DNA REPLICATION 5 4 Phosphate 3 DNA structure Nitrogenous base 1 Deoxyribose 2 Nucleotide DNA strand = DNA polynucleotide 2004 Biology Olympiad Preparation Program 2 2004 Biology Olympiad Preparation Program 3 DNA structure Semiconservative replication dsdna is antiparallel. Hydrogen bonds hold the two chains together. Native form of dsdna in cells is the double helix. It is very stable. Conservative parental DNA intact, copy is entirely new. Dispersive daughter molecules contain a mix of old and newly made DNA. 2004 Biology Olympiad Preparation Program 4 Semiconservative daughter molecules contain one old, one new strand of DNA. 2004 Biology Olympiad Preparation Program 5 Origin of replication DNA replication begins at origins of replication. The DNA double helix opens up to form a small bubble. Helicase unwinds the double helix at the ends of the bubble. Single-stranded binding proteins hold the two strands apart. 2004 Biology Olympiad Preparation Program 6 Replication bubbles and forks Replication fork Y-shaped region where new strands of DNA are elongating. 2 replication forks per replication bubble. DNA replication proceeds in both directions of each replication bubble. Multiple bubbles speed up DNA replication. They grow and eventually fuse. 2004 Biology Olympiad Preparation Program 7 1
Prokaryote bubbles Priming Prokaryotes have circular chromosomes, and only have one origin of replication Primase attaches and synthesises a short RNA strand complimentary to one of the DNA strands. Primase works 5 to 3. Required because DNA polymerase cannot initiate its own strand of DNA. 2004 Biology Olympiad Preparation Program 8 2004 Biology Olympiad Preparation Program 9 Elongation leading strand DNA polymerase adds DNA nucleotides to the 3 end of the RNA primer. Elongation lagging strand 5 3 5 3 DNA synthesis always occurs in the 5 to 3 direction. New DNA strand is lengthened by DNA polymerase through complimentary base pairing with the template strand. This is the leading strand. 2004 Biology Olympiad Preparation Program 10 3 Lagging strand cannot be made continuously DNA polymerase can only add nucleotides to a free 3 end. Lagging strand is made of fragments that are linked together. Leading & lagging strands are made at the same time. 5 2004 Biology Olympiad Preparation Program 11 Elongation lagging strand Simultaneous synthesis Primase synthesises an RNA primer. DNA polymerase adds DNA nucleotides to the 3 end of the primer. Process continues, and Okazaki fragments are made. Another DNA polymerase replaces the RNA primer with DNA, and DNA ligase seals gaps, forming the completed lagging strand. 2004 Biology Olympiad Preparation Program 12 Leading strand is made continuously. Lagging strand is synthesised in fragments which are then joined. 2004 Biology Olympiad Preparation Program 13 2
DNA replication Proteins involved in DNA replication Double helix unwinding, providing ssdna templates Helicase Single-stranded binding protein Leading strand Priming Elongation Replacement of RNA primer Primase DNA polymerase DNA polymerase Lagging strand Priming for Okazaki fragment Elongation of fragment Replacement of RNA primer Joining of fragments Primase DNA polymerase DNA polymerase Ligase 2004 Biology Olympiad Preparation Program 14 2004 Biology Olympiad Preparation Program 15 Proof-reading DNA polymerisation DNA polymerase proof-reads each nucleotide as it is added, against the template strand. If it finds an incorrectly paired nucleotide, it removes it and resumes new strand synthesis. DNA polymerase activities: 5 3 polymerase synthesis 3 5 exonuclease proof-reading 5 3 exonuclease removing primers Repairing DNA damage Cells continuously monitor and repair their genetic material. Many repair mechanisms take advantage of base-pairing of DNA. Nucleotide excision repair (left) endonuclease cuts, DNA polymerase fills, ligase seals. Mismatch repair involves enzymes similar to NER. 2004 Biology Olympiad Preparation Program 16 2004 Biology Olympiad Preparation Program 17 The end-replication problem DNA polymerase removes the RNA primer but needs a free 3 end from which to polymerise the primer replacement at the end, this is not available. After repeated replications, the ends of daughter DNA strands gets increasing shorter. Telomeres Special repetitive nucleotide sequences at the ends of chromosomes prevent gene erosion. Telomerase produces a 3 overhang so that successive DNA replications do not reduce overall chromosome length. 2004 Biology Olympiad Preparation Program 18 2004 Biology Olympiad Preparation Program 19 3
DNA replication, in summary Deoxyribonucleic acid is a polymer of nucleotides. Nitrogenous bases in DNA are adenine, guanine, cytosine and thymine. DNA is a double helix in its native form. Replication of DNA occurs in a semiconservative fashion daughter dsdna contain one old and one new strand DNA replication begins at origin(s) of replication where helicase unwinds DNA and single-stranded binding proteins hold the ssdna strands apart. Replication bubbles contain two replication forks. Eukaryotic DNA replication involves multiple bubbles, while prokaryotic involves one. DNA replication, in summary Primase synthesises an RNA primer to which DNA polymerase can add nucleotides. DNA polymerase adds nucleotides to a free 3 end of the primer by base pairing rules. DNA synthesis occurs in the 5 3 direction. The leading strand is synthesised continuously. The lagging strand is composed of Okazaki fragments made by primase, DNA polymerase, and sealed with ligase. It is synthesised in fragments. Leading and lagging strands are synthesised simultaneously. DNA polymerase proof-reads what it polymerises. DNA polymerase has 5 3 polymerase activity, 3 5 exonuclease activity and 5 3 exonuclease activity. 2004 Biology Olympiad Preparation Program 20 2004 Biology Olympiad Preparation Program 21 DNA replication, in summary All cells continuously monitor and repair DNA damage. Nucleotide excision repair is conducted by endonucleases, DNA polymerases and ligases. The inability of DNA polymerase to stick nucleotides on to 5 ends of existing nucleic acid molecules means that linear chromosomes shorten after successive replications. Telomeres are a solution to this problem. They are nonencoding repetitive DNA sequences at ends of eukaryotic chromosomes. Telomerase lengthens telomeres. It is found active in germ-line cells and cancerous cells. GENE EXPRESSION 2004 Biology Olympiad Preparation Program 22 2004 Biology Olympiad Preparation Program 23 One gene one polypeptide The flow of genetic information Study of auxotrophs lead to the one gene one enzyme hypothesis. One gene one polypeptide hypothesis. Not all proteins are enzymes, and not all proteins are made up of only 1 polypeptide chain. Transcription DNA to mrna Translation mrna to polypeptide. 2004 Biology Olympiad Preparation Program 24 2004 Biology Olympiad Preparation Program 25 4
The genetic code 20 amino acids but only 4 nucleotides! Need 3-letter code: 4 3 = 64 combinations, enough to encode 20 amino acids. Triplet code. Template strand transcribed to mrna, codons translated by ribosomes to polypeptide. 2004 Biology Olympiad Preparation Program 26 The genetic code Highly conserved. Redundant but not ambiguous. Reading frame important: THE CAT ATE THE RAT HEC ATA TET HER ATX 2004 Biology Olympiad Preparation Program 27 Transcription initiation RNA polymerase is responsible for transcription. Works 5 to 3 downstream. RNA pol attaches to the promoter, and transcription is ended at the terminator. DNA portion that is transcribed is the transcription unit. Transcription factors bind to the promoter before RNA pol binds. 2004 Biology Olympiad Preparation Program 28 Transcription elongation RNA polymerase moves along the DNA, synthesising new RNA in the 5 3 direction using complementary base pairing rules. This direction is with reference to the newly produced DNA. Double-helix reforms after RNA pol has passed, and RNA strand peels away from template DNA. 2004 Biology Olympiad Preparation Program 29 Transcription termination Transcribed terminator sequence acts as the termination signal. RNA pol drops off as does the newly synthesised pre-mrna. Alterations of mrna ends Coding segment codes for polypeptide, flanked by the start and stop codons and untranslated regions (UTRs). Modified guanine cap attached to 5 end, poly adenine (poly-a) tail added to 3 end. Facilitate ribosome attachment, assist export from nucleus, helps protect mrna from degradation. 2004 Biology Olympiad Preparation Program 30 2004 Biology Olympiad Preparation Program 31 5
RNA splicing Removal of non-coding regions in the coding segment. Small nuclear ribonucleoproteins (snrnps) + other proteins = spliceosome. Introns excised, and exons ligated to form completed mrna. Export Eukaryotes make mrna in the nucleus. mrna must be transported out of nucleus through nuclear pores to cytoplasm where translation can take place. 2004 Biology Olympiad Preparation Program 32 2004 Biology Olympiad Preparation Program 33 trna AminoacyltRNA synthetase Transfer RNA (trna) transfers amino acids to ribosomes. trna molecules differ in their anticodon sequence, which bind to a complementary codon on mrna. Amino acids are joined to their own trna by aminoacyl-trna synthetase. 2004 Biology Olympiad Preparation Program 34 Ribosomes in depth Ribosomes small and large subunits, made of protein and rrna. trna fits into binding sites when its anticodon base pairs with an mrna codon in that site. P site holds the trna attached to the growing polypeptide. A site holds the trna carrying the next amino acid to be added. E site discharged trnas exit here. 2004 Biology Olympiad Preparation Program 35 Translation initiation Small ribosomal subunit binds upstream of the start codon. Moves downstream, finds start codon (nearly always AUG). Initiator trna binds to the start codon, bearing methionine. Large ribosomal subunit binds, forming the initiation complex. Initiator trna sits in the P site. Translation elongation Appropriate trna enters the A site, its anticodon base pairing with the codon exposed in the site. Peptide bond formation is catalysed by the ribosome. Ribosome moves 1 codon downstream, translocating the trna. P trna moves to E and leaves, A trna moves to P, and A site is open to next trna. 2004 Biology Olympiad Preparation Program 36 2004 Biology Olympiad Preparation Program 37 6
Translation termination UAA, UAG, UGA act as stop codons, and do not code for amino acids. Post-translational modification Modifications may be needed after translation is complete to make a functional protein from the polypeptide(s). Release factor (a protein) binds to the stop codon in the A site. The translation assembly falls apart, releasing the completed polypeptide chain. 2004 Biology Olympiad Preparation Program 38 Attachment of sugars, lipids, phosphate groups (phosphorylation), etc. Removal of parts of the polypeptide. Joining polypeptides together. 2004 Biology Olympiad Preparation Program 39 Polyribosomes Signal peptides Once a ribosome has moved past the start codon, another one can attach multiple ribosomes can translate the same mrna simultaneously. These strings of ribosomes are called polyribosomes, or polysomes. 2004 Biology Olympiad Preparation Program 40 Signal peptide targets the polypeptide for the ER. Taken by another protein to the ER. Polypeptide is fed into the ER and folds into its final conformation. 2004 Biology Olympiad Preparation Program 41 Roles of different types of RNA mrna Carries genetic information from DNA to ribosomes. Prokaryote vs eukaryote gene expression RNA polymerase and ribosomes are different. trna rrna Primary transcript snrna SRP RNA Translates mrna codons into amino acids. Found in ribosomes. Precursor to mrna, trna or rrna, and may be processed by cleavage or splicing. Found in spliceosomes. Plays a role in signal peptide recognition. 2004 Biology Olympiad Preparation Program 42 Eukaryotes rely on transcription factors to initiate transcription. Translation and transcription are coupled in prokaryotes no nucleus. 2004 Biology Olympiad Preparation Program 43 7
Gene expression, in summary One gene codes for one polypeptide. Genetic information flows from DNA to RNA to polypeptide. The genetic code is redundant but not ambiguous. Reading frame of codons is important. Transcription copies an RNA message (in the form of mrna) from DNA. Transcriptional initiation requires transcription factors attaching to the promoter before RNA polymerase binds (eukaryotes). Post-transcriptional modifications in eukaryotes include a 5 modified G-cap and 3 poly-a tail. Pre-mRNA is spliced in eukaryotes by spliceosomes, removing introns and ligating exons. Gene expression, in summary mrna is exported out of the nucleus in eukaryotes before translation can begin. Translation interprets the mrna message (in codons) to polypeptides by way of ribosomes and specific trna. Translation is initiated at the start codon, AUG, coding for the initiator trna, carrying methionine. The ribosome catalyses peptide bond formation as trnas bring amino acids to the ribosome. Stop codons (UAA, UAG, UGA) code for a protein release factor that causes the translation assembly to fall apart. Polypeptides may undergo post-translational modifications before becoming a functional protein. 2004 Biology Olympiad Preparation Program 44 2004 Biology Olympiad Preparation Program 45 Gene expression, in summary Many ribosomes can translate a single mrna transcript at once in polyribosomes. Signal peptides target polypeptides for specific destinations in eukaryotes. Many types of RNA exist in cellular metabolic machinery, especially in gene expression. Prokaryotic and eukaryotic RNA polymerase and ribosomes are different. Prokaryotic transcription and translation are coupled. Mutations Mendelian genetics Non-Mendelian genetics Next time When? 2004 Biology Olympiad Preparation Program 46 2004 Biology Olympiad Preparation Program 47 8