divided into three steps: 1. Denaturation step occurs at 94–97 °C, separation of the two strands of the double stranded template dsDNA. 2. Annealing step occurs at 50–70 °C, primers hybridize to the template strands at their complementary sequences, and by this the multipliable section is determined. 3. Extension step occurs 72 °C, DNA polymerase builds up the new strands using the dNTP building blocks. 9 University of Babylon /College of Pharmacy PCR 2024-2025/ Semester I/ 3rd year 10 University of Babylon /College of Pharmacy 2024-2025/ Semester I/ 3rd year PCR Substances required for PCR:  Double-stranded DNA template (e.g. from the studied cell).  Synthetic oligonucleotide primers in excess amounts. By hybridization to their complementary sequences, they will determine the termini of the newly synthesized DNA section.  DNA „building blocks” - dNTP-s (dCTP, dATP, dGTP and dTTP ) in equal amounts.  Buffer and Mg(II) ions to ensure the optimal reaction conditions.  Heat-stable DNA polymerase (Taq polymerase DNA synthesizing enzyme originates from the Thermus aquaticus bacteria that live in thermal water).  There are about 37 types of PCR, The common PCR types are Real-Time PCR (Quantitative PCR (qPCR)); Repetitive sequence-based PCR; Reverse Transcriptase PCR (RT-PCR); Reverse-Transcriptase Real-Time PCR (RT-qPCR) 11 University of Babylon /College of Pharmacy DNA Transcription 2024-2025/ Semester I/ 3rd year  DNA transcription produces a single-stranded RNA molecule that is complementary to one strand of DNA.  Transcription is performed by enzymes called RNA polymerases, which link nucleotides to form an RNA strand (using a DNA strand as a template). 12  Transcription has three stages: initiation, elongation, and termination University of Babylon /College of Pharmacy DNA Transcription 2024-2025/ Semester I/ 3rd year  Relationship between the sequences of an RNA transcript and its gene, in which the coding and template strands are shown with their polarities. The RNA transcript with a 5′ to 3′ polarity is complementary to the template strand with its 3′ to 5′ polarity.  Note that the sequence in the RNA transcript and its polarity is the same as that in the coding strand, except that the U of the transcript replaces the T of the gene. 13 University of Babylon /College of Pharmacy DNA Transcription 2024-2025/ Semester I/ 3rd year Messenger RNA (mRNA)  This class is the most heterogeneous in size and stability.  All members of the class function as messengers conveying the information in a gene to the protein synthesizing machinery, where each serves as a template on which a specific sequence of amino acids is polymerized to form a specific protein molecule.  Messenger RNAs, particularly in eukaryotes, have some unique chemical characteristics.  5′ terminal of mRNA is “capped” by a 7-methylguanosine triphosphate that is linked to an adjacent 2′-O-methyl ribonucleoside at its 5′-hydroxyl through the three phosphates.  mRNA molecules frequently contain internal 6-methyladenylates and other 2′-O-ribose methylated nucleotides.  Cap is involved in the recognition of mRNA by the translating machinery, and it probably helps stabilize the mRNA by preventing the attack of 5′-exonucleases.  Protein-synthesizing machinery begins translating the mRNA into proteins beginning downstream of the 5′ or capped terminal.  Other end of most mRNA molecules, the 3′-hydroxyl terminal, has an attached polymer of adenylate residues 20– 250 nucleotides in length. 14 University of Babylon /College of Pharmacy DNA Transcription  Specific function of the poly(A) “tail” at the 3′-hydroxyl terminal of mRNAs is not fully understood, but it seems that it maintains the intracellular stability of the specific mRNA by preventing the attack of 3′-exonucleases.  Some mRNAs, including those for some histones, do not contain poly(A).  Poly(A) tail, because it will form a base pair with oligodeoxythymidine polymers attached to a solid substrate like cellulose, can be used to separate mRNA from other species of RNA, including mRNA molecules that lack this tail.  The relationship between the sequences of an RNA transcript and its gene, in which the coding and template strands are shown with their polarities.  RNA transcript with a 5′ to 3′ polarity is complementary to the template strand with its 3′ to 5′ polarity. Note that the sequence in the RNA transcript and its polarity is the same as that in the coding strand, except that the U of the transcript replaces the T of the gene.  In mammalian cells, including cells of humans, the mRNA molecules present in the cytoplasm are not the RNA products immediately synthesized from the DNA template but must be formed by processing from a precursor molecule before entering the cytoplasm.  In mammalian nuclei, the immediate products of gene transcription constitute a fourth class of RNA molecules.  These nuclear RNA molecules are very heterogeneous in size and are quite large. 15 University of Babylon /College of Pharmacy DNA Transcription 2024-2025/ Semester I/ 3rd year  The cap structure attached to the 5′ terminal of most eukaryotic messenger RNA molecules. A 7-methylguanosine triphosphate (black) is attached at the 5′ terminal of the mRNA (shown in blue), which usually contains a 2′ -O-methylpurine nucleotide.  These modifications (the cap and methyl group) are added after the mRNA is transcribed from DNA.  The heterogeneous nuclear RNA (hnRNA) molecules may have a molecular weight in excess of 107, whereas the molecular weight of mRNA molecules is generally less than 2 ×106.  hnRNA molecules are processed to generate the mRNA molecules which then enter the cytoplasm to serve as templates for protein synthesis. 16 University of Babylon /College of Pharmacy DNA Translation 2024-2025/ Semester I/ 3rd year  Expression of genetic information in DNA into the form of an mRNA transcript.  This is subsequently translated by ribosomes into a specific protein molecule. 17 University of Babylon /College of Pharmacy DNA Translation 2024-2025/ Semester I/ 3rd year  Translation is the way genetic code contained in mRNA is decoded to produce a specific sequence of amino acids in a polypeptide chain.  In this process, the ribosome translates the mRNA produced from DNA into a chain of specific amino acids.  This chain of amino acids leads to protein synthesis 18 University of Babylon /College of Pharmacy DNA Translation 2024-2025/ Semester I/ 3rd year  Translation proceeds in three phases:  Initiation: The ribosome assembles around the target mRNA. The first tRNA is attached at the start codon.  Elongation: The last tRNA validated by the small ribosomal subunit (accommodation) transfers the amino acid. It carries to the large ribosomal subunit which binds it to one of the preceding admitted tRNA (transpeptidation). The ribosome then moves to the next mRNA codon to continue the process (translocation), creating an amino acid chain.  Termination: When a stop codon is reached, the ribosome releases the polypeptide. The ribosomal complex remains intact and moves on to the next mRNA to be translated. 19 University of Babylon /College of Pharmacy 2024-2025/ Semester I/ 3rd year Translation 20 University of Babylon /College of Pharmacy DNA Translation 2024-2025/ Semester I/ 3rd year Transfer RNA (tRNA)  tRNA molecules vary in length from 74 to 95 nucleotides.  The tRNA molecules serve as adapters for the translation of the information in the sequence of nucleotides of the mRNA into specific amino acids.  There are at least 20 species of tRNA molecules in every cell, at least one (and often several) corresponding to each of the 20 amino acids required for protein synthesis.  Although each specific tRNA differs from the others in its sequence of nucleotides.  The primary structure ie, the nucleotide sequence of all tRNA molecules allows extensive folding and intrastrand complementarity to generate a secondary structure that appears like a cloverleaf.  All tRNA molecules contain four main arms. The acceptor arm terminates in the nucleotides CpCpAOH.  These three nucleotides are added posttranscriptionally.  tRNA-appropriate amino acid is attached to the 3′-OH group of the A moiety of the acceptor arm. The D, T C, and extra arms help define a specific tRNA.  Although tRNAs are quite stable in prokaryotes, they are somewhat less stable in eukaryotes.  Opposite is true for mRNAs, which are quite unstable in prokaryotes but generally stable in eukaryotic organisms. 21 University of Babylon /College of Pharmacy DNA Translation 2024-2025/ Semester I/ 3rd year  Typical aminoacyl tRNA in which the amino acid (aa) is attached to the 5′ CCA terminal.  The anticodon, T,C, and dihydrouracil (D) arms are indicated, as are the positions of the intramolecular hydrogen bonding between these base pairs. 22 University of Babylon /College of Pharmacy DNA Translation 2024-2025/ Semester I/ 3rd year Ribosomal RNA (rRNA)  A ribosome is a cytoplasmic nucleoprotein structure that acts as the machinery for the synthesis of proteins from the mRNA templates.  On the ribosomes, the mRNA and tRNA molecules interact to translate into a specific protein molecule information transcribed from the gene.  In active protein synthesis, many ribosomes are associated with an mRNA molecule in an assembly called the polysome.  Components of the mammalian ribosome, which has a molecular weight of about 4.2 × 106 and a sedimentation velocity of 80S (Svedberg units).  Mammalian ribosome contains two major nucleoprotein subunits—a larger one with a molecular weight of 2.8 × 106 (60S) and a smaller subunit with a molecular weight of 1.4 × 106 (40S).  The 60S subunit contains a 5S ribosomal RNA (rRNA), a 5.8S rRNA, and a 28S rRNA; there are also probably more than 50 specific polypeptides. 23 University of Babylon /College of Pharmacy DNA Translation 2024-2025/ Semester I/ 3rd year  40S subunit is smaller and contains a single 18S rRNA and approximately 30 distinct polypeptide chains. All of the ribosomal RNA molecules except the 5S rRNA are processed from a single 45S precursor RNA molecule in the nucleolus.  5S rRNA is independently transcribed. The highly methylated ribosomal RNA molecules are packaged in the nucleolus with the specific ribosomal proteins.  In the cytoplasm, the ribosomes remain quite stable and capable of many translation cycles.  The functions of the ribosomal RNA molecules in the ribosomal particle are not fully understood, but they are necessary for ribosomal assembly and seem to play key roles in the binding of mRNA toribosomes and its translation. Recent studies suggest that an rRNA component performs the peptidyl transferase activity and thus is an enzyme (a ribozyme). Small stable RNA  A large number of discrete, highly conserved, and small stable RNA species are found in eukaryotic cells.  Majority of these molecules are complexed with proteins to form ribonucleoproteins and are distributed in the nucleus, in the cytoplasm, or in both.  They range in size from 90 to 300 nucleotides and are present in 100,000–1,000,000 copies per cell. Small nuclear RNAs (snRNAs), a subset of these RNAs, are significantly involved in mRNA processing and gene regulation.  Several snRNAs, U1, U2, U4, U5, and U6 are involved in intron removal and the processing of hnRNA into mRNA.  U7 snRNA may be involved in production of the correct 3′ ends of histone mRNA which lacks a poly(A) tail.  U4 and U6 snRNAs may also be required for poly(A) processing. 24 University of Babylon /College of Pharmacy  A simplified presentation of recombinant DNA technology up to the protein purification  Generally, the first step of the synthesis of a protein is the construction of the functional gene of the protein of interest by recombinant DNA technology.  Biological tools in chemistry research field.  This technology involves two or more DNA molecules from different organisms being recombined together and then introduced into a host organism – usually a bacterial cell.  This procedure also allows to change the genetic material to introduce mutations and thereby change desired amino acids in a protein.  New gene is ligated with a carrier DNA (vector, plasmid), cloned in bacteria and used for protein expression. 25