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A Zero-Knowledge Based Introduction to Biology Cory McLean 26 Sep 2008 Thanks to George Asimenos Cells: Building Blocks of Life cell, nucleus, cytoplasm, mitochondrion © 1997-2005 Coriell Institute for Medical Research 2 DNA: “Blueprints” for a cell • Genetic information encoded in long strings of double-stranded DNA • DeoxyriboNucleic Acid comes in only four flavors: Adenine, Cytosine, Guanine, Thymine 3 Nucleotide deoxyribose, nucleotide, base, A, C, G, T, purine, pyrimidine, 3’, 5’ purines to previous nucleotide O O P O- H O Thymine (T) Cytosine (C) C H C H C 3’ to base O C H Adenine (A) Guanine (G) 5’ H C H pyrimidines H to next nucleotide Let’s write “AGACC”! 4 “AGACC” (backbone) 5 “AGACC” (DNA) deoxyribonucleic acid (DNA) 3’ 5’ 3’ 5’ 6 DNA is double stranded strand, reverse complement 5’ 3’ 3’ 5’ DNA is always written 5’ to 3’ AGACC or GGTCT 7 DNA Packaging histone, nucleosome, chromatin, chromosome, centromere, telomere telomere centromere nucleosome DNA chromatin H1 ~146bp H2A, H2B, H3, H4 8 The Genome The genome is the full set of hereditary information for an organism Humans bundle two copies of the genome into 46 chromosomes in every cell = 2 * (1-22 + X/Y) 9 Building an Organism DNA cell Every cell has the same sequence of DNA Subsets of the DNA sequence determine the identity and function of different cells 10 From DNA To Organism ? Proteins do most of the work in biology, and are encoded by subsequences of DNA, known as genes. 11 RNA ribose, ribonucleotide, U purines to previous ribonucleotide O O P O- H O Uracil (U) C H Cytosine (C) C H C 3’ to base O C H Adenine (A) Guanine (G) 5’ H C OH to next ribonucleotide H pyrimidines TU 12 Genes & Proteins gene, transcription, translation, protein Double-stranded DNA 5’ 3’ TAGGATCGACTATATGGGATTACAAAGCATTTAGGGA...TCACCCTCTCTAGACTAGCATCTATATAAAACAGAA ATCCTAGCTGATATACCCTAATGTTTCGTAAATCCCT...AGTGGGAGAGATCTGATCGTAGATATATTTTGTCTT 3’ 5’ (transcription) Single-stranded RNA AUGGGAUUACAAAGCAUUUAGGGA...UCACCCUCUCUAGACUAGCAUCUAUAUAA (translation) protein 13 Gene Transcription promoter 5’ 3’ G A T T A C A . . . C T A A T G T . . . 3’ 5’ 14 Gene Transcription transcription factor, binding site, RNA polymerase 5’ 3’ G A T T A C A . . . C T A A T G T . . . 3’ 5’ Transcription factors: a type of protein that binds to DNA and helps initiate gene transcription. Transcription factor binding sites: Short sequences of DNA (6-20 bp) recognized and bound by TFs. RNA polymerase binds a complex of TFs in the promoter. 15 Gene Transcription 5’ 3’ 3’ 5’ The two strands are separated 16 Gene Transcription 5’ 3’ 3’ 5’ An RNA copy of the 5’→3’ sequence is created from the 3’→5’ template 17 Gene Transcription 5’ 3’ pre-mRNA 5’ G A T T A C A . . . 3’ 5’ C T A A T G T . . . G A U U A C A . . . 3’ 18 RNA Processing 5’ cap, polyadenylation, exon, intron, splicing, UTR, mRNA 5’ cap exon poly(A) tail intron mRNA 5’ UTR 3’ UTR 19 Gene Structure introns 5’ 3’ promoter 5’ UTR exons 3’ UTR coding non-coding 20 How many? (Human Genome) • Exons per gene: ~ 8 on average (max: 148) • Nucleotides per exon: 170 on average (max: 12k) • Nucleotides per intron: 5,500 on average (max: 500k) • Nucleotides per gene: 45k on average (max: 2,2M) 21 From RNA to Protein • Proteins are long strings of amino acids joined by peptide bonds • Translation from RNA sequence to amino acid sequence performed by ribosomes • 20 amino acids 3 RNA letters required to specify a single amino acid 22 Amino acid amino acid H N H O C C H R OH Alanine Arginine Asparagine Aspartate Cysteine Glutamate Glutamine Glycine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Proline Serine Threonine Tryptophan Tyrosine Valine There are 20 standard amino acids 23 Proteins N-terminus, C-terminus to previous aa N H O C C to next aa H R H N-terminus (start) from 5’ OH C-terminus (end) 3’ mRNA 24 Translation ribosome, codon P site A site mRNA The ribosome (a complex of protein and RNA) synthesizes a protein by reading the mRNA in triplets (codons). Each codon is translated to an amino acid. 25 Translation A site P site mRNA U C A G U C A G UUU Phenylalanine (Phe) UCU Serine (Ser) UAU Tyrosine (Tyr) UGU Cysteine (Cys) U UUC Phe UCC Ser UAC Tyr UGC Cys C UUA Leucine (Leu) UCA Ser UAA STOP UGA STOP A UUG Leu UCG Ser UAG STOP UGG Tryptophan (Trp) G CUU Leucine (Leu) CCU Proline (Pro) CAU Histidine (His) CGU Arginine (Arg) U CUC Leu CCC Pro CAC His CGC Arg C CUA Leu CCA Pro CAA Glutamine (Gln) CGA Arg A CUG Leu CCG Pro CAG Gln CGG Arg G AUU Isoleucine (Ile) ACU Threonine (Thr) AAU Asparagine (Asn) AGU Serine (Ser) U AUC Ile ACC Thr AAC Asn AGC Ser C AUA Ile ACA Thr AAA Lysine (Lys) AGA Arginine (Arg) A AUG Methionine (Met) or START ACG Thr AAG Lys AGG Arg G GUU Valine (Val) GCU Alanine (Ala) GAU Aspartic acid (Asp) GGU Glycine (Gly) U GUC Val GCC Ala GAC Asp GGC Gly C GUA Val GCA Ala GAA Glutamic acid (Glu) GGA Gly A GUG Val GCG Ala GAG Glu GGG Gly G 26 Translation 5’ ...AUUAUGGCCUGGACUUGA... UTR Met Start Codon Ala Trp 3’ Thr Stop Codon 27 Translation 5’ ...AUUAUGGCCUGGACUUGA... 3’ 28 Translation Met 5’ Trp Ala ...AUUAUGGCCUGGACUUGA... 3’ 29 Errors? mutation • What if the transcription / translation machinery makes mistakes? • What is the effect of mutations in coding regions? 30 Reading Frames reading frame G C U U G U U U A C G A A U U A G G C U U G U U U A C G A A U U A G G C U U G U U U A C G A A U U A G G C U U G U U U A C G A A U U A G 31 Synonymous Mutation synonymous (silent) mutation, fourfold site G G C U U G U U U A C G A A U U A G G C U U G U U U A C G A A U U A G Ala Cys Leu Arg Ile G C U U G U U U G C G A A U U A G Ala Cys Leu Arg Ile 32 Missense Mutation missense mutation G G C U U G U U U A C G A A U U A G G C U U G U U U A C G A A U U A G Ala Cys Leu Arg Ile G C U U G G U U A C G A A U U A G Ala Trp Leu Arg Ile 33 Nonsense Mutation nonsense mutation A G C U U G U U U A C G A A U U A G G C U U G U U U A C G A A U U A G Ala Cys Leu Arg Ile G C U U G A U U A C G A A U U A G Ala STOP 34 Frameshift frameshift G C U U G U U U A C G A A U U A G G C U U G U U U A C G A A U U A G Ala Cys Leu Arg Ile G C U U G U U A C G A A U U A G Ala Tyr Cys Glu Leu 35 Gene Expression Regulation Regulation, signal transduction • When should each gene be expressed? • Regulate gene expression Examples: – Make more of gene A when substance X is present – Stop making gene B once you have enough – Make genes C1, C2, C3 simultaneously • Why? Every cell has same DNA but each cell expresses different proteins. • Signal transduction: One signal converted to another – Cascade has “master regulators” turning on many proteins, which in turn each turn on many proteins, ... 36 Gene Regulation Gene expression is controlled at many levels: DNA chromatin structure Transcription Post-transcriptional modification RNA transport Translation mRNA degradation Post-translational modification 37 Transcription Regulation • Much gene regulation occurs at the level of transcription. • Primary players: – Binding sites (BS) in cis-regulatory modules (CRMs) – Transcription factor (TF) proteins – RNA polymerase II • Primary mechanism: – TFs link to BSs – Complex of TFs forms – Complex assists or inhibits formation of the RNA polymerase II machinery 38 Tx Factor Binding Sites • Short, degenerate DNA sequences recognized by particular TFs • For complex organisms, cooperative binding of multiple TFs required to initiate transcription Binding Sequence Logo 39 Transcription Regulation Mechanisms enhancer, silencer, insulator Transcription Factor Specificity: Enhancer: Silencer: Insulator: 40 Unicellular vs. Multicellular unicellular multicellular 41 Non-coding RNAs • RNAs transcribed from DNA but not translated into protein • Structural ncRNAs: Conserved secondary structure (A-U, C-G, G-U) • Involved in gene regulation 42 Summary • All hereditary information encoded in doublestranded DNA • Each cell in an organism has same DNA • DNA RNA protein • Proteins have many diverse roles in cell • Gene regulation diversifies protein products within different cells 43 The end? 44