BIOL_BCHM_111_23_topic 5.pdf

TOPIC 5: Life requires the expression and transmission of genetic information Be familiar with the experiments used to determine the nature of genetic material Many proteins work together in DNA replication – Case study – PCR can also be used to copy DNA Genes specify proteins via transcription (syn...

TOPIC 5: Life requires the expression and transmission of genetic information Be familiar with the experiments used to determine the nature of genetic material Many proteins work together in DNA replication – Case study – PCR can also be used to copy DNA Genes specify proteins via transcription (synthesis of RNA using a DNA template) and translation (RNA directed synthesis of a protein) Mutations can affect protein structure and function – Case study – how can we use CRISPR/Cas9 to change genetic information (and should we)? How do we store information? What are some of the features of a storage device? How can we retrieve the information? How can we make a copy? How much information do we store? HIV (virus) SARS-CoV-2 E. coli (bacteria) Saccharomyces cerevisia (yeast) Tetraodon nigroviridis (puffer fish) Populus trichocarpa (tree) Homo sapiens (mammal) Protopterus aethiopicus (lungfish) Paris japonica (plant) 9,749 bp 29,900 4,600,000 12,100,000 385,000,000 480,000,000 3,200,000,000 130,000,000,000 150,000,000,000 2.4 kB 7.5 kB 115 kB 605 kB 193 MB 240 MB 1.6 GB 65 GB 75 GB What sort of information do we store? DNA molecule Gene 2 Gene 1 Gene 3 DNA template strand TRANSCRIPTION mRNA Codon TRANSLATION Protein Amino acid What can we find out from your DNA? What can we find out from your DNA? What can we find out from your DNA? What can we find out from your DNA? Ability to Match Musical Pitch Asparagus Odor Detection Back Hair (available for men only) Bald Spot (available for men only) Bitter Taste Bunions Cheek Dimples Cilantro Taste Aversion Cleft Chin Dandruff Earlobe Type Early Hair Loss (available for men only) Earwax Type Eye Color Fear of Heights Fear of Public Speaking Finger Length Ratio Flat Feet Freckles Hair Photobleaching (hair lightening from the sun) Hair Texture Hair Thickness Ice Cream Flavor Preference Light or Dark Hair Misophonia (hatred of the sound of chewing) Mosquito Bite Frequency Motion Sickness Newborn Hair Photic Sneeze Reflex Red Hair Skin Pigmentation Stretch Marks Sweet vs. Salty Toe Length Ratio Unibrow Wake-Up Time Widow's Peak What can we find out from your DNA? The Central Dogma DNA molecule Gene 2 Gene 1 Gene 3 DNA template strand TRANSCRIPTION mRNA Codon TRANSLATION Protein Amino acid How did people identify DNA as a genetic material? Frederick Griffiths (1928) Living S cells (control) Mouse dies Living R cells (control) Mouse healthy Heat-killed S cells (control) Mouse healthy Mixture of heatkilled S cells and living R cells Mouse dies Living S cells Oswald Avery, Maclyn McCarty & Colin MacLeod (1944) Heat killed S cells DNase RNase Protease + living R cells + living R cells + living R cells Alfred Hershey & Martha Chase (1952) Phage Radioactive protein Empty protein shell Radioactivity (phage protein) in liquid Bacterial cell Batch 1: radioactive sulfur (35S) DNA Phage DNA Centrifuge Pellet (bacterial cells and contents) Radioactive DNA Batch 2: radioactive phosphorus (32P) Centrifuge Pellet Radioactivity (phage DNA) in pellet A Structure for Deoxyribonucleic acid A Structure for Deoxyribonucleic acid The structure needs to be chemically sound A Structure for Deoxyribonucleic acid A double helix with antiparallel strands A Structure for Deoxyribonucleic acid Strands are held together by H-bonds between bases A Structure for Deoxyribonucleic acid Strands are complementary - A binds to T, C binds to G A Structure for Deoxyribonucleic acid A Structure for Deoxyribonucleic acid DNA Source Adenine Thymine Guanine Cytosine Calf thymus 1.7 1.6 1.2 1.0 Beef spleen 1.6 1.5 1.3 1.0 Yeast 1.8 1.9 1.0 1.0 Tubercle Bacillus 1.1 1.0 2.6 2.4 A Structure for Deoxyribonucleic acid How do we copy something? “It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material.” Watson & Crick, Nature, 1953 How do we pull the strands apart? Use helicase & topoisomerase The separated strands form a replication bubble Replication fork How do we make a polymer? Use DNA polymerase DNA polymerase cannot join the first two nucelotides Primase makes short RNA primers DNA can only go in one direction The lagging stand contains Okazaki fragments Origin of replication Okazaki Fragments Lagging strand Leading strand Primer Lagging strand Leading strand Overall directions of replication How do we get rid of the RNA primer? DNA polymerase has exonuclease activity, DNA ligase fills the gap Many proteins are involved in DNA replication! PCR (Polymerase Chain Reaction) PCR can amplify DNA without as many enzymes Replication of an enveloped RNA virus Replication of an enveloped RNA virus Minus strand A U G C U C A G U A C G How do we test for Coronavirus: PCR Problem: Extraction of RNA can be tricky, as it’s less stable than DNA Replication of a retrovirus How do we test for Coronavirus: PCR Real-time PCR How do we test for Coronavirus: PCR https://www.sciencedirect.com/science/article/pii/S0031302521000854?via%3Dihub How do we test for Coronavirus: RAT How do we test for Coronavirus: RAT Test solution Marker antibody Antigen Sample pad Antibody for nucleocapsid protein Antibody for marker T C Absorption pad How do we test for Coronavirus: RAT Test solution Sample pad Antibody for nucleocapsid protein Antibody for marker T C Absorption pad How do we test for Coronavirus: RAT Test solution Sample pad Antibody for nucleocapsid protein Antibody for marker T C Absorption pad How do we test for Coronavirus: RAT vs PCR Expression of genes Genes specify proteins via transcription and translation Gene: a discrete unit of hereditary information consisting of a specific nucleotide sequence in DNA (or RNA, in some viruses) Campbell & Reece definition The human genome has 3,200,000,000 base pairs encoding 20,687 protein encoding genes (~1.5 %) Expression of genes Expression of genes Why use RNA, why not use DNA directly as a template? Expression of genes Why use RNA, why not use DNA directly as a template? Nuclear envelope DNA TRANSCRIPTION DNA TRANSCRIPTION Pre-mRNA RNA PROCESSING mRNA mRNA Ribosome TRANSLATION Polypeptide TRANSLATION (a) Bacterial cell Ribosome Polypeptide (b) Eukaryotic cell RNA and proteins are constantly made and degraded http://book.bionumbers.org/how-fast-do-rnas-and-proteins-degrade/ Expression of genes There are several different types of RNA DNA Transcription rRNA (85%) mRNA (4%) Translation Protein tRNA (10%) Transcription is the DNA-directed synthesis of RNA Initiation of gene expression How do we know where to start? Initiation of gene expression How do we know where to start? Elongation of the RNA strand RNA polymerase adds RNA nucleotides Termination of elongation Specific sequences trigger dissociation of RNA polymerase Processing of transcript 5′ G Region that includes protein-coding segments AAA…AAA P P P 5′ Cap 3′ 5′ UTR Start codon Stop codon Poly-A tail Translation is the RNA-directed synthesis of a protein N’- Cys Ser Ser Gly Leu His -C’ The genetic code translates nucleotides into amino acids Read 5’ -> 3’ Why 3? Non overlapping No punctuation The genetic code translates nucleotides into amino acids the code is unambiguous the code is degenerate the code is ordered The genetic code is ordered The genetic code is ordered tRNA is the adaptor molecule 3 Amino acid attachment site 5 5 3 Amino acid attachment site Hydrogen bonds Hydrogen bonds 3 Anticodon Anticodon 5 Anticodon We need to match the correct amino acid amino acid tRNA synthetase tRNA A U A Complementary tRNA anticodon ATP AMP + 2 P i Ribosomes are the machinery involved in protein synthesis Ribosomes have three binding sites How does the ribosome know where to start? Correct binding is required for the right reading frame Elongation of the polypeptide chain Elongation of the polypeptide chain Termination of protein synthesis Mutations can affect protein structure and function Mutations are changes in the genetic material from replication errors or DNA damage Mutations can affect protein structure and function Mutations are changes in the genetic material from replication errors or DNA damage Mutations can affect protein structure and function Small scale mutations include substitutions or insertions & deletions Mutations can affect protein structure and function Many mutations are associated with disease Mutations can affect protein structure and function Many mutations are associated with disease COVID mutations Alpha variant 69del, 70del, 144del, (E484K*), (S494P*), N501Y, A570D, D614G, P681H, T716I, S982A, D1118H (K1191N*) Beta variant D80A, D215G, 241del, 242del, 243del, K417N, E484K, N501Y, D614G, A701V Delta variant T19R, (V70F*), T95I, G142D, E156-, F157-, R158G, (A222V*), (W258L*), (K417N*), L452R, T478K, D614G, P681R, D950N COVID mutations COVID mutations N501Y Asn -> Tyr Asparagine Tyrosine A570D Ala -> Asp Alanine Aspartic acid COVID mutations Deletion of 69/70 Some form of antiviral drug? A key Covid-19 antiviral used to treat vulnerable New Zealanders has been officially deemed useless, but health officials failed to highlight the change with doctors and pharmacists who prescribe the drug. Molnupiravir was one of two antivirals approved for use in the community but was always considered about half as effective as its counterpart, Paxlovid. But because molnupiravir interacted with fewer other drugs than Paxlovid, it was still prescribed in an estimated 30% of patients. But on February 24, following international research stating the drug could fuel mutations of the virus, Te Whatu Ora’s expert advisory group on Covid-19 medicines updated official guidelines to say molnupiravir was no longer recommended “due to lack of clinical benefit”. Some form of antiviral drug? Remdesivir is an inhibitor of RNA dependant RNA polymerases Paxlovid (Nirmatrelvir/ritonavir) Some form of antiviral drug? Molnupiravir reduces accuracy of RNA dependant RNA polymerases Antibodies How to make a vaccine How to make an attenuated or inactivated vaccine How to make a genetic vaccine