Chapter 10

1. Chapter 10 0 Molecular Biology of the Gene

2. Discovery of the Role of DNA A. 1928 - Frederick Griffith discovers transformation in bacteria : * discovered that “something” was able to transform harmless (“R”non – virulent) bacteria into harmful (“S”virulent) bacteria studied: pneumococcus S type with capsule and R type without capsule

3. Discovery of the Role of DNA (cont’d) • 1944 -Oswald Avery and colleagues show that DNA can transform bacteria • 1952 - Alfred Hershey and Martha Chase use bacteriophage to confirm that DNA – not protein- is the genetic material

4. Hershey-Chase Experiment: Infected cells make more virus by injecting their DNA animation 35-S 1 32-P

5. Discovery of the Role of DNA (cont’d) D. 1953 - James Watson and Francis Crick propose a structural model for the DNA molecule Based On: X-Ray crystallographyimages prepared by Maurice Wilkins and Rosalind Franklin Chargraff’s Rule: # of Adenines = # of Thymines # Guanines = # of Cytosines

6. DNA and RNA are Polymers of Nucleotides • Both are nucleic acidsmade of long chains of nucleotide monomers • A nucleotide (building block of a nucleic acid) has 3 parts: • A phosphate (PO4-)group that is negatively charged • A 5-carbon sugar(deoxyribose in DNA or ribose in RNA) • Anitrogen-containing base

7. Thymine, Cytosine, Adenine, and Guanine DNA (deoxyribonucleic acid) bases: purines pyrimidines • Pyrimidines: single ring bases • Purines: double ring bases • Complimentary binding pattern: • Adenine + Thymine (share 2 hydrogen bonds) • Cytosine + Guanine (share 3 hydrogen bonds)

8. RNA: ribonucleic acid Similar to DNA except: • Sugar in RNA = ribose • Base “uracil” instead of thymine • Single stranded Figure 10.2C, D

9. Twist The Structure of DNA Two polynucleotide strands wrapped around each other in a double helix • A sugar-phosphate backbone held together by phosphodiester bonds • Steps made of hydrogen-bound bases (A=T, C G)

10. DNA REPLICATION: Starts with the separation of DNA strands • Enzymes use each strand as a template to assemble new nucleotides into complementary strands…“semi-conservative” (Meselson & Stahl 1958) • Portions to be replicated must untwist first

11. DNA segments unwind Helicase splits H bonds between bases, unzip DNA Binding proteins keep unzipped DNA apart Primase makes a short RNA primer because DNA polymerase can only extend a nucleotide chain, not start one. DNA polymerase adds complimentary nucleotides to parent strand RNase H cuts out original primers DNA polymerase fills in gap of removed primers DNA ligase glues S/P backbone where needed DNA replication begins at specific sites on double helix replication forks Animation/tutorial 9. Two identical double helices • Topoisomerase: prevents further coiling at replication fork

12. A Structural Problem with DNA Replication • Each strand of the double helix is oriented in the opposite direction (“anti-parallel”) • “prime” #’s refer to carbons in the sugar • At one end, the 3’ carbon has an (OH) and at the opposite, a 5’ carbon has the PO4- • Why does this matter? DNA polymerase can only add nucleotides to the 3’ end (at the OH). A daughter strand can only grow from 5’  3’ • Therefore, only one daughter strand is made continuously (leading strand) • The other strand (lagging strand) is made in a series of short pieces (Okazaki fragments), later connected by DNA ligase 3’ 5’ 3’ 5’ 3’ 5’ DNA Polymerase moves 3’5’ Daughter strand is made 5’3’ 3’ 5’ Animation/tutorial

13. When DNA can repair mistakes and when it can’t DNA Repair enzymes work like a spell checker • Cut out wrong sequences • Undamaged strand is template • Only 2 or 3 stable changes per year Mutations: some severe, others are not • Inheritable changes occur in gametogenesis • Now the “wrong” sequences are copied • Ex: cystic fibrosis (CF): a deletion of 3 nucleotides in a certain gene • Ex: sickle cell anemia: one nucleotide substitution in the hemoglobin gene • Mutagen: a mutation causing substance (can break DNA) • Ex: x-rays, radioactivity, nicotine

14. Protein Synthesis: the transfer of information from: DNA  RNA  Proteins “gene expression”: A gene is a linear sequence of many nucleotides. 3 Types: • Structural genes: have info to make proteins • Regulatory genes: code for proteins which are on/off switches for other genes • Genes that code for tRNA, rRNA, histones DNA vs. RNA • single stranded • A U C G • ribose sugar • 3 types of RNA: messenger, transfer, ribosomal • double stranded • A T C G • deoxyribose sugar mRNA (messenger):copies DNA’s message in nucleus  brings it to cytoplasm tRNA (transfer):carries amino acids to mRNA so protein can be made rRNA (ribosomal):major part of the ribosome. Helps link amino acids from tRNA’s together  assemble protein

15. Protein Synthesis is Two Steps: • Transcription: The DNA of the gene is transcribed into mRNA • Translation: decoding the mRNA and assembling the protein

16. Transcription: Eukaryote • DNA sequence (message for protein) is transcribed by mRNA • Only one strand (bottom; non-coding strand) is needed as a template • Steps: RNA polymerase splits H bonds in DNA section RNA polymerase travels along bottom strand of DNA. RNA nucleotides join in a complimentary pattern (A=U, C=G) A termination signal is reached, transcription is over mRNA strip detaches from DNA, DNA helix closes up mRNA is processed: Intronsare cut out, Exonsare glued together, cap (7-methyl guanine) and tail (Poly-Adenine) are added. Mature mRNA leaves nucleus through pores cytoplasm for next step spliceosome: SNRP’s and other proteins scan the mRNA - cut out introns and glue exons together! bioflix video

17. Translation: the synthesis of proteins using mRNA, tRNA and ribosomes • The Genetic Code: the language in which instructions for proteins are written in the nucleotide sequences • Each triplet of mRNA nucleotides is a “codon” because it will “code” for 1 amino acid • Ex: AUG GUC CCU AAU CCU Met – Val – Pro – Asn – Pro • Original top strand of DNA (coding strand): ATG GTC CCT AAT CCT • Only difference: U is substituted for T • Use the Genetic Codechart to “decode” mRNA message

18. The Genetic Code is the Rosetta Stone of Life • Nearly allorganisms use exactly the same genetic code • More than one codon for most amino acids = degenerate nature…a change (mutation) in gene does not always mean a different amino acid. • what does CAU code for? ACU? UAU? GCC? • how many codons for Leu? • what is special about AUG and it’s amino acid, Methionine? • what is special about UAA, UAG, and UGA? HIStidine…THReonine…TYRosine…ALAnine

19. An exercise in translating the genetic code: non-coding strand Step 1: fill in the corresponding DNA bases for dark blue strand (bottom, non-coding) Step 2: Transcribe the dark blue strand into mRNA (pink) Step 3: Translate the codons into correct amino acids (use chart) G A A A T G T G T T T A coding strand transcription translation

20. An exercise in translating the genetic code: answers Step 1: fill in corresponding DNA bases to dark blue strand (non-coding) Step 2: Transcribe the dark blue strand into mRNA (pink) Step 3: Translate the codons into correct amino acids (use chart)

21. How Does Translation Happen? Need: tRNAs and ribosomes (rRNA) tRNA: single stranded RNA, folded up • 2 parts: anticodon and aa attachment site • Ribosome: 2 protein subunits and ribosomal RNA • allows aa’s to attach by making peptide bonds • travels along mRNA strip, tRNA’s join and bring correct amino acids • 3 sites on ribosome: • P site – where protein is growing • Asite – where new tRNA’s and amino acids join • E site – where empty tRNA’s exit ribosome • Translocation: as ribosome moves, tRNA’s move from A site to P site. “A” site is now open for new tRNA with attached amino acid to join • Confused? Watch bioflix video A E P animation

22. Putting It All Together:

23. Mutations: • changes in DNA base sequence • caused by errors in DNA replication, recombination, or by mutagens Mutations can change the message of genes • substituting, inserting, or deleting nucleotides also alters a gene point ex: point mutation (substitution) may or may not alter amino acid sequence deletion ex: frame-shift mutation: most devastating to protein structure. (caused by deletion or insertion) **the entire codon reading frame shifts

24. The Role of Viruses and Mutations in DNA Viral DNA may become part of the host chromosome Viruses are “genes in a box of proteins” (capsid). * Proteins on capsid determine attachability to specific host cell (ex: polio, hepatitis, adenovirus) Viruses (non-cellular) cannot reproduce on their own, must use a host cell’s machinery to replicate, transcribe, and translate their genetics into more viruses Two types of viral replication cycles: lytic and lysogenic 1. Lytic: viral replication causes lysis (bursting) of host cell new viral particles released (ex: flu, common cold) 2. Lysogenic: viral replication without host cell destruction initially. - Viral DNA is inserted into host chromosome (now called prophage). -Host cells divide, more copies of prophage are made. -Incorporation of viral genes into host DNA causes breaks, mutations. -Some viruses are cancer causing because of this. (HIV, HPV) • environmental signals switch “on” prophage genes, viral replication begins, followed by host cell destruction (lysis)

25. Lytic Virusesvs.Lysogenic Viruses

26. Retroviruses: Make a DNA copy of their RNA * HIV contains 2 copies of its RNA * HIV carries an enzyme “reverse transcriptase” and can make a DNA copy of it’s RNA (reverse order than normal DNA  RNA) * Reverse Transcriptase : 1. Uses the RNA as a template to make a DNA strand 2. Then adds a second, complementary DNA strand 3. Double stranded DNA enters the host nucleus and inserts itself into the chromosomal DNA, becoming a provirus (analogous to prophage). • 4. Occasionally the provirus is transcribed into RNA and then… • 5. Translated into viral proteins • New viruses assembled from these components leave the cell and infect others • * HIV infects and eventually kills several kinds of white blood cells of our immune system

28. Bonus: Pick any 2 of your choice • What kind of bacteria were used in Griffith’s experiment? • During what specific part of the cell cycle will RNA polymerase be used? • In what direction are daughter strands synthesized (5’  3’ OR 3’  5’)? • During what year did Watson and Crick figure out the structure of the DNA molecule? • What is the name of the modified guanine cap placed on processed mRNA? • What name is given to the complex of proteins and SNRP’s in charge of processing the mRNA strip?

29. Bonus: Pick any 4 of your choice • What kind of cancer does HPV cause? • The tail of a mature mRNA is made up of about 100 (A’s, U’s, C’s, G’s) ? • What words did the letters “R” and “S” stand for in Griffith’s experiment? • Which parent strand of DNA (5’  3’ OR 3’  5’) results in Okazaki fragments in it’s replicated daughter strand? • During what specific part of the cell cycle will RNA polymerase be used? • DNA polymerase moves from (5’  3’ OR 3’  5’)? • What is the name of the enzyme which removes RNA primers? • During what year did Watson and Crick figure out the structure of the DNA molecule?

30. 1.How many nucleotide pairs are there in the DNA code inside every cell of your body?? 3 BILLION!

31. 1. If you were to write out the entire genetic code for a human, how many 500 page phone books would you need to write the equivalent of ?? 200!!

32. 1. How long would it take to recite the genetic code if you were to recite one letter every second, 24 hours a day?? 100 years !!

33. 1. If we were to recite our individual codes one letter per second, how long would it be before you would encounter a difference between your DNA and the person sitting next to you??? 9 minutes !!

34. 1. What percentage of a human’s DNA is identical to a chimpanzee’s DNA?? 98%!!

35. 1. How many genes does the average human have?? only 30,000

36. 1. On what chromosome is the largest human gene found?? X – dystrophin gene

37. What cells (when mature) contain NO DNA)?? RBC’s, WBC’s, heart, or lung RBC’s

38. 1. How many cells are there in the human body?? 100 trillion !!

39. 1. If all the DNA from all those cells were placed end-to-end, how many times to and from the SUN would it stretch?? SIX times !!