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Lecture PowerPoint to accompany Inquiry into Life Twelfth Edition Sylvia S. Mader Chapter 24 GCU DNA, RNA Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 1 24.1 DNA Structure and Replication • Hershey-Chase Experiments (1952) – Demonstrated that DNA is the genetic material that directs all cells – DNA stands for Deoxyribonucleic Acid 2 Hershey-Chase Experiments http://highered.mcgraw-hill.com/olc/dl/120076/bio21.swf 3 24.1 DNA Structure and Replication • Structure of DNA – James Watson and Francis Crick determined the structure of DNA in 1953 – DNA is a chain of nucleotides – Each nucleotide is a complex of three subunits • Phosphoric acid (phosphate) • A pentose sugar (deoxyribose) • A nitrogen-containing base (adenine, guanine, cytosine, thymine) 4 24.1 DNA Structure and Replication H • Structure of DNA – Four Possible Bases • Adenine (A) - a purine N Sugar • Cytosine (C) - a pyrimidine – Complimentary Base Pairing N N Adenine (A) • Guanine (G) - a purine • Thymine (T) - a pyrimidine N H N O N N Sugar O CH3 H N N O Sugar Thymine (T) H H N N H N N N H H Guanine (G) O N Sugar Cytosine (C) • Adenine (A) always pairs with Thymine (T) • Guanine (G) always pairs with Cytosine (C) • http://highered.mcgrawhill.com/sites/dl/free/0072835125/126997/animation 12.html 5 Overview of DNA Structure 6 24.1 DNA Structure and Replication • Replication of DNA – Semi-conservative replication • Each daughter DNA molecule consists of one new chain of nucleotides and one from the parent DNA molecule – The two daughter DNA molecules will be identical to the parent molecule A C T A G T G A T C A C T A G T G A T C (a) The parent molecule has two (b) The first step in replication is complementary strands of DNA. separation of the two DNA Each base is paired by hydrogen strands. bonding with its specific partner, A with T and G with C. A C T A G T G A T C A C T A G T G A T C (c) Each parental strand now serves as a template that determines the order of nucleotides along a new, complementary strand. A C T A G T G A T C A C T A G T G A T C (d) The nucleotides are connected to form the sugar-phosphate backbones of the new strands. Each “daughter” DNA molecule consists of one parental strand and one new strand. 7 24.1 DNA Structure and Replication • Replication of DNA – Before replication begins, the two strands of the parent molecule are hydrogen-bonded together between the bases – DNA helicase (enzyme) unwinds and “unzips” the doublestranded DNA – New complementary DNA nucleotides fit into place along divided strands by complementary base pairing. These are positioned and joined by DNA polymerase (enzyme) – DNA ligase (enzyme) repairs any breaks in the sugar-phosphate backbone – The Two double helix molecules identical to each other and to the original DNA molecule 8 Overview of DNA Replication http://highered.mc grawhill.com/sites/dl/fr ee/0072835125/1 26997/animation1 6.html 9 Ladder Configuration and DNA Replication 10 24.2 Gene Expression • Gene: A segment of DNA that specifies the amino acid sequence of a polypeptide • DNA does not directly control protein synthesis, instead its information is transcribed into RNA • The “Central Dogma”: 11 24.2 Gene Expression • RNA (ribonucleic acid) 12 24.2 Gene Expression • Three Classes of RNA – Messenger RNA (mRNA) • Takes a message from DNA to the ribosomes • strand – Ribosomal RNA (rRNA) • Makes up ribosomes (along with proteins) • globular – Transfer RNA (tRNA) • Transfers amino acids to ribosomes • Hairpin shape 13 24.2 Gene Expression • Gene Expression Requires Two Steps: • Transcription – Is the synthesis of RNA under the direction of DNA – Produces messenger RNA (mRNA) • Translation – Is the actual synthesis of a polypeptide, which occurs under the direction of mRNA – Occurs on ribosomes http://highered.mcgrawhill.com/sites/dl/free/0072835125 /126997/animation1.html 14 Ribonucleic Acid • Why would the cell want to have an intermediate between DNA and the proteins it encodes? – The DNA can then stay pristine and protected, away from the caustic chemistry of the cytoplasm. – Gene information can be amplified by having many copies of an RNA made from one copy of DNA. – Regulation of gene expression can be effected by having specific controls at each element of the pathway between DNA and proteins. – The more elements there are in the pathway, the more opportunities there are to control it in different circumstances. 15 24.2 Gene Expression • Transcription – During transcription, a segment of the DNA serves as a template for the production of an RNA molecule – Messenger RNA (mRNA) • RNA polymerase (enzyme) binds to a promoter (“start” sequence) • DNA helix is opened so complementary base pairing can occur • RNA polymerase joins new RNA nucleotides in a sequence complementary to that on the DNA, in a 5’ to 3’ direction 16 Transcription of DNA to form mRNA 17 Messenger RNA • mRNA - of the 64 possible 3-base combinations: – 61 code for the twenty different amino acids – 3 code for "stop"; i.e. chain termination • Specific nucleotide sequences call for “start” of transcription (usually AUG = methionine) = PROMOTOR sequence • “stop” of mRNA synthesis = TERMINATION sequence (UAA, UGA, UAG) • Finished mRNA strands are ~500-10,000 nucleotides long 18 • During transcription – The gene determines the sequence of bases along the length of an mRNA molecule Gene 2 DNA molecule Gene 1 Gene 3 DNA strand 3 (template) A C C A A A C C G A G T 5 TRANSCRIPTION mRNA U G G U U U G G C U C A 5 3 Codon TRANSLATION Protein Figure 17.4 Trp Amino acid Phe Gly Ser 19 24.2 Processing of mRNA • After Transcription • Primary “Pre-”mRNA must be modified into mature mRNA – Introns are intragene segments (often, junk) – Exons are the portion of a gene that is expressed • Intron sequences are removed, and a poly-A tail is added – Ribozyme splices exon segments together – http://highered.mcgrawhill.com/sites/dl/free/0072835125/126997/animation20.html 20 mRNA Processing pre-RNA must be modified before translation 21 The Functional and Evolutionary Importance of Introns • The presence of introns – Allows for alternative RNA splicing – Animations of RNA processing: http://highered.mcgrawhill.com/olcweb/cgi/pluginpop.cgi?it=swf::535::535::/sites/dl/free/00 72437316/120077/bio25.swf::Processing%20of%20Gene%20Infor mation%20-%20Prokaryotes%20versus%20Eukaryotes – http://highered.mcgrawhill.com/olcweb/cgi/pluginpop.cgi?it=swf::535::535::/sites/dl/free/00 72437316/120077/bio30.swf::How%20Spliceosomes%20Process% 20RNA 22 24.2 Gene Expression • Translation – The Genetic Code • Triplet code: each 3-nucleotide unit of a mRNA molecule is called a codon • There are 64 different mRNA codons – 61 code for particular amino acids » Redundant code; some amino acids have numerous code words » Provides some protection against mutations – 3 are stop codons signal polypeptide termination 23 Messenger RNA Codons 24 Overview of Gene Expression Protein synthesis http://highered.mcgrawhill.com/olcweb/cgi/plugi npop.cgi?it=swf::535::53 5::/sites/dl/free/0072437 316/120077/micro06.sw f::Protein%20Synthesis 25 24.2 Gene Expression • Transfer RNA – tRNA transports amino acids to the ribosomes (creates polypeptide chain) – Single stranded nucleic acid that correlates a specific nucleotide sequence with a specific amino acid – Amino acid binds to one end, the opposite end has an anticodon – the order of mRNA codons determines the order in which tRNA brings in amino acids Protein synthesis http://highered.mcgrawhill.com/olcweb/cgi/pluginp op.cgi?it=swf::535::535::/si tes/dl/free/0072437316/12 0077/micro06.swf::Protein %20Synthesis 26 Transfer RNA: Amino Acid Carrier 27 rRNA – Ribosomal RNA is the most abundant type of RNA in cells – Ribosomes: comprised of subunits 2/3 RNA, 1/3 protein • Two populations of ribosomes are evident in cells, Free and bound • Free ribosomes in the cytosol initiate the synthesis of all proteins 28 • The ribosome has three binding sites for tRNA – The P site – The A site – The E site P site (Peptidyl-tRNA binding site) A site (AminoacyltRNA binding site) E site (Exit site) Large subunit E mRNA binding site Figure 17.16b P A Small subunit (b) Schematic model showing binding sites. A ribosome has an mRNA binding site and three tRNA binding sites, known as the A, P, and E sites. This schematic ribosome will appear in later diagrams. 29 • Concept 17.4: Translation is the RNAdirected synthesis of a polypeptide: a closer look Quicktime movie: http://carbon.cudenver.edu/~bstith/transla.MOV Narrated animation: http://highered.mcgrawhill.com/olcweb/cgi/pluginpop.cgi?it=swf::535::535::/sites/dl/free/0072437316/ 120077/micro06.swf::Protein%20Synthesis Interactive practice: http://learn.genetics.utah.edu/content/begin/dna/transcribe/ 30 24.2 Gene Expression • Ribosome and Ribosomal RNA – Ribosome has a binding site for mRNA and for 2 tRNAs – Facilitates complementary base pairing – Ribosome moves along mRNA and new tRNAs come in and line up in order – This brings amino acids in line in a specific order to form a polypeptide – Several ribosomes may move along the same mRNA • Multiple copies of a polypeptide may be made • The entire complex is called a polyribosome 31 Polyribosomes • A number of ribosomes can translate a single mRNA molecule simultaneously – Forming a polyribosome Completed polypeptide Growing polypeptides Incoming ribosomal subunits Start of mRNA (5 end) End of mRNA (3 end) (a) An mRNA molecule is generally translated simultaneously by several ribosomes in clusters called polyribosomes. Ribosomes mRNA 0.1 µm Figure 17.20a, b (b) This micrograph shows a large polyribosome in a prokaryotic cell (TEM). 32 Translation (Building a polypeptide) requires Three Steps: – Initiation (requires energy) – Elongation (requires energy) – Termination Amino end Growing polypeptide Next amino acid to be added to polypeptide chain tRNA 3 mRNA 5 Codons (c) Schematic model with mRNA and tRNA. A tRNA fits into a binding site when its anticodon base-pairs with an mRNA codon. The P site holds the tRNA attached to the growing polypeptide. The A site holds the tRNA carrying the next amino acid to be added to the polypeptide chain. Discharged tRNA leaves via the E site. 33 Summary of Gene Expression 34 24.2 Gene Expression • Genes and Gene Mutations – A gene mutation is a change in the sequence of bases within a gene. – Gene mutations can lead to malfunctioning proteins in cells. 35 24.2 Gene Expression • Genes and Gene Mutations – Causes of Mutations • Errors in replication – Rare – DNA polymerase “proofreads” new strands and errors are cleaved out • Mutagens – Environmental influences – Radiation, UV light, chemicals – Rate is still fairly low because DNA repair enzymes monitor and repair DNA 36 Transposons Transposons • “jumping genes” • Can move to new locations and disrupt gene sequences 37 Types of Gene Mutations Point Mutations – The substitution of one nucleotide for another • Missense mutations – a point mutation in which a single nucleotide is changed, resulting in a codon that codes for a different amino acid – Missense mutations are responsible for about 75% of the mutations in the p53 gene. Mutations of this gene are responsible for about 30-50% of cancers in humans • Silent mutations • Nonsense mutations – mutations that change an amino acid to a stop codon 38 Types of Gene Mutations • Frameshift Mutations – One or more nucleotides are inserted or deleted – Results in a polypeptide that codes for the wrong sequence of amino acids – Codons must be read in the correct reading frame for the specified polypeptide to be produced 39 DNA Technology • In laboratory experiments – Genes can be transcribed and translated after being transplanted from one species to another • Called “Recombinant DNA” technology • Can be produced via “Genetic Engineering” (laboratory manipulation) 40 Overview: Understanding and Manipulating Genomes • One of the greatest achievements of modern science has been the sequencing of the human genome, which was largely completed by 2003 • DNA sequencing accomplishments – Have all depended on advances in DNA technology, starting with the invention of methods for making recombinant DNA – DNA sequencing animation 41 DNA Cloning • Concept 20.1: DNA cloning permits production of multiple copies of a specific gene or other DNA segment • To work directly with specific genes – Scientists have developed methods for preparing well-defined, gene-sized pieces of DNA in multiple identical copies, a process called gene cloning 42 24.3 DNA Technology • The Cloning of a Gene – Cloning: Production of many identical copies of an organism through some asexual means. – Gene Cloning: The production of many identical copies of a single gene – Two Ways to Clone a Gene: – Recombinant DNA – Polymerase Chain Reaction 43 Using Restriction Enzymes to Make Recombinant DNA • Bacterial restriction enzymes – Cut DNA molecules at a limited number of specific DNA sequences, called restriction sites 44 Restriction Enzymes and Sticky Ends Step through animation of cut/splice using EcoRI Narrated animation 45 Cloning of a Human Gene / Recombinant DNA – Restriction enzymes breaks open a plasmid vector at specific sequence of bases “sticky ends” – Foreign DNA that is to be inserted is also cleaved with same restriction enzyme so ends match – Foreign DNA is inserted into plasmid DNA and “sticky ends” pair up – DNA ligase seals them together – Narrated animation of “Cloning a Gene” 46 24.3 DNA Technology • Polymerase Chain Reaction – Amplifies a targeted DNA sequence – Requires DNA polymerase, a set of primers, and a supply of nucleotides • Primers are single stranded DNA sequences that start replication process – – – – Amount of DNA doubles with each replication cycle Process is now automated Narrated animation Step by step animation 47 Polymerase Chain Reaction (PCR) 48 24.3 DNA Technology • DNA Fingerprinting – Permits identification of individuals and their relatives – Based on differences between sequences in nucleotides between individuals – RFLPs : restriction fragment length polymorphisms – Narrated animation – Detection of the number of repeating segments (called repeats) are present at specific locations in DNA • Different numbers in different people • PCR amplifies only particular portions of the DNA • Procedure is performed at several locations to identify repeats 49 DNA Fingerprints DNA fragments (after digest with restriction enzymes) can be separated through gel ELECTROPHORESIS See How: Step-by-step electrophoresis Another walk-through explanation 50 Forensic Evidence • DNA “fingerprints” obtained by analysis of tissue or body fluids found at crime scenes Defendant’s blood (D) Blood from defendant’s clothes 4 D Jeans g 8 shirt Victim’s blood (V) g V – Can provide definitive evidence that a suspect is guilty or, more specifically, not guilty – Is a specific pattern of bands of RFLP markers on a gel Figure 20.17 51 DNA fingerprinting Can also be used in establishing paternity Figure: Electrophoresis of PCRamplified DNA fragments. (1) Father. (2) Child. (3) Mother. The child has inherited some, but not all of the fingerprint of each of its parents, giving it a new, unique fingerprint. http://en.wikipedia.org/wiki/Polymerase_chain_reaction#Paternity_testing 52 24.3 DNA Technology • Biotechnology – Biotechnology uses natural biological systems to create a product or to achieve a goal desired by humans. – “Model Organisms” favored for genetics research – Transgenic organisms have a foreign gene inserted into their DNA 53 – Have been engineered to be pharmaceutical “factories” Figure 20.18 “Pharm” Animals 54 Pharmaceutical Products 55 24.3 DNA Technology • Transgenic Bacteria – Medical Uses: Production of Insulin, Human Growth Hormone, Hepatitis B Vaccine – Agricultural Uses: Bacteria that protects plants from freezing, bacteria that protect plant roots from insects – Environmental: Bacteria that degrade oil (clean up after oil spills), bacteria that remove sulfur from coal 56 Environmental Cleanup • Genetic engineering can be used to modify the metabolism of microorganisms – So that they can be used to extract minerals from the environment or degrade various types of potentially toxic waste materials 57 24.3 DNA Technology • Transgenic Plants – Plants have been engineered to secrete a toxin that kills insects (ex: Bt corn) – Plants have been engineered to be resistant to herbicides (ex: Roundup Ready) 58 Agricultural Applications • DNA technology – Is being used to improve agricultural productivity and food quality 59 Genetic Engineering in Plants • Agricultural scientists – Have already endowed a number of crop plants with genes for desirable traits Bt corn (right) 60 24.3 DNA Technology • Transgenic Animals – Fish, cows, pigs, rabbits and sheep have been engineered to produce human growth hormone in order to increase size of the animals 61 Animal Husbandry and “Pharm” Animals • Transgenic animals – Contain genes from other organisms – Sometimes called “chimeras” – Fig 1. transgenic mouse lines expressing GFP known as “green mice.” 62 Animal Husbandry and “Pharm” Animals • “Knockout” mice http://www.nca-nl.org/English/Newsletters/Nb13/nl13txt.html Since cancer is a multistage process, it is obvious that transgenic or knock out animals, which have already undergone one step in the cancer process, may be a sensitive alternative for the standard bioassay. A number of mice models have been developed: either possessing an inactivated tumor suppressor gene (p53), an activated oncogene (Tg.AC), over-expression of a (human) oncogene (rasH2) or being deficient in nucleotide excision repair (Xpa, de Vries et al., 1995). These mice models have several advantages: • the number of animals needed for one study is 120 instead of 400-500 • the duration of the study is 6-9 instead of 24 months leading to less distress of the animals • the transgenic mouse model is considered more discriminating hence improving the accuracy and reliability of human carcinogen identification. 63 Safety and Ethical Questions Raised by DNA Technology • The potential benefits of genetic engineering – Must be carefully weighed against the potential hazards of creating products or developing procedures that are harmful to humans or the environment • Today, much public concern about possible hazards – Centers on genetically modified (GMOs) organisms used as food – Gene “escape” 64 Transgenic Animals 65