DNA: The Genetic Code Story PDF

 DNA The Genetic Code Story Presented by Marwa Hasan Soliman  CONTENT The Genetic Code Story  RNA Replication, Transcription, and Translation are the three main proc...

 DNA The Genetic Code Story Presented by Marwa Hasan Soliman  CONTENT The Genetic Code Story  RNA Replication, Transcription, and Translation are the three main processes used by all cells to maintain their genetic information Central Dogma and to convert the genetic information encoded in DNA into gene products, which are either RNAs or proteins, depending on the gene. Gene Expression (Protein Synthesis) Transcription & Translation  Mendel’s 1900, Gene is a unit of heredity. Definition a molecular entity capable of replication, transcription, translation, and mutation. What is a Gene? Genes are composed of DNA arranged on chromosomes. Some genes encode structural or regulatory RNAs. Other genes encode proteins. Protein-encoding genes specify the sequences of amino acids, which are the building blocks of proteins. In the nucleus of each cell, the DNA molecule is packaged into thread-like structures called chromosomes. Each chromosome is made up of DNA tightly coiled many times around proteins called histones that support its structure. Chromosomes are not visible in the cell’s nucleus—not even under a microscope—when the cell is not dividing. However, the DNA that makes up chromosomes becomes more tightly packed during cell division and is then visible under a microscope. Most of what researchers know about chromosomes was learned by observing chromosomes during cell division. Each chromosome has a constriction point called the centromere, which divides the chromosome into two sections, or “arms.” The short arm of the chromosome is labeled the “p arm.” The long arm of the chromosome is labeled the “q arm.” The location of the centromere on each chromosome gives the chromosome its characteristic shape, and can be used to help describe the location of specific genes. In the nucleus of each cell, the DNA molecule is packaged into thread-like structures called chromosomes. Each chromosome is made up of DNA tightly coiled many times around proteins called histones that support its structure. Chromosomes are not visible in the cell’s nucleus—not even under a microscope—when the cell is not dividing. However, the DNA that makes up chromosomes becomes more tightly packed during cell division and is then visible under a microscope. Most of what researchers know about chromosomes was learned by observing chromosomes during cell division. Each chromosome has a constriction point called the centromere, which divides the chromosome into two sections, or “arms.” The short arm of the chromosome is labeled the “p arm.” The long arm of the chromosome is labeled the “q arm.” The location of the centromere on each chromosome gives the chromosome its characteristic shape, and can be used to help describe the location of specific genes.  Ribonucleic acid (RNA), unlike DNA, is usually single-stranded. RNA mostly exists in the single-stranded form, but there are special RNA Properties of RNA viruses that are double-stranded. A nucleotide in an RNA chain will contain ribose (the five-carbon sugar), one of the four nitrogenous bases (A, U, G, or C), and a phosphate group.  Types of RNA 1. According to Coding ✓ Coding RNA (mRNA) ✓ Non coding RNA (tRNA, rRNA, lncRNA, miRNA, snoRNA, siRNA, snRNA,piRNA) 1. According to Function ✓ Main RNA (mRNA, tRNA, rRNA) ✓ Processing (snRNA) ✓ Regulatory 3. According to size  Types of RNA 1. According to Function ✓ Coding RNA (mRNA) Three main types of RNA are involved in protein synthesis. They are messenger RNA (mRNA), transfer RNA (tRNA), and ribosomal RNA (rRNA). Messenger RNA (mRNA) carries the genetic code from DNA in a form that can be recognized to make proteins. The coding sequence of the mRNA determines the amino acid sequence in the protein produced. Once transcribed from DNA, eukaryotic mRNA briefly exists in a form called “precursor mRNA (pre- mRNA)” before it is fully processed into mature mRNA. This processing step, which is called “RNA splicing”, removes the introns— non-coding sections of the pre-mRNA. There are approximately 23,000 mRNAs encoded in the human genome.  Types of RNA 1. According to Function ✓ Non coding RNA (tRNA, rRNA, lncRNA, miRNA, snoRNA, siRNA, snRNA,piRNA) Ribosomal RNA (rRNA): Ribosomal RNA is the catalytic component of ribosomes. In the cytoplasm, rRNAs and protein components combine to form a nucleoprotein complex called the ribosome which binds mRNA and synthesizes proteins (also called translation). Transfer RNA (tRNA): Transfer RNA is a small RNA chain of about 80 nucleotides. During translation, tRNA transfers specific amino acids that correspond to the mRNA sequence into the growing polypeptide chain at the ribosome.  Types of RNA Main RNA Messenger RNA (mRNA) is an intermediate between a protein-coding gene and its protein product. If a cell needs to make a particular protein, the gene encoding the protein will be turned “on,” meaning an RNA-polymerizing enzyme will come and make an RNA copy, or transcript, of the gene’s DNA sequence. The transcript carries the same information as the DNA sequence of its gene. However, in the RNA molecule, the base T is replaced with U. Ribosomal RNA (rRNA) is a major component of ribosomes, where it helps mRNA bind in the right spot so its sequence information can be read out. Transfer RNAs (tRNAs) are also involved in protein synthesis, but their job is to act as carriers – to bring amino acids to the ribosome, ensuring that the amino acid added to the chain is the one specified by the mRNA. Processing RNA Small Nuclear RNA Small nuclear RNAs (snRNA) are non-coding RNAs that are responsible for splicing introns. The snRNAs join with proteins to form small nuclear ribonucleoproteins (snRNP), Small Nucleolar and Small cytoplasmic. Regulatory RNA (miRNAs and siRNAs) Some types of non-coding RNAs (RNAs that do not encode proteins) help regulate the expression of other genes. Such RNAs may be called regulatory RNAs. For example, microRNAs (miRNAs) and small interfering RNAs siRNAs are small regulatory RNA molecules about 22 nucleotides long. They bind to specific mRNA molecules (with partly or fully complementary sequences) and reduce their stability or interfere with their translation, providing a way for the cell to decrease or fine-tune levels of these mRNAs TRANSCRIPTION  Enzyme of transcription is RNA polymerase. RNA enzymes are Blind Enzymes in Eukaryotes. Transcription factors (proteins) help RNA to determine end and start. Outlines about The templet strand (non-coding) 3′ ----- 5′ is the strand from which RNA is transcribed will be complement to the transcript RNA. transcription The non-templet (coding) 5′----- 3′ identical to mRNA transcript, except that T is replaced with U Transcription of several genes on chromosome Transcription by RNA Polymerase Proceeds in a Series of Steps RNA polymerase proceeds through three phases: Initiation. Elongation. Termination. A. TRANSCRIPTION in Prokaryotes Transcription in Prokaryotes The prokaryotes, which include bacteria and archaea, are mostly single- celled organisms that, by definition, lack membrane-bound nuclei and other organelles. A bacterial chromosome is a covalently closed circle that, unlike eukaryotic chromosomes, is not organized around histone proteins. The central region of the cell in which prokaryotic DNA resides is called the nucleoid. In addition, prokaryotes often have abundant plasmids, which are shorter circular DNA molecules that may only contain one or a few genes. Plasmids can be transferred independently of the bacterial chromosome during cell division and often carry traits such as antibiotic resistance. Prokaryotes do not have membrane-enclosed nuclei. Therefore, the processes of transcription, translation, and mRNA degradation can all occur simultaneously. The intracellular level of a bacterial protein can quickly be amplified by multiple transcription and translation events occurring simultaneously on the same DNA template. 1.Initiation of Transcription in Prokaryotes Prokaryotic RNA Polymerase Promoter  1. Initiation of Transcription in Prokaryotes Prokaryotic RNA Polymerase Prokaryotes use the same RNA polymerase to transcribe all of their genes. In E. coli, the polymerase is composed of five polypeptide subunits, two of which are identical. Four of these subunits, denoted α, α, β, and β′ comprise the polymerase core enzyme. These subunits assemble every time a gene is transcribed, and they disassemble once transcription is complete. Each subunit has a unique role; the two α-subunits are necessary to assemble the polymerase on the DNA; the β-subunit binds to the ribonucleoside triphosphate that will become part of the nascent “recently born” mRNA molecule ; and the β′ binds the DNA template strand. The fifth subunit, σ, is involved only in transcription initiation. It confers transcriptional specificity such that the polymerase begins to synthesize mRNA from an appropriate initiation site. Without σ, the core enzyme would transcribe from random sites and would produce mRNA molecules that specified protein gibberish. The polymerase comprised of all five subunits is called the holoenzyme (a holoenzyme is a biochemically active compound comprised of an enzyme and its coenzyme).  1. Initiation of Transcription in Prokaryotes A promoter is a DNA sequence onto which the transcription machinery binds and initiates transcription. In most cases, promoters exist upstream of the genes they regulate. The specific sequence of a promoter is very important because it determines whether the corresponding gene is transcribed all the time, some of the time, or infrequently. Although promoters vary among prokaryotic genomes, a few elements are conserved. At the -10 and -35 regions upstream of the initiation site, there are two promoter consensus sequences, or regions that are similar across all promoters and across various bacterial species. The -10 consensus sequence, called the -10 region, is TATAAT. The -35 sequence, TTGACA, is recognized and bound by σ. Once this interaction is made, the subunits of the core enzyme bind to the site. The A–T-rich -10 region facilitates unwinding of the DNA template, and several phosphodiester bonds are made. The transcription initiation phase ends with the production of abortive transcripts, which are polymers of approximately 10 nucleotides that are made and released. 1. Transcription Initiation Involves Three Defined Steps The first step is the initial binding of polymerase to a promoter to form what is called a closed complex. In this form, the DNA remains double-stranded, and the enzyme is bound to one face of the helix. In the second step of initiation, the closed complex undergoes a transition to the open complex in which the DNA strands separate over a distance of 13 bp around the start site to form the transcription´bubble. 3rd step is Abortive phase. The phase of initial transcription followed by promoter escape.  2 &3.Elongation and Termination of Transcription in Prokaryotes Elongation The transcription elongation phase begins with the release of the σ subunit from the polymerase. The dissociation of σ allows the core enzyme to proceed along the DNA template, synthesizing mRNA in the 5′ to 3′ direction. Termination Rho-dependent terminators have rather will-defined RNA elements called rut sites (Rho UtilizaTion Site), and for them to work requires the action of the Rho factor. Rho-independent terminators, also called intrinsic termination because they need no other factors to work, consist of two sequence elements: a short inverted repeat (of 20 nucleotides) followed by a stretch of about eight A:T base pairs. When polymerase transcribes an inverted repeat sequence, the resulting RNA can form a stem-loop structure (often called a “hairpin”) by base-pairing with itself. Formation of the hairpin causes termination by disrupting the elongation complex. B. TRANSCRIPTION in Eukaryotes  Genes are stored deep inside a cell, in a locked room called the nucleus. Transcription in These mRNA transcripts escape the nucleus and travel to the Eukaryotic cells ribosomes, where they deliver their protein assembly instructions. RNA polymerase in Eukaryotes The Three Eukaryotic RNA Polymerases (RNAPs) RNA polymerase I synthesizes all of the rRNAs except for the 5S rRNA molecule. RNA polymerase II is located in the nucleus and synthesizes all protein-coding nuclear pre-mRNAs RNA polymerase III This polymerase transcribes a variety of structural RNAs that includes the 5S pre-rRNA, transfer pre-RNAs (pre-tRNAs), and small nuclear pre-RNAs. Transcription promoter in Eukaryotes  1. Transcription initiation Eukaryotes require proteins, called transcription factors (TF), to first bind to the promoter region and then help recruit the appropriate polymerase. The completed assembly of transcription factors and RNA polymerase bind to the promoter, forming a transcription pre-initiation complex (PIC). The TATA box, as a core promoter element, is the binding site for a transcription factor known as TATA-binding protein (TBP), which is itself a subunit of another transcription factor: Transcription Factor II D (TFIID). After TFIID binds to the TATA box via the TBP, five more transcription factors and RNA polymerase combine around the TATA box in a series of stages to form a pre-initiation complex. One transcription factor, Transcription Factor II H (TFIIH), is involved in separating opposing strands of double-stranded DNA to provide the RNA Polymerase access to a single-stranded DNA template.  2 &3.Transcription Elongation and Termination in Eukaryotes Elongation All RNA Polymerases travel along the template DNA strand in the 3′ to 5′ direction and catalyze the synthesis of new RNA strands in the 5′ to 3′ direction. Termination In the case of protein-encoding genes, the cleavage site which determines the “end” of the emerging pre-mRNA occurs between an upstream AAUAAA sequence and a downstream GU-rich sequence separated by about 40-60 nucleotides in the emerging RNA. Once both of these sequences have been transcribed, a protein called Cleavage and polyadenylation specificity factor (CPSF) in humans binds the AAUAAA sequence, and a protein called CStF (cleavge stimulation factor) in humans binds the GU- rich sequence, It is involved in the cleavage of the 3' signaling region from a newly synthesized pre-messenger RNA (mRNA) molecule. CstF is recruited by cleavage and polyadenylation specificity factor (CPSF) and assembles into a protein complex on the 3' end to promote the synthesis of a functional polyadenine tail, which results in a mature mRNA molecule ready to be exported from the cell nucleus to the cytosol for translation. The Poly(A) Polymerase enzyme which catalyzes the addition of a 3′ poly-A tail on the pre-mRNA.  mRNA Proccesing Once RNA polymerase is done, the mRNA transcript has to be processed before it can make its journey out of the nucleus and to the ribosome. Processing has two phases: protection and splicing 1. Protection The 5’ end of a single G nucleotide is attached to the 5’ end of the transcript. This is called the 5’ cap. At the 3’ end of the transcript, a long sequence of A nucleotides are attached. This is called the poly-A tail. 2. Splicing All RNA Polymerases travel along the template DNA strand in the 3′ to 5′ direction and catalyze the synthesis of new RNA strands in the 5′ to 3′ direction, adding new nucleotides to the 3′ end of the growing RNA strand.  The Genetic Code Is Degenerate and Universal The genetic code is degenerate as there are 64 possible nucleotide triplets, which is far more than the number of amino acids. These nucleotide triplets are called codons; they instruct the addition of a specific amino acid to a polypeptide chain. codon: a sequence of three adjacent nucleotides, which encode for a specific amino acid during protein synthesis or translation Sixty-one of the codons encode twenty different amino acids. Most of these amino acids can be encoded by more than one codon. Three of the 64 codons terminate protein synthesis and release the polypeptide from the translation machinery. These triplets are called stop codons. T R A N S L AT I O N Translation is the process by which mRNA is decoded and translated to produce a polypeptide sequence, known as a protein. This method of synthesizing proteins is directed by the mRNA and accomplished with the help of a ribosome, a large complex of ribosomal RNAs (rRNAs) and proteins. Transfer RNA, or tRNA, translates the sequence of codons on the mRNA strand. Translation in Eukaryotes Differences between Prokaryotes and Eukaryotes Translation  Gene regulation is the process of turning genes on and off. These include 1. structural and chemical changes to the genetic material. 2. binding of proteins to specific DNA elements to regulate transcription 3. or mechanisms that modulate translation of mRNA. Gene regulation is the process by which the cell determines which genes will be active and which genes will not be active. And gene regulation is what makes a cell decide to become a red blood cell, or a neuron, or a hepatocyte in the liver, or a muscle cell. Genes have transcription factors, which are proteins that bind to DNA, near these genes. And those transcription factors actually help the RNA machinery get there and transcribe that gene in those cells, and those tissues, transcription factors, rather, are expressed specifically in those tissues. There are also factors expressed in those tissues that will be suppressors that can turn a gene off.  ‫َو َف ْو َق ُكل ِّ ذِي عِ ْل ٍم َعلِيم‬ ‫َ‬ ‫َٰ‬ ‫ت َو ْاْلَ ْر ِ‬ ‫َ‬ ‫َ‬ ‫َّ‬ ‫َ‬ ‫ْ‬ ‫َ‬ ‫ض أَ ْك َب ُر ِمنْ َخ ْل ِق ال َّن ِ‬ ‫اس َول ِكنَّ أكث َر الن ِ‬ ‫اس َل َي ْعل ُمونَ‬ ‫َل َخ ْل ُق ال َّ‬ ‫س َم َاوا ِ‬  PCR Polymerase Chain Reaction RNA DNA Protein  CONTENT PCR Introduction Principle of the PCR The reaction conditions PCR product detection and analysis Types of PCR Real- Time PCR and COVID Overview of molecular techniques based on PCR technology/Applications DNA What if we want to replicate the DNA outside the cell?  INTRODUCTION Mullis 1983 Polymerase Chain Reaction (PCR) was invented by Mullis in 1983 and patented in 1985. Its principle is based on the use of DNA polymerase which is an in vitro replication of specific DNA sequences. This method can generate tens of billions of copies of a particular DNA fragment (the sequence of interest, DNA of interest) from a DNA extract (DNA template). It is possible to selectively replicate it in very large numbers.  Why PCR? PCR DNA extracted from an organism or sample containing DNAs of various origins is not directly analyzable. It contains many mass of nucleotide sequences. It is therefore necessary to isolate and purify the sequence or sequences that are of interest. PCR can therefore select one or more sequences and amplify them by replication to tens of billions of copies. Once the reaction is complete, the amount of matrix DNA that is not in the area of interest will not have varied. In contrast, the amount of the amplified sequence(s) (the DNA of interest) will be very big.  Types of sample PCR makes it possible to obtain, by in vitro replication, multiple copies of a DNA fragment from an extract. Matrix DNA can be genomic DNA as well as complementary DNA (cDNA)obtained by Reverse transcriptase (RT-PCR) from a DNA RNA messenger RNA extract.  Principle PCR This amplification is based on the replication of a double-stranded DNA/cDNA template. The polymerase chain reaction is carried out in a reaction mixture which comprises the DNA extract (template DNA), Taq polymerase, the primers, and the four deoxyribonucleoside triphosphates (dNTPs) in excess in a buffer solution. The tubes containing the mixture reaction are subjected to repetitive temperature cycles several tens of times in the heating block of a thermal cycler, which is based on the nature of DNA interaction with heating. Thermal Cycler (apparatus which has an enclosure where the sample tubes are deposited and in which the temperature can vary, very quickly and precisely, from 0 to 100°C.  Process PCR It is broken down into three phases: Denaturation phase Hybridization (annealing) phase with primers. Elongation phase. The products of each synthesis step serve as a template for the following steps, thus exponential amplification is achieved. PCR Polymerase Chain Reaction The Denaturation It is the separation of the two strands of DNA, obtained by raising the temperature. The first period is carried out at a temperature of 94°C, called the denaturation temperature. At this temperature, the matrix DNA, which serves as matrix during the replication, is denatured: the hydrogen bonds cannot be maintained at a temperature higher than 80°C and the double-stranded DNA is denatured into single-stranded DNA (single-stranded DNA). PCR  The Annealing The second step is hybridization (annealing). It is carried out at a temperature generally between 40 and 70°C, called primer hybridization temperature. Decreasing the temperature allows the hydrogen bonds to reform and thus the complementary strands to hybridize. The primers, short single-strand sequences complementary to regions that flank the DNA to be amplified, hybridize more easily than long strand matrix DNA. The higher the hybridization temperature, the more selective the hybridization, the more specific it is. PCR The Elongation phase The third period is carried out at a temperature of 72°C, called elongation temperature. It is the synthesis of the complementary strand. At 72°C, Taq polymerase binds to primed single-stranded DNAs and catalyzes replication using the deoxyribonucleoside triphosphates present in the reaction mixture. The regions of the template DNA downstream of the primers are thus selectively synthesized. Each cycle theoretically doubles the amount of DNA present in the previous cycle. It is recommended to add a final cycle of elongation at 72°C, especially when the sequence of interest is large (greater than 1 kilobase), at a rate of 2 minutes per kilobase. PCR makes it possible to amplify sequences whose size is less than 6 kilobases. The PCR reaction is extremely rapid, it lasts only a few hours (2–3 hours for a PCR of 30 cycles). PCR Typical PCR Temperature Profile The Reaction Conditions  The Reaction Conditions The volumes of reaction medium vary between 10 and 100 μl. The buffer solution with Taq polymerase. Concentrated 10 times, its formula is approximately the following: 100 mM Tris-HCl, pH 9.0; 15 mM MgCl2, 500 mM KCl, It is possible to add detergents (Tween 20, Triton X-100) or glycerol in order to increase the conditions of stringency that make it harder and therefore more selective hybridization of the primers. This approach is generally used to reduce the level of nonspecific amplifications due to the hybridization of the primers on sequences without relationship with the sequence of interest. dNTPs (deoxyribonucleoside triphosphates) provide both the energy and the nucleotides needed for DNA synthesis during the chain polymerization. They are incorporated in the reaction medium in excess, that is, about 200 μM final. The maximum DNA quantity may in no case exceed 2 μg, the amounts used are in the range of 10–500 ng of template DNA, also DNA matrix is not too degraded. It is also important that the DNA extract is not contaminated with inhibitors of the polymerase chain reaction (detergents, EDTA, phenol, proteins, etc.) PCR  The Reaction Conditions POLYMERASE DNA polymerase allows replication. We use a DNA polymerase purified or cloned from of an extremophilic bacterium, Thermus aquaticus, which lives in hot springs and resists temperatures above 100°C. This polymerase (Taq polymerase) has the characteristic remarkable to withstand temperatures of around 100°C, which are usually sufficient to denature most proteins. Thermus aquaticus finds its temperature of comfort at 72°C, optimum temperature for the activity of its polymerase. High-fidelity A 3´→ 5´ proofreading exonuclease domain is intrinsic to most DNA polymerases. It allows the enzyme to check each nucleotide during DNA synthesis and excise mismatched nucleotides in the 3´ toP5´Cdirection. R  The Reaction Conditions POLYMERASE Hot-start PCR enzymes Platinum II Taq Hot-Start DNA polymerase AmpliTaq Gold DNA polymerase High-fidelity PCR enzymes Platinum SuperFi II DNA Polymerase AccuPrime Taq DNA Polymerase High Fidelity Long-range PCR enzymes Platinum SuperFi II DNA Polymerase GC-rich PCR enzymes Platinum SuperFi II DNA Polymerase Platinum II Taq DNA Polymerase Multiplex PCR enzymes Platinum SuperFi II DNA Polymerase Platinum Multiplex PCR Master Mix Direct PCR enzymes Platinum Direct PCR Universal Master Mix Standard PCR enzymes Taq DNA polymerase AmpliTaq DNA polymerase PCR  The Reaction Conditions PRIMERS Depending on the reaction volume chosen, the primer concentration may vary between 10 and 50 pmol per sample. To achieve selective amplification of nucleotide sequences from a DNA extract by PCR, it is essential to have least one pair of oligonucleotides. These oligonucleotides, which will serve as primers for replication, are synthesized chemically and must be PCR the best possible complementarity with both ends of the sequence of interest that one wishes to amplify. One of the primers is designed to recognize complementarily a sequence located upstream of the fragment 5′–3′ strand DNA of interest; the other to recognize, always by complementarity, a sequence located upstream complementary strand (3′–5′) of the same fragment DNA. Primers are single-stranded DNAs whose hybridization on sequences flanking the sequence of interest will allow its replication so selective. The size of the primers is usually between 10 and 30 nucleotides in order to guarantee a sufficiently specific hybridization on the sequences of interest of the matrix DNA.  The Reaction Conditions PRIMERS Primer Design Specific to sequence of interest Length 18-30 nucleotides Annealing temperature 50°C-70°C - Ideally 58°C-63 ° C GC content 40-60% 3' end critical (new strand extends from here) GC clamp (G or C at 3' terminus) Inner self complementarity: - Hairpins