PHTH1011 007 Nucleic Acids & Protein Synthesis - Amarakoon.pdf

1 NUCLEIC ACIDSSTRUCTURE AND FUNCTION & PROTEIN SYNTHESIS LECTURER: ICOLYN I. AMARAKOON, MPhil, PhD [email protected] 2 Use all the resources provided - download the lecture notes -use the online resources (video links) provided -attend lectures and READ -ask questions of your lecturer...

1 NUCLEIC ACIDSSTRUCTURE AND FUNCTION & PROTEIN SYNTHESIS LECTURER: ICOLYN I. AMARAKOON, MPhil, PhD [email protected] 2 Use all the resources provided - download the lecture notes -use the online resources (video links) provided -attend lectures and READ -ask questions of your lecturer If you do not understand something, it will not miraculously become clear to you or go away if you ignore it. Take responsibility for your learning and ask for help. Objectives 3  Describe the 3 main components of nucleic acids  Describe the main functions of DNA and RNA  Discuss the basic monomeric structure of DNA and RNA  Demonstrate individual uniqueness by virtue of unique DNA. Why study nucleic acids? 4  DNA is the blueprint for the individuality of an organism. The organism relies upon the information stored in its DNA for the management of every biochemical process.  The life, growth and unique features of the organism depend on its DNA. Overview 5 EXPLORING OUR MOLECULAR SELVESANIMATION VIDEO https://vimeo.com/175257248 Significance 6  Life depends on the ability of cells:  to store, retrieve and translate the genetic instructions required to make and maintain a living organism.  This hereditary information is passed on  from a cell to its daughter cells at cell division,  from one generation of an organism to the next through the organism's reproductive cells. STRUCTURE AND FUNCTION OF DNA & RNA 7 Discovery of DNA Structure 8  One of the most important discoveries in biology  Good illustration of science in action:  Missteps in the path to a discovery  Value of knowledge  Value of collaboration  Cost of sharing your data too early Watson and Crick 9 Franklin and Wilkins 10 Components of Nucleic acids 11 Deoxyribonucleic Acid: DNA is the hereditary material in humans Ribonucleic Acid: RNA is the genetic material that transcribes DNA's instructions and translates these instructions to proteins  DNA and RNA are polymers [polynucleotides]  Monomer unit is a nucleotide. Nucleotide 12 A nucleotide consists of: a 5-carbon sugar (pentose) – deoxyribose for DNA and ribose for RNA  a purine or a pyrimidine base containing nitrogen attached to the sugar  and a phosphate group. 5-carbon sugar (Pentose) 13 Nitrogen bases 14 Nitrogen bases 15 Nucleotide Pentose ring 16 RNA: Ribonucleic Acid 17  Chemically, RNA is very similar to DNA. There are some main differences:  – RNA uses ribose instead of deoxyribose in its backbone.  – RNA uses the base Uracil (U) instead of Thymine (T). U is also complementary to A.  – RNA tends to be single-stranded.  Functional differences between RNA and DNA  – DNA single function, RNA many functions  Example of types of RNA: mRNA, tRNA, rRNA Components of Nucleic Acids 18 DNA & RNA backbone 19 DNA/RNA backbone :  a polymer with an alternating sugar-phosphate sequence.  The deoxyribose sugars are joined at both the 3'-hydroxyl and 5'-hydroxyl groups to phosphate groups in ester links, also known as "phosphodiester" bonds DNA & RNA backbone 20 DNA Helix 21  DNA is a double stranded macromolecule.  Two polynucleotide chains, held together by weak bonds, form a DNA molecule Features of the DNA Double Helix  Two DNA strands form a helical spiral, winding around a helix axis in a right-handed spiral  The two polynucleotide chains run in opposite directions 22 Features of the DNA Double Helix  The sugar-phosphate backbone of the two DNA strands spirals around the helix axis  The bases are on the inside of the helix, stacked like the steps of a spiral staircase. 23 THE DOUBLE HELIX. C Hydrogen bond G 3 end C G G 5 end G C A T C 3.4 nm A T G C G G C A T 1 nm C T C G A G C G A G C C T T A A T 3 end T A (a) Key features of DNA structure 0.34 nm 5 end (b) Partial chemical structure BASE PAIRS RULES 25  Adenine always base pairs with Thymine (or Uracil if RNA)  A’ forms 2 hydrogen bonds with T on the opposite strand  Cytosine always base pairs with Guanine.  G’ forms 3 hydrogen bonds with C on the opposite strand There is exactly enough room for one purine and one pyrimidine base between the two polynucleotide strands of DNA. The standard A=T and G≡C base pairs have very similar geometries and there is exactly enough room for one purine and one pyrimidine base between the strands of DNA. incorrectly paired bases can exclude them from the active site (Blue shade) 26 27 What is a Gene? 28  A gene is the basic physical and functional unit of heredity.  Genes, which are made up of DNA, act as instructions to make molecules called proteins.  Every person has two copies of each gene, one inherited from each parent What is a chromosome? 29  In the nucleus the DNA molecule is packaged into thread-like structures called chromosomes.  Chromosomes are not visible in the cell’s nucleus—not even under a microscope—when the cell is not dividing. DNA Gene 30 Chromosome Nucleic Acids Concept Map 31 32 REPLICATION: The process of copying a parental DNA molecule to form two daughter DNA molecules Objectives 33  Describe the functions of DNA replication proteins- DNA polymerase, RNA primase, helicase, DNA ligase  Illustrate and discuss the DNA replication fork and the coping of the anti-parallel strands  Describe the function of RNA polymerase.  Discuss the phases of the transcription cycle  Have a sense of the sequential flow of information as illustrated by the central dogma of molecular biology. OVERVIEW 34  DNA is situated in the nucleus, organized as chromosomes  Every cell must contain the genetic information and the DNA is duplicated before a cell divides (Replication)  When proteins are needed, the genes are transcribed into RNA (Transcription)  RNA is processed then transported out of the nucleus  Outside the nucleus, the proteins are built based upon the code in the RNA (Translation) The Central Dogma of Molecular biology 35  The flow of genetic information : DNA → RNA → Protein DNA REPLICATION TRANSCRIPTION TRANSLATION  A gene is expressed in two steps: DNA is transcribed to RNA  RNA is translated into protein  DNA: ‘the book of life’ 36  DNA is the physical carrier of inheritance  How is DNA, inherited from one cell to its daughter cells and from one generation to another?  REPLICATION  Replication "copying followed by cell division machine“ DNA repair system DNA replication 37  One of the single strands of the double-stranded DNA molecules is the template for the generation of the other.  DNA replication is semi-conservative.  Each of the two molecules double-stranded molecules resulting from replication contains one old and one new single strand. The Meselson-Stahl Experiment 38  The Meselson-Stahl experiment was about the origin of the two strands in each of the daughter genomes  Bacterial cells were grown on a medium containing only 15N isotope until all their DNA became fully 15N labeled  Cells were then switched to 14N medium and allowed to divide once  CsCl density gradient centrifugation was used to determine the mass of genomic DNA before and after each round of replication 39 40 The MeselsonStahl Experiment Animation http://highered.mcgrawhill.com/sites/dl/free/0072 437316/120076/bio22.swf DNA Replication is Semi-conservative 41  The Meselson-Stahl experiment showed that the nitrogen used for the synthesis of new dsDNA becomes equally divided between the two daughter genomes  This suggests a semi-conservative replication mechanism 42 Replication of DNA suggested by Watson and Crick. The preexisting or "parent" strands become separated, and each is the template for biosynthesis of a complementary "daughter" strand (in pink). Steps in DNA Replication 43  Initiation : this involves the assembly of a replication fork (bubble) at an origin of replication sequence of DNA  Elongation : this is the addition of bases -Parental strands unwind and daughter strands are synthesized.  Termination: the duplicated strands separate from each other. REPLICATION: Initiation 44  Helicase is the enzyme that splits the two strands by uncoiling the double stranded twisted DNA. The unwinding of the two strands is the starting point.  Origin of replication is the initiation point where the splitting starts.  Replication Fork is the open bubble structure that was created. REPLICATION: Initiation cont. 45  The RNA Primase binds at the initiation point of the 3'- 5' parent chain  RNA Primase can attract RNA nucleotides which bind to the DNA nucleotides of the 3'-5' strand due to the hydrogen bonds between the bases  The primers (starters) are the RNA nucleotides for the binding of DNA nucleotides. Replication Fork 46 REPLICATION: Elongation 47 The elongation process is different for the 5'-3' and 3'-5' template  3'-5' Template (leading strand )  The 5'-3' proceeding daughter strand -that uses a 3'-5' template- is called leading strand because DNA Polymerase ä can "read" the template and continuously adds nucleotides  DNA polymerase read the template in 3’-5’ direction to produce a matching stand 5’- 3’ REPLICATION: Elongation 48  5'-3‘ Template (lagging strand)  The 5'-3' template cannot be "read" by DNA Polymerase is called lagging strand.  RNA primase adds RNA primers. DNA polymerase å reads the template and lengthens  Okazaki fragments are the fragments separated by a gap between two RNA primers  The DNA Pol I (exonuclease) removes the RNA primers. The gaps are filled by the action of   DNA Polymerase (adds complementary nucleotides ) DNA Ligase (adds phosphate to the phosphate - sugar backbone) REPLICATION: Termination 49  This process happens when the DNA Polymerase reaches the end of the strands  The ends of DNA consists of non-coding DNA that contains repeat sequences and are called telomeres, which are removed in every cycle of replication  The DNA replication is not completed before a mechanism of repair fixes possible errors  Enzymes like nucleases remove the wrong nucleotides and the DNA Polymerase fills the gaps Enzymes of DNA Replication 50  Helicase: Unwounds DNA Double Helix  RNA Primase: Attaches RNA primers to the replicating strands.  DNA Polymerase : Binds to the 5' - 3' strand to create the     daughter leading strand. DNA Polymerase : Binds to the 3' - 5' strand to create discontinuous segments starting from different RNA primers. Exonuclease (DNA Polymerase): Finds removes RNA Primers DNA Ligase: Adds phosphate in the remaining gaps of the phosphate - sugar backbone Nucleases: Remove wrong nucleotides from the daughter strand DNA Replication Animation 51 HOW NUCLEOTIDES ARE ADDED IN DNA REPLICATION: https://youtu.be/gSebRSfo23Y Summary: DNA Replication 52 RNA TRANSCRIPTION 53 TRANSCRIPTION - The process by which a DNA template is copied to an RNA strand TRANSCRIPTION 54  The first step in expressing a gene  Occurs in the nucleus  DNA-directed RNA synthesis  An RNA copy of DNA is made.  This RNA serves as a messenger between the nucleus and the cytoplasm (mRNA). Major Classes of RNA 55  mRNA - messenger RNA  carries the genetic information that will be expressed ultimately as proteins.  tRNA - transfer RNA  is the adapter molecule. It recognizes the codons of the mRNA at one end, and covalently bonded to the appropriate amino acid, on the other end.  rRNA - ribosomal RNA  is found in the ribosomes which are the ‘protein factories’. Types of RNA involved in protein synthesis Messenger RNA [mRNA] -the template Ribosomal RNA [rRNA] -structural component of the ribosome Transfer RNA [tRNA] - the adapter 56 RNA TRANSCRIPTION 57  The DNA never leaves the nucleus, instead the genetic code (the genes) is copied into RNA which then in turn is decoded (translated) into proteins in the cytoplasm RNA works as a sort of throw-away version of DNA - good for limited work but not for long-term storage. regulate the rate of protein synthesis RNA TRANSCRIPTION 58 The RNA transcription process also occurs in three stages: initiation chain elongation termination Where does transcription initiate? 59  RNA polymerase recognizes the beginning of a gene  It is directed to the start site of transcription by one of its subunits  the RNA Polymerase-Promoter Complex (RNA transcription factors) binds to the promoter gene in the DNA  finding of the start sequence for the RNA polymerase  The promoter is unidirectional sequence (TATA) on one strand of the DNA that tells the RNA polymerase both where to start and in which direction to go RNA TRANSCRIPTION 60 RNA TRANSCRIPTION: Elongation 61  Elongation begins when RNA polymerase "reads" the template DNA  The RNA polymerase recruits rNTPs (ribonucleic nucleotides triphosphates)  Synthesis is single stranded and proceeds in the 5' to 3' direction  there is no need for Okazaki fragments. RNA TRANSCRIPTION 62 63 Transcription Animation 64 https://youtu.be/vLz2A1cjPH8 65 PROTEIN SYNTHESIS FROM DNA TO PROTEINS https://youtu.be/gG7uCskUOrA Objectives 66  Illustrate and discuss the functions of the ribosome and charged tRNAs.  Outline the phases of translation: initiation, elongation and termination  Evaluate and interpret the genetic code  Discuss and demonstrate the central dogma of molecular biology mRNA PROCESSING 67 mRNA undergo three major modifications before it leaves the nucleus and be translated into a protein  Capping - a special nucleotide is attached at the 5' end of the mRNA  Poly(A)-tail - a special enzyme attaches a chain of 150-200 adenine nucleotides to the 3' end of the pre-mRNA directly after transcription  Splicing - The removal of non-coding sequences, introns, from the pre-mRNA to create mRNA 68 mRNA TRANSPORT 69  The mRNA has to exit the nucleus to the cytoplasm for protein synthesis through channels called the nuclear pore complex. RNA TRANSLATION 70 TRANSLATION : the synthesis of proteins directed by a mRNA template OVERVIEW 71  The information contained in the nucleotide sequence of the mRNA is read as three letter words (triplets), called codons. Each word stands for one amino acid  Translation is dependent on two main components  the ribosome - the cellular factory responsible for the protein synthesis  the tRNA - specialised RNA molecule that carries an amino acid at one end and has a triplet of nucleotides, an anticodon, at the other end Types of RNA involved in protein synthesis Messenger RNA [mRNA] -the template Ribosomal RNA [rRNA] -structural component of the ribosome Transfer RNA [tRNA] - the adapter 72 tRNA Structure 73 Transfer–RNA (tRNA) 74  Main functions of tRNA: To recognise and read the codon of the mRNA to fetch correct amino acid  transfer a specific amino acid to the growing polypeptide chain at the ribosomal site Ribosomal Binding sites 75 4 binding sites for RNA on the ribosome: one for mRNA and three for tRNA  mRNA site - binds the mRNA for protein synthesis  A site (acceptor site)- binds to the tRNA holding the new amino acid to be added to the polypeptide chain.  P site (the peptidyl site) - binds to the tRNA holding the growing polypeptide chain of amino acids  E site (exit site) - serves as a threshold, the final transitory step, where the discharged tRNAs leave the ribosome. Ribosome Structure 76 Ribosomes are composed of two subunits, one small and one large. Four binding sites are located on the ribosome RNA Translation: Initiation 77  small subunit of a ribosome charged with a tRNA + the amino acid methionine encounters an mRNA, it attaches and starts to scan for a start signal.  finds the start sequence AUG, the codon (triplet) for the amino acid methionine, large subunit joins the small one to form a complete ribosome  protein synthesis is initiated Ribosomal Assembly and Initiation 78  both ribosomal subunits (small and large) assemble at the start codon (towards the 5' end of the mRNA) Ribosomal Assembly and Initiation 79  Once the small subunit associates with an mRNA molecule, the two subunits come together, creating a compactor that keeps the mRNA and tRNA in stable and proper orientation for protein synthesis RNA Translation: Elongation Overview 80  A new tRNA+amino acid enters the ribosome, at the next codon downstream of the AUG codon. If its anticodon matches the mRNA codon it base pairs and the ribosome link the two amino acids together.  The ribosome then moves one triplet forward and a new tRNA+amino acid can enter the ribosome and the procedure is repeated Translation Elongation: Codon recognition 81  The initial tRNA with attached amino acid is in the P site. A new tRNA, corresponding to the next codon on the mRNA, binds to the A site Translation Elongation: Peptide bonds 82  A peptide bond is formed between the incoming amino acid and the end of the polypeptide chain on the tRNA in the P site. The polypeptide chain is transferred to the tRNA in the A site during this process Translation Elongation: Translocation 83  The ribosome moves one codon down the mRNA, positioning the next codon to be read into the A site, and moving the tRNA with the growing polypeptide chain into the P site. Translation: Elongation 84 The process is repeated: the tRNA at the P site holds the peptide chain, and a new tRNA binds to the A site. The peptide chain is transferred onto the amino acid attached to the A site tRNA. the ribosome moves down one codon, displacing the empty P site tRNA and moving the tRNA with the peptide chain from the A site to the P site Translation: Termination 85  When the ribosome reaches the stop codons, eg.UGA, there are no corresponding tRNAs to that sequence. Instead termination proteins (release factors) bind to the ribosome and stimulate the release of the polypeptide chain, and the ribosome dissociates from the mRNA. Translation: Termination 86  When the ribosome is released from the mRNA, its large and small subunit dissociate  The small subunit can now be loaded with a new tRNA+methionine and start translation once again Post-Translation process 87  Amino acids may be cleaved from the polypeptide, or the entire chain may be cut into multiple pieces  New polypeptides usually fold themselves spontaneously into their active conformation.  Proteins have sugars, phosphate groups, fatty acids, and other molecules covalently attached to certain amino acids in the endoplasmic reticulum.  Many proteins are targeted to specific organelles within the cell. Targeting is accomplished through “signal sequences” on the polypeptide. Post-Translation process 88 THE GENETIC CODE 89 Genetic code 90  The nucleotide sequence of the mRNA is composed of four different     nucleotides whereas a protein is built up from 20 amino acids. To allow the four nucleotides to specify 20 different amino acids, the nucleotide sequence is interpreted in codons, groups of three nucleotides. These codons have their corresponding anticodon in the tRNA. each anticodon is linked to one particular amino acid. each codon specifies one amino acid. This is referred to as the genetic code Genetic code 91 GENETIC CODE  The sentence in the DNA language is a series of words that gives a sequence of amino acids because of the triplet nature of the DNA language DNA sentence AACGTATCGCAT would be read as a polypeptide chain composed of the amino acids  the leucine-histidine-serine-valine GENETIC CODE  the nucleotide DNA triplets ATT, ATC, and ACT are analogous to a full stop (.) in ending a sentence—all three signal the end of a polypeptide chain.  DNA, just like a book, is organized into chapters. The book for humans consists of 23 different chapters or chromosomes. Dictionary of tRNA codons & their amino acids (symbols & abbreviations) 94 PARTIALLY SOLVED MESSAGE 95 GIVEN: DNA code message --> GAA SOLUTION (steps 1-4) 1. mRNA (built to match the DNA -------> CUU 2. tRNA (determined by matching ---------> GAA 3. Amino acids carried by L each tRNA (according to e dictionary, below)----- ----> u 4. Symbols of amino acids:--> L TAG AAA CTT ACT TAG AGC ATT CCT GCC CTT CGA TGC ATC message, letter AUC UUU GAA UGA letters (bases) UAG AAA CUU ACU I P G s h l o e u I F E - for letter AUC UCG..................... with those in mRNA) UAG........................ I s o I........ Activity: Genetic Message 96  Decode this message: CTA TTA CGA ACT TAG AGC ATT GAA TAG AAA CTT ATC SUMMARY 97 Student Learning Assessment  1. A nucleotide is made of three parts: a ___________________ group, a five carbon __________________, and a nitrogen containing _____________________  2. In a single strand of DNA, the phosphate group binds to the __________ of the next group.  3. The 5' end of a single DNA strand contains a free __________________, while the 3' end contains a free __________________________.  4. Chromosomes consist of what two substances: ___________ & ___________  5. Write out the complete name for DNA: ________________  6. Purines have _________ rings, and pyrimidines have ____________ ring.  7. In DNA, thymine is complementary to ________ ; cytosine is complementary to_____________  8. In a strand of DNA, the percentage of thymine is 30 %. What is the percentage of cytosine in the same DNA strand? _________________  9. Number the steps of DNA replication in the correct order (1, 2, 3)  _______Daughter strands are formed using complementary base pairing.  ______ DNA unwinds  ______ The DNA of the daughter strands winds with together with its parent strand.  10. Why is DNA replication called "semi-conservative"?_____________________  11. What enzyme unwinds and unzips the parent strand? ________________  12. The junction between separated strands is called the ____________________  13. What enzyme synthesizes the new DNA strand? _______________________  14. What enzyme binds fragments of DNA on the lagging strand? _____________  15. DNA polymerase only travels in the ________ to _________ direction. 98 OTHER RESOURCES 99  The Central Dogma: DNA to proteins, is a 30 min video - https://youtu.be/QvNdzLALvkI (in this video up to about the 15 min mark is related to this series of lectures) Questions??, Queries, ‘Not so sure about that,’ Please say that again, ‘Does that mean….’