Molecular Biology Background

1. Molecular Biology Background Debasis Mitra Florida Tech Credit: Pevezner text-site dmitra

2. Section1: What is Life made of? dmitra

3. 2 types of cells: Prokaryotes v.s.Eukaryotes dmitra

4. Life begins with Cell • A cell is a smallest structural unit of an organism that is capable of independent functioning • All cells have some common features dmitra

5. Prokaryotes and Eukaryotes • According to the most recent evidence, there are three main branches to the tree of life. • Prokaryotes include Archaea (“ancient ones”) and bacteria. • Eukaryotes are kingdom Eukarya and includes plants, animals, fungi and certain algae. dmitra

6. Prokaryotes and Eukaryotes, continued dmitra

7. Overview of organizations of life • Nucleus = library • Chromosomes = bookshelves • Genes = books • Almost every cell in an organism contains the same libraries and the same sets of books. • Books represent all the information (DNA) that every cell in the body needs so it can grow and carry out its vaious functions. dmitra

8. Chromosomes Organism Number of base pair number of Chromosomes --------------------------------------------------------------------------------------------------------- Prokayotic Escherichia coli (bacterium) 4x106 1 Eukaryotic Saccharomyces cerevisiae (yeast) 1.35x107 17 Drosophila melanogaster(insect) 1.65x108 4 Homo sapiens(human) 2.9x109 23 Zea mays(corn) 5.0x109 10 dmitra

9. Bio-molecules • Nucleic acids (DNA, RNA): Library of life • Proteins: Workhorse of life • Fatty acids, carbohydrates, and other supporting molecules dmitra

10. DNA • DNA has a double helix structure which composed of • sugar molecule • phosphate group • and a base (A,C,G,T) • DNA always reads from 5’ end to 3’ end for transcription replication 5’ ATTTAGGCC 3’ 3’ TAAATCCGG 5’ dmitra

11. DNA, RNA, and the Flow of Information Replication Transcription Translation dmitra

12. Proteins Functions • Structural • Enzymes • Information exchange (e.g., across cell walls) • Transporting other molecules (e.g., oxygen to cells) • Activating-deactivating genes • Etc. dmitra

13. Proteins • Amino acids • Protein is a chain of “residues” • 20 to 5000 long, typically a few hundred long dmitra

14. Protein structure • Important for its function • Primary structure: sequence • Secondary structure: a few topological features • Tertiary structure: 3D folding • Quaternary structure: Protein complex dmitra

15. Protein Folding • Proteins tend to fold into the lowest free energy conformation. • Proteins begin to fold while the peptide is still being translated. • Proteins bury most of its hydrophobic residues in an interior core to form an α helix. • Most proteins take the form of secondary structures α helices and β sheets. • Molecular chaperones, hsp60 and hsp 70, work with other proteins to help fold newly synthesized proteins. • Much of the protein modifications and folding occurs in the endoplasmic reticulum and mitochondria. dmitra

16. Protein Folding (cont’d) • The structure that a protein adopts is vital to it’s chemistry • Its structure determines which of its amino acids are exposed carry out the protein’s function • Its structure also determines what substrates it can react with dmitra

17. Nucleic acids • Two types: • DNA: Deoxy-ribonucleic acid • RNA: Ribonucleic acid dmitra

18. Nucleic acids • Sugar molecule chain forms the base of the polymer • Two types of sugar: ribose (RNA), 2’-deoxyribose (DNA) dmitra

19. Nucleic acids: DNA • 4 types of bases connected to sugar molecules: Adenine (a), Guanine (g), Thymine (t) and Cytosine (c) • A and T forms strong bonds, and so do G and C dmitra

20. The Purines The Pyrimidines

21. DNA • DNA has a double helix structure which composed of • sugar molecule • phosphate group • and a base (A,C,G,T) • DNA always reads from 5’ end to 3’ end for transcription replication 5’ ATTTAGGCC 3’ 3’ TAAATCCGG 5’ dmitra

22. Nucleic acids: DNA • Double stranded: two strands of sugar molecule-chains • Each strand is directed: 5’ to 3’ • Attached inside by base-pairings (a-t and g-c) dmitra

23. Double helix of DNA

24. 1 Biologist 1 Physics Ph.D. Student 900 words Nobel Prize Discovery of DNA • DNA Sequences • Chargaff and Vischer, 1949 • DNA consisting of A, T, G, C • Adenine, Guanine, Cytosine, Thymine • Chargaff Rule • Noticing #A#T and #G#C • A “strange but possibly meaningless” phenomenon. • Wow!! A Double Helix • Watson and Crick, Nature, April 25, 1953 • Rich, 1973 • Structural biologist at MIT. • DNA’s structure in atomic resolution. Crick Watson dmitra

25. Nucleic acids: DNA • Each strand is complementary and reverse to the other • If s=agacgt reverse(s)=tgcaga reverse-complement(s)=acgtct Double-strand: 5’--agacgt->3’ 3’<-t ctgca—5’ dmitra

26. Nucleic acids: DNA • 3D structure is helical • Double-stranded helix: like step ladder • Each unit is a base pair (sugar-base-base-sugar) • DNA’s in cells are chromosomes (human chromosome ~3*(10^9) bp long) • Squeezed 3D structure in cell may have functional importance – not well studied dmitra

27. DNA Replication dmitra

28. Nucleic acids: RNA • Replace t with u (uracil) as base • May or may not be (mostly not) double stranded • Functions: Information storage like DNA, sometimes workhorse like proteins • Possible evolutionary precursor to DNA and protein dmitra

29. Genetic code • Proteins do almost all the works!! • Information for coding proteins are stored on DNA’s (or RNA’s): genes • Three consecutive bases on a gene codes an amino acid, or the STOP code: codon • The table is called genetic code dmitra

30. Cell Information: Instruction book of Life • DNA, RNA, and Proteins are examples of strings written in either the four-letter nucleotide of DNA and RNA (A C G T/U) • or the twenty-letter amino acid of proteins. Each amino acid is coded by 3 nucleotides called codon. (Leu, Arg, Met, etc.) dmitra

31. Overview of DNA to RNA to Protein • A gene is expressed in two steps • Transcription: RNA synthesis • Translation: Protein synthesis dmitra

32. Central Dogma of Biology The information for making proteins is stored in DNA. There is a process (transcription and translation) by which DNA is converted to protein. By understanding this process and how it is regulated we can make predictions and models of cells. Assembly Protein Sequence Analysis Sequence analysis Gene Finding

33. Transcription • Genes are transcribed to proteins: typically one gene to one protein • Genes are subsequenes on chromosomes started by a promoter region, ended around a stop codon dmitra

34. Transcription • Steps: • DNA is split over gene after promoter is recognized (may have other regulatory regions upstream) • mRNA is copied from the gene • Exons are spliced out from the mRNA keeping the introns only • Ribosome (rRNA and protein complex) works on mRNA dmitra

35. Transcription • The process of making RNA from DNA • Catalyzed by “transcriptase” enzyme • Needs a promoter region to begin transcription. • ~50 base pairs/second in bacteria, but multiple transcriptions can occur simultaneously http://ghs.gresham.k12.or.us/science/ps/sci/ibbio/chem/nucleic/chpt15/transcription.gif dmitra

36. Definition of a Gene • Regulatory regions: up to 50 kb upstream of +1 site • Exons: protein coding and untranslated regions (UTR) 1 to 178 exons per gene (mean 8.8) 8 bp to 17 kb per exon (mean 145 bp) • Introns: splice acceptor and donor sites, junk DNA average 1 kb – 50 kb per intron • Gene size: Largest – 2.4 Mb (Dystrophin). Mean – 27 kb. dmitra

37. Translation • tRNA are attached to codons on mRNA • On the other end the tRNA attracts appropriate amino acid • Amino acids are zipped up • No tRNA for STOP codon • Every step is facilitated by appropriate enzyme Central Dogma of biology dmitra

38. Translation, continued • Catalyzed by Ribosome • Using two different sites, the Ribosome continually binds tRNA, joins the amino acids together and moves to the next location along the mRNA • ~10 codons/second, but multiple translations can occur simultaneously http://wong.scripps.edu/PIX/ribosome.jpg dmitra

39. Revisiting the Central Dogma • In going from DNA to proteins, there is an intermediate step where mRNA is made from DNA, which then makes protein • This known as The Central Dogma • Why the intermediate step? • DNA is kept in the nucleus, while protein sythesis happens in the cytoplasm, with the help of ribosomes dmitra

40. The Central Dogma (cont’d) dmitra

41. Open Reading Frame • Three reading frames in a strand • Complementary strand may have another three frames dmitra

42. Types of chromosomes • Procaryotes (bacteria, blue algae): circular • Eucaryotes (has nuclear wall): diploid (human has 23 pairs) • Homologous genes and alleles (e.g., human hemoglobin of type A, B, and O) • Haploid chromosomes in Eucaryote sex cells dmitra

43. DNA Sequencing • A DNA fragment is split at each position starting from one end • Four tubes: one containing molecules ending with G, one with A, one with T and another one with C • Electrophoresis separates each chunk of different size in each tube [page 22] • Information is recombined to sequence the DNA chunk • Can be done for the size of only ~1K bp long chunk dmitra

44. DNA Sequencing • Human DNA is ~10^9 bp long • Restriction enzyme cuts at restriction sites (a product of genetic engineering) [page 18] • After sequencing, information from fragments need to be recombined to get the broader picture dmitra

45. DNA Sequencing • Depends on finding restriction site/enzyme for fragmenting DNA of appropriate size • Privately funded Tiger project (Celera now) used heat and vibration to create fragments • Recombining information is no longer trivial because fragment’s location is no longer known • Needed Fragment assembly algorithm dmitra

46. DNA Sequencing • Needs multiple copies of DNAs • Recombinant DNA by biologically copying them within host organisms • Polymerase Chain Reaction: heat and tear two strands of DNA, then let each strand attract nucleic acids to form double stranded DNA, repeat dmitra