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Introduction to Bioinformatics Molecular Biology Primer 1 Genetic Material • DNA (deoxyribonucleic acid) is the genetic material • Information stored in DNA – the basis of inheritance – distinguishes living things from nonliving things • Genes – various units that govern living thing’s characteristics at the genetic level 2 Nucleotides • Genes themselves contain their information as a specific sequence of nucleotides found in DNA molecules • Only four different bases in DNA molecules – – – – Guanine (G) Adenine (A) Thymine (T) Cytosine (C) Base P Sugar • Each base is attached to a phosphate group and a deoxyribose sugar to form a nucleotide. • The only thing that makes one nucleotide different from another is which nitrogenous base it contains 3 Purine: Pyrimidine: Nucleoside 4 Nucleotides • Complicated genes can be many thousands of nucleotides long • All of an organism’s genetic instructions, its genome, can be maintained in millions or even billions of nucleotides 5 Orientation • Strings of nucleotides can be attached to each other to make long polynucleotide chains • 5’ (5 prime) end – The end of a string of nucleotides with a 5' carbon not attached to another nucleotide • 3’ (3 prime) end – The other end of the molecule with an unattached 3' carbon 6 5’ 4’ 1’ 3’ 2’ 7 Base Pairing • Structure of DNA – Double helix – Seminal paper by Watson and Crick in 1953 – Rosalind Franklin’s contribution • Information content on one of those strands essentially redundant with the information on the other – Not exactly the same—it is complementary • Base pair – G paired with C (G C) – A paired with T (A = T) 8 9 Base Pairing • Reverse complements – 5' end of one strand corresponding to the 3' end of its complementary strand and vice versa • Example – one strand: 5'-GTATCC-3' the other strand: 3'-CATAGG-5' 5'-GGATAC-3' • Upstream: Sequence features that are 5' to a particular reference point • Downstream: Sequence features that are 3' to a particular reference point 5' 3' Upstream Downstream 10 DNA Structure 11 DNA Structure 12 Chromosome • Threadlike "packages" of genes and other DNA in the nucleus of a cell 13 14 Chromosome • Different kinds of organisms have different numbers of chromosomes • Humans – 23 pairs – 46 in all 15 Central Dogma of Molecular Biology • • • • DNA: information storage Protein: function unit, such as enzyme Gene: instructions needed to make protein Central dogma 16 Central Dogma of Molecular Biology • Central dogma reverse transcription (reverse transcriptase) replication (DNA polymerase) • DNA obtained from reverse transcription is called complementary DNA (cDNA) Difference between DNA and cDNA will be discussed later 17 Central Dogma of Molecular Biology • RNA (ribonucleic acid) – Single-stranded polynucleotide – Bases • • • • A G C U (uracil), instead of T • Transcription (simplified …) – A A, G G, C C, T U DNA Base P Sugar H RNA Base P Sugar OH 18 19 20 DNA Replication (DNA DNA) 21 DNA Replication (DNA DNA) 22 DNA Replication Animation 23 Courtesy of Rob Rutherford, St. Olaf University Transcription (DNA RNA) • Messenger RNA (mRNA) – carries information to be translated • Ribosomal RNA (rRNA) – the working “spine” of the ribosome • Transfer RNA (tRNA) – the “decoder keys” that will translate nucleic acids to amino acids 24 Transcription Animation 25 Courtesy of Rob Rutherford, St. Olaf University Peptides and Proteins • mRNA Sequence of amino acids connected by peptide bond • Amino acid sequence – Peptide: < 30 – 50 amino acids – Protein: longer peptide 26 27 28 Genetic Code – Codon Codon: 3-base RNA sequence Stop codons Start codon 29 List of Amino Acids A C D E F G H I K L Amino acid Alanine Cysteine Aspartic Acid Glutamic Acid Phenylalanine Glycine Histidine Isoleucine Lysine Leucine Symbol Ala Cys Asp Glu Phe Gly His Ile Lys Leu Codon GC* UGU, UGC GAU, GAC GAA, GAG UUU, UUC GG* CAU, CAC AUU, AUC, AUA AAA, AAG UUA, UUG, CU* 30 List of Amino Acids Amino acid M Methionine N Asparagine P Proline Q Glutamine R Arginine S Serine T Threonine V Valine W Tryptophan Y Tyrosine Symbol Met Asn Pro Gln Arg Ser Thr Val Trp Tyr 20 letters, no B J O U X Z Codon AUG AAU, AAC CC* CAA, CAG CG*, AGA, AGG UC*, AGU, AGC AC* GU* UGG UAU, UAC 31 Codon and Reading Frame • • • • • 4 AA letters 43 = 64 triplet possibilities 20 (< 64) known amino acids Wobbling 3rd base Redundant Resistant to mutation Reading frame: linear sequence of codons in a gene • Open Reading Frame (ORF), definition varies: – a reading frame that begins with a start codon and end at a stop codon – a series of codons in a DNA sequence uninterrupted by the presence of a stop codon a potential protein-coding region of DNA sequence 32 Open Reading Frame • Given a nucleotide sequence – How many reading frames? __ • __ forward and __ backward • Example: Given a DNA sequence, 5’-ATGACCGTGGGCTCTTAA-3’ – – – – ATG ACC GTG GGC TCT TAA M T V G S * TGA CCG TGG GCT CTT AA * P W A L GAC CGT GGG CTC TTA A D R G L L Figure out the three backward reading frames • In random sequence, a stop codon will follow a Met in ~20 AAs • Substantially longer ORFs are often genes or parts of them 33 Translation (RNA Protein) 34 Translation Animation 35 Courtesy of Rob Rutherford, St. Olaf University Gene Expression • Gene expression – Process of using the information stored in DNA to make an RNA molecule and then a corresponding protein • Cells controlling gene expression by – reliably distinguishing between those parts of an organism’s genome that correspond to the beginnings of genes and those that do not – determining which genes code for proteins that are needed at any particular time. 36 Promoter • The probability (P) that a string of nucleotides will occur by chance alone if all nucleotides are present at the same frequency P = (1/4)n, where n is the string’s length • Promoter sequences – Sequences recognized by RNA polymerases as being associated with a gene • Example – Prokaryotic RNA polymerases scan along DNA looking for a specific set of approximately 13 nucleotides marking the beginning of genes – 1 nucleotide that serves as a transcriptional start site – 6 that are 10 nucleotides 5' to the start site, and – 6 more that are 35 nucleotides 5' to the start site – What is the frequency for the sequence to occur? 37 Gene Regulation • Regulatory proteins – Capable of binding to a cell’s DNA near the promoter of the genes – Control gene expression in some circumstances but not in others • Positive regulation – binding of regulatory proteins makes it easier for an RNA polymerase to initiate transcription • Negative regulation – binding of the regulatory proteins prevents transcription from occurring 38 Promoter and Regulatory Example • Low tryptophan concentration RNA polymerase binds to promoter genes transcribed • High tryptophan concentration repressor protein becomes active and binds to operator blocks the binding of RNA polymerase to the promoter • Tryptophan concentration drops repressor releases its tryptophan and is released from DNA 39 polymerase again transcribes genes Gene Structure 40 Exons and Introns 41 Exons and Introns Example 42 Protein Structure and Function • Genes encode the recipes for proteins 43 Protein Structure and Function • Proteins are amino acid polymers 44 Proteins: Molecular Machines Proteins in your muscles allows you to move: myosin and actin 45 Proteins: Molecular Machines Digestion, catalysis (enzymes) Structure (collagen) 46 Proteins: Molecular Machines Signaling (hormones, kinases) Transport (energy, oxygen) 47 Protein Structures 48 Information Flow in Nucleated Cell 49 Point Mutation Example: Sickle-cell Disease • Wild-type hemoglobin DNA 3’----CTT----5’ • Mutant hemoglobin DNA 3’----CAT----5’ mRNA 5’----GAA----3’ mRNA 5’----GUA----3’ Normal hemoglobin ------[Glu]------ Mutant hemoglobin ------[Val]-----50 51 image credit: U.S. Department of Energy Human Genome Program, http://www.ornl.gov/hgmis. Thinking about the Human Genome 50% is high copy number repeats About 10% is transcribed (made into RNA) Only 1.5% actually codes for protein 98.5% Junk DNA 52 Thinking about the Human Genome ~ 3 X 109 bps (3 billion base pairs) If each base were one mm long… 2000 miles, across the center of Africa Average gene about 30 meters long Occur about every 270 meters between them Once spliced the message would only be ~1 meter long 53