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) 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 Sugar P Base 3

Nucleoside Purine: Pyrimidine: 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

1’ 2’ 3’ 4’ 5’ 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

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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

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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 Sugar P Base Sugar P Base H OH DNA RNA 18

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DNA Replication (DNA  DNA) 21

DNA Replication (DNA  DNA) 22

DNA Replication Animation Courtesy of Rob Rutherford, St. Olaf University 23

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 Courtesy of Rob Rutherford, St. Olaf University 25

Peptides and Proteins mRNA  Sequence of amino acids connected by peptide bond Amino acid sequence –Peptide: < 30 – 50 amino acids –Protein: longer peptide 26

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29 Genetic Code – Codon Stop codons Start codon Codon: 3-base RNA sequence

List of Amino Acids Amino acid Symbol Codon A Alanine Ala GC* C Cysteine Cys UGU, UGC D Aspartic Acid Asp GAU, GAC E Glutamic Acid Glu GAA, GAG F Phenylalanine Phe UUU, UUC G Glycine Gly GG* H Histidine His CAU, CAC I Isoleucine Ile AUU, AUC, AUA K Lysine Lys AAA, AAG L Leucine Leu UUA, UUG, CU* 30

List of Amino Acids Amino acid Symbol Codon M Methionine Met AUG N Asparagine Asn AAU, AAC P Proline Pro CC* Q Glutamine Gln CAA, CAG R Arginine Arg CG*, AGA, AGG S Serine Ser UC*, AGU, AGC T Threonine Thr AC* V Valine Val GU* W Tryptophan Trp UGG Y Tyrosine Tyr UAU, UAC 20 letters, no B J O U X Z 31

Codon and Reading Frame 4 AA letters  4 3 = 64 triplet possibilities 20 (< 64) known amino acids Wobbling 3 rd 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 Courtesy of Rob Rutherford, St. Olaf University 35

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  polymerase again transcribes genes 39

Gene Structure 40

Exons and Introns 41

Exons and Introns Example 42

Protein Structure and Function Genes encode the recipes for proteins 43

44 Protein Structure and Function Proteins are amino acid polymers

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’ mRNA 5’----GAA----3’ Normal hemoglobin [Glu] Mutant hemoglobin DNA 3’----CAT----5’ mRNA 5’----GUA----3’ Mutant hemoglobin [Val]

image credit: U.S. Department of Energy Human Genome Program, 51

50% is high copy number repeats About 10% is transcribed (made into RNA) Only 1.5% actually codes for protein 98.5% Junk DNA Thinking about the Human Genome 52

Thinking about the Human Genome ~ 3 X 10 9 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