DNA Replication, Transcription, and Protein Synthesis PDF

DNA REPLICATION, TRANSCRIPTION AND PROTEIN SYNTHESIS DEPARTMENT OF MEDICAL BIOCHEMISTRY (ABUAD) Dr. A. B. AKAWA  DNA STRUCTURE  DNA usually exists as a double stranded structure, with both strands coiled together to form the characteristic double helix. ...

DNA REPLICATION, TRANSCRIPTION AND PROTEIN SYNTHESIS DEPARTMENT OF MEDICAL BIOCHEMISTRY (ABUAD) Dr. A. B. AKAWA  DNA STRUCTURE  DNA usually exists as a double stranded structure, with both strands coiled together to form the characteristic double helix.  Each single strand of DNA is a chain of four types of nucleotides having the bases: Adenine, Cytosine, Guanine and Thymine (commonly noted as A, C, G and T).  Adenine pairs with thymine with the formation of TWO hydrogen bonds while:  Cytosine pairs with Guanine with the formation of THREE hydrogen bonds.  A purine must always pair with a pyrimidine.  A purine cannot pair with another purine because the strands would be very close to each other and in a pyrimidine pair, there would be one hydrogen not bound to anything, making the DNA unstable.  DNA strands have a directionality and the different ends of a single strand are called the 3’ end and the 5’ end with the direction of the naming going 5’ to the 3’ region. Polynucleotide Chains  DNA REPLICATION  DNA replication is a biological process that occurs in all living organisms and which involves copying of DNA.  It is the basis for biological inheritance.  If a cell does not replicate DNA at all, each daughter cell would only obtain half of a complete genome after cell division occurs.  The process starts when one double stranded DNA molecule produces two identical copies of the molecule.  Each strand of the original double-stranded DNA molecule serves as a template for the production of the complementary strand, a process referred to as Semi- Conservative Replication.  In a cell, DNA replication begins at specific locations in the genome called ORIGINS.  Unwinding of DNA at the origin and synthesis of new strands forms a replication fork.  The synthesis of the new strands of DNA is done by an enzyme called DNA POLYMERASE by adding nucleotides matched to the template strand. Semi conservative DNA replication  The strands of the helix are anti-parallel with one being 5’ to 3’ then the opposite strand 3’ to 5’  Directionality has consequences in DNA synthesis, because DNA polymerase can synthesize DNA in only one direction by adding nucleotides to the 3’ end of a DNA strand. The Replication Fork  The replication fork is a structure that forms within the long helical DNA during DNA replication and it is the area where DNA is being actively copied during DNA replication.  It is produced by enzymes called Helicases that break the hydrogen bonds and hold the DNA strands together in a helix.  The resulting structure has two branching "prongs", each one made up of a single strand of DNA. These two strands serve as the template for the Leading and Lagging strands, which will be created as DNA polymerase matches complementary nucleotides to the templates; the templates may be properly referred to as the leading strand template and the lagging strand template. S/N Enzyme Function in DNA replication DNA helicase Unwinds the DNA double helix at the replication fork 2. DNA primase Provides a starting point of RNA or DNA for DNA polymerase to begin synthesis of the new DNA strand 3. DNA Polymerase Builds a new duplex DNA strand by adding nucleotides in the 5’ to 3’ direction. Also performs proof reading and error correction. 4. DNA Clamp A protein which prevents DNA polymerase III from dissociating from the DNA parent strand 5. Single-stranded DNA Bind to ssDNA and prevent the DNA double helix from re- binding proteins annealing after DNA helicase unwinds it thus maintaining the (SSB) strand separation. 6. Topo-isomerase Relaxes the DNA from its super-coiled nature 7. DNA gyrase Relieves strain of unwinding by DNA helicase 8. DNA Ligase Re-anneales the semi-conservative strand and joins okazaki fragment of the lagging strand 9. Telomerase Lengthens telomeric DNA by adding repetitive nucleotide sequences to the end of eukaryotic chromosomes  DNA is read by DNA polymerase in the 3′ to 5′ direction, meaning the new strand is synthesized in the 5' to 3' direction.  Since the leading and lagging strand templates are oriented in opposite directions at the replication fork, a major issue is how to achieve synthesis of new lagging strand DNA, whose direction of synthesis is opposite to the direction of the growing replication fork. Leading strand  The leading strand is the strand of new DNA which is synthesized in the same direction as the growing replication fork. This sort of DNA replication is continuous.  Lagging strand  The lagging strand is the strand of new DNA whose direction of synthesis is opposite to the direction of the growing replication fork.  Because of its orientation, replication of the lagging strand is more complicated as compared to that of the leading strand.  As a consequence, the DNA polymerase on this strand is seen to "lag behind" the other strand.  The lagging strand is synthesized in short, separated segments.  On the lagging strand template, a primase "reads" the template DNA and initiates synthesis of a short complementary RNA primer.  A DNA polymerase extends the primed segments, forming Okazaki fragments.  The RNA primers are then removed and replaced with DNA, and the fragments of DNA are joined by DNA ligase. Replication Fork Diagrammatic Representations  DNA POLYMERASE  DNA polymerases are a family of enzymes that carry out all forms of DNA replication. However, a DNA polymerase can only extend an existing DNA strand paired with a template strand.  To begin synthesis, a short fragment of DNA or RNA called primer must be created and paired with the template DNA strand.  DNA polymerase then synthesizes a new strand of DNA extending the 3’ end of an existing nucleotide chain, adding new nucleotides matched to the template strand one at a time via the creation of phosphodiester bonds.  When a nucleotide is being added to a growing DNA strand, two of the phosphates are removed and the energy produced creates a phosphodiester- bond that attaches the remaining phosphates to the growing chain.  In general, DNA polymerases are extremely accurate, making less than one mistake for every 107 nucleotides added.  Even some DNA polymerases also have proofreading ability: they can remove nucleotides from the end of a strand in order to correct mismatched bases.  DNA REPAIR MECHANISMS  Even after proofreading, some mistakes may be retained in the print. Similarly, a few defects may remain in the DNA. These are called Mutations.  Mutations are due to a change in the base sequence of DNA. These may result from faulty replication or repair of DNA.  Various physical and chemical agents produce base alterations. However, these errors must be appropriately corrected immediately.  An agent which will increase DNA damage or cell proliferation can cause increased rate of mutations also. Such substances are called MUTAGENS. X-ray, Gamma ray, UV-rays, acridine orange etc are well known mutagens.  It is worthy of note that mutations may not be lethal (deadly or fatal) but may alter the regulatory mechanisms. Such a mutation in a somatic cell may result in uncontrolled cell division leading to cancer.  Any substance causing increased rate of mutation can also increase the probability of cancer. Thus all carcinogens are mutagens.  General Mechanism of DNA repair  The original template DNA usually contains methylated residues (N6-Methyl adenine and 5- methyl cytosine) but the newly synthesized strand will not have methylated bases.  This makes it easy for enzymes to recognize the original (correct) DNA strand.  However, mismatched bases are then identified in the newly synthesized strand and removed along with a few bases around that area.  A small segment of DNA with correct base sequence is then synthesized by DNA polymerase beta.  Then the gap or nick is sealed by DNA ligase.  Diseases Associated with DNA Repair  Xeroderma pigmentosum (XP)  Ataxia Telangectasia Xeroderma Pigmentosum  It is a genetic disorder in which there is a decreased ability to repair DNA damage such as that caused by ultraviolet (UV) light.  Symptoms may include a severe sunburn after only a few minutes in the sun, freckling in sun-exposed areas, dry skin and changes in skin pigmentation. Nervous system problems, such as hearing loss. poor coordination, loss of intellectual function and seizures, may also occur. Complications include a high risk of skin cancer with about half having skin cancer by age 10 without preventative efforts, and cataracts.  There may be a higher risk of other cancers such as brain cancers XP is autosomal recessive, with mutations in at least nine specific genes able to result in the condition. Normally, the damage to DNA which occurs in skin cells from exposure to UV light is repaired by nucleotide excision repair.  In people with xeroderma pigmentosum, this damage is not repaired.  As more abnormalities form in DNA, cells malfunction and eventually become cancerous or die.  Diagnosis is typically suspected based on symptoms and confirmed by genetic testing.  There is no cure for XP.  Treatment involves completely avoiding the sun.  This includes protective clothing, sunscreen and dark sunglasses when out in the sun.  Retinoid creams may help decrease the risk of skin cancer. Vitamin D supplementation is generally required.  If skin cancer occurs, it is treated in the usual way.  The life expectancy of those with the condition is about 30 years less than normal.  The disease affects about 1 in 100,000 worldwide.  It occurs equally commonly in males and females. XP manifestations and features  Ataxia Telangectasia  Ataxia–telangiectasia (AT or A–T), also referred to as ataxia telangiectasia syndrome or Louis–Bar syndrome, is a rare, neurodegenerative disease causing severe disability.  Ataxia refers to poor coordination and telangiectasia to small dilated blood vessels, both of which are hallmarks of the disease. A–T affects many parts of the body:  It impairs certain areas of the brain including the cerebellum, causing difficulty with movement and coordination.  It weakens the immune system, causing a predisposition to infection.  It prevents repair of broken DNA, increasing the risk of cancer.  A–T has an autosomal recessive pattern of inheritance.  A–T is caused by a defect in the ATM gene, named after this disease, which is involved in the recognition and repair of damaged DNA. Heterozygotes will not experience the characteristic symptoms but it has been reported they have higher risks of cancer and heart disease. The prevalence of A–T is estimated to be as high as 1 in 40,000 to as low as 1 in 300,000 people.  Inhibitors of DNA Replication  DNA synthesis inhibitors are a group of antibiotics that target the synthesis of DNA in bacteria and other organisms.  Some antibacterial, antiviral and many chemotherapeutic drugs inhibit replication.  Many antibacterial drugs like Novobiocin, Nalidixic acid and Ciprofloxacin inhibit the prokaryotic enzyme, topoisomerase (DNA gyrase).  These topoisomerase inhibitors are widely used for the treatment of urinary tract and other infections.  Camptothecin, an antitumor drug, inhibits human topoisomerase.  Certain anticancer and antiviral drugs inhibits elongation of DNA chain by incorporating certain nucleotide analogs, e.g. 2, 3 deoxyinosine. CENTRAL DOGMA OF MOLECULAR BIOLOGY  The important role of DNA in transfer of information in living cells is called the central dogma of molecular biology where the information available in the DNA is passed to a messenger RNA which is then used for synthesis of a particular protein.  This process is defined in three major steps.  Replication  Transcription  Translation Replication:- This involves copying of parent DNA to form daughter or new synthesized DNA molecules having nucleotide sequences identical to those of the parent DNA. Transcription:- In this process, the genetic message in DNA are re-written in the form of RNA. Here, the genetic information of DNA is transcribed (copied) to the messenger RNA (mRNA). During transcription, the message from the DNA is copied in the language of nucleotides (ACGU) Translation:- Here, the genetic message transcribed or coded by mRNA is translated by the ribosomes into the protein synthesizing structure in the cytoplasm. During translation, the nucleotide sequence is translated to the language of amino acid sequence (20 STANDARD AMINO ACIDS)  TRANSCRIPTION  In transcription, the genetic information of DNA is transcribed to the messenger RNA (mRNA).  Here, transcription is defined as the synthesis of RNA from DNA that results in the transfer of the information stored in doubled stranded DNA into a single stranded RNA which is used to direct the synthesis of its proteins. Stages of Transcription  Initiation  Elongation  Termination  Post-transcriptional Processing  In mRNA, (transcription stage) Uracil (U) replaces Thymine (T) and it is at this stage that we refer to the 4 letter NUCLEOTIDES, A, G, C, T which becomes A, G, C, U.  Template Strand 3’ C-A-G-T-T-A-G-G-C 5’ Coding Strand 5’ G-T-C-A-A-T-C-C-G 3’ mRNA Transcript 5’ G-U-C-A-A-U-C-C-G 3’  Template strand is transcribed to give rise to mRNA and has the complementary sequence of mRNA.  Coding Strand is the DNA strand whose base sequence corresponds to the base sequence of the RNA transcript produced (although with thymine replaced by uracil). It is complementary to the template strand  REVERSE TRANSCRIPTION  Generally speaking, genes are made up of DNA. However, the genetic materials of some animals and plant viruses are made up of RNA.  Retroviruses is a subgroup of RNA viruses.  The Human-Immunodeficiency virus (HIV) causing AIDs is a retrovirus.  Here, the RNA acts as a template for the synthesis of new DNA molecule i.e synthesis of a DNA molecule from an RNA template by the help of the enzyme REVERSE TRANSCRIPTASE and it is an RNA dependent DNA Polymerase.  Some viruses referred to as retroviruses having RNA as their genetic material and can synthesize double stranded DNA from their genomic RNA by a process known as REVERSE TRANSCRIPTION.  REVERSE TRANSCRIPTION-PCR  Reverse transcription PCR (RT-PCR) uses mRNA rather than DNA as the starting template.  First, the enzyme reverse transcriptase uses the mRNA template to produce a complementary single-stranded DNA called cDNA in a process known as reverse transcription.  Next, DNA polymerase is used to convert the single-stranded cDNA into double-stranded DNA.  These DNA molecules can now be used as templates for a PCR reaction as described below.  The value of RT-PCR is that it can be used to determine if a mRNA species is present in a sample or to clone a cDNA sequence for a subsequent experiment. LINK BETWEEN TRANSCRIPTION AND REVERSE TRANSCRIPTION  POLYMERASE CHAIN REACTION (PCR) (IN-VITRO DNA REPLICATION)  PCR is an in-vitro DNA amplification procedure in which millions of copies of a particular sequence of DNA can be produced within a few hours.  Karry Mullis invented this ingenious method in the year 1989 and was awarded a nobel prize in 1993.  PCR allows scientist to take a very small sample of DNA and amplify it to a large enough amount to study in detail.  PCR is now a common and often indispensable technique used in medical laboratory and clinical laboratory research for a broad variety of applications including biomedical research and criminal forensics.  PCR methods rely on thermal cycling which exposes DNA samples to repeated cycles of heating and cooling to permit different temperature- dependent reactions, using two main reagents known as the primer and a DNA polymerase.  STEPS OF POLYMERASE CHAIN REACTION  DNA strands are first SEPARATED (melted) by heating at 95°C for 15 seconds to 2 minutes.  The primers are ANNEALED by cooling to 50°C and the primers hybridize with their complementary single stranded DNA produced in step 1  New DNA strands are SYNTHESIZED by Taq Polymerase. Taq Polymerase is an enzyme derived from the bacteria, Thermus acquaticus and they are found in hot environments. Therefore, the enzyme is not denatured at high temperature.  The PCR is allowed to take place at 72°C for 30 seconds in the presence of all four deoxyribonucleotide triphosphate (DNTPs) and at this time, both strands of DNA are now duplicated.  The steps of 1, 2, 3 and 4 are then repeated in each cycle and at this time, the DNA strands are already doubled. Thus, 20 cycles provide 1 million times amplification. These cycles are generally repeated by automated equipment called Thermocycler or Tempcycler.  NOTABLE APPLICATIONS OF PCR  Polymerase Chain Reaction (PCR) has many applications, including:  Medical diagnostics: PCR is used to diagnose infectious diseases and determine paternity.  Forensics: PCR is used to analyze genetic markers for forensic applications.  Agriculture: PCR is used to study the diversity of species in ecosystems to understand how they function.  Biomedical research: PCR is used to study gene expression, mutagenesis, methylation analysis, and sequencing. Dentistry: PCR is used to diagnose infectious agents that cause maxillofacial infections, such as caries, periodontal disease, endodontic infections, and oral cancer. Archaeology: PCR is used to analyze DNA from archaeological specimens. Tissue typing: PCR is used to detect mutations relevant for tissue typing.  TRANSLATION (PROTEIN BIOSYNTHESIS)  The DNA is transcribed to mRNA which is translated into protein with the help of Ribosomes.  The information needed to direct the synthesis of protein is contained in the mRNA in the form of a GENETIC CODE.  The genetic code is the system of nucleotide sequences of mRNA that determines the sequence of amino acids in protein.  It is a triplet sequence and it represents the codon for each amino acid e.g the codon for phenylalanine is UUU.  Nirenberg was awarded the nobel prize in 1968 for deciphering the genetic code  There are a total of 64 codons with the exception of the STOP codon known as UGA, UAA and UAG.  FEATURES OF GENETIC CODE  Triplet Codon:- The codes are on the mRNA. Each codon is a consecutive sequence of three bases on the mRNA. E.g UUU codes for phenylalanine  Non Overlapping:- The codes are consecutive. Therefore, the starting point is extremely important. The codes are read one after the other in a continuous manner e.g AUG, CAU, GAU, GCA etc.  Non Punctuated:- There is no punctuation between the codons. It is consecutive or continuous.  Degenerate:- In the genetic code table, it was shown that 61 codes stand for the 20 amino acids. So one amino acid has more than one codon. E.g Serine has 6 codons while glycine has 4 codons. This is called degeneracy of codons.  Unambiguous:- Though the codons are degenerate, they are unambiguous or without any doubtful meaning. That is, one codon stands only for one amino acid.  Universal:- The codons are the same for the same amino acid in all species. The same for Elephant and E-coli  Wobbling Hypothesis Proposed by Crick in 1966 According to this hypothesis, the base in the first position of anti- codon on tRNA is usually an abnormal base, like Inosine, pseudouridine, tyrosine, etc. These abnormal bases are able to pair with more than one type of nitrogenous base in the third position of the codon on mRNA. eg. Inosine (I) can pair with A, C, or U. This base is called a Wobble base or fluctuating base. Wobble occurs at position 1 of the anti-codon and position 3 of the codon. The wobble hypothesis states that the third position (3') of the codon on mRNA and the first position (5') of the anti-codon on tRNA are bound less tightly than the other pair and therefore, offer unusual base combinations. In order to maintain consistency of nucleic acid nomenclature, “I’’ is used for hypoxanthine because hypoxanthine is the nucleobase of inosine The four main wobble base pairs are guanine-uracil (G-U), hypoxanthine-adenine (I-A) hypoxanthine-uracil (I-U) and hypoxanthine-cytosine (I-C)  A single amino acid may be specified by many codons, i.e. called degeneracy. Degeneracy is due to the last base in a codon, which is known as a wobble base. Thus the first two codons are more important in determining the amino acid and the one differs without affecting the coding known as the Wobble hypothesis. Thus according to this, in codon-anticodon pairing, the third base of the tRNA anticodon does not have to pair with a complementary codon. It is called a wobble base and this position is called the wobble position. The actual base pairing occurs in the first two positions only  The pathway of protein biosynthesis is called translation Translation is the process by which ribosomes convert the information carried by mRNA in the form of genetic code to the synthesis of a new protein. The process of translation can be conveniently divided into 5 stages  Activation of amino acid  Initiation  Elongation  Termination  Post-translational processing  Inhibitors of Protein Synthesis Streptomycin It binds to the 30S subunit of prokaryotes at the A site, thereby inhibits chain elongation by preventing the binding of additional aminoacyl tRNA Tetracycline Binds to the 30S subunit and inhibits binding of aminoacyl tRNA to mRNA in prokaryotes Chloramphenicol Binds to the 50S ribosomal subunit and blocks the peptidyl transferase reaction in prokaryotes Erythromycin Binds to the 50S ribosomal subunit that inhibits the translocation reaction in prokaryotes Lincomycin and Binds to the 50S subunit and inhibits peptidyl transferase, thereby, preventing peptide bond formation in prokaryotes Puromycin Causes premature chain termination in both prokaryotes and eukaryotes Cycloheximide Inhibits peptidyl transferase activity of the 60S ribosomal subunit in eukaryotes