Introduction to Bioinformatics Part 1 of 2

Transcription

1 Introduction to Bioinformatics Part 1 of 2 CS January 28, 2004 Georges Grinstein, Ph.D. grinstein@cs.uml.edu Copyright notice Many of the images in this PowerPoint presentation are from Bioinformatics and Functional Genomics by Jonathan Pevsner (ISBN ). Copyright 2003 by John Wiley & Sons, Inc. The book has a homepage at including hyperlinks to the book chapters. The initial draft of the slides are form Pevsner course at Johns opkins.

2 Lead Course and Research Assistants Mary Beth Smrtic ongli Li owie Goodell recorder Any others from the IVPR lab Who is taking this course? People with very diverse backgrounds in biology and chemistry People with diverse backgrounds in computer science and biostatistics NOTE: Most people have a favorite gene, protein, or disease

3 What are the goals of the course? To provide an introduction to bioinformatics with a focus on the National Center for Biotechnology Information (NCBI) and EBI To focus on the analysis of DNA, RNA and proteins To introduce you to the analysis of genomes To combine theory and practice to help you solve research problems Themes throughout the course Textbooks Web sites Literature references Gene/protein families Computer labs Algorithms

4 Themes throughout the course: textbooks The textbooks (required) are Bioinformatics and Functional Genomics (Pevsner) and contains content, lab exercises, and quizzes Introduction to Computational Molecular Biology (Setubal and Meidanis) The following books are highly recommended Durbin, Eddy, Krogh, and Mitchison Biological Sequence Analysis: Probabilistic Models of Proteins and Nucleic Acids Clark and Russell Molecular Biology Made Simple and Fun Themes throughout the course: web sites The course website is: The textbook website is: This has 1000 URLs, organized by chapter The site offers a 15% discount on book purchases The principal website we will explore is NCBI:

5 Themes throughout the course: Literature references You are encouraged to read original source articles, which will be provided. Although articles are not required, they will enhance your understanding of the material. You can also obtain articles through PubMed and through the WelDoc service at Welch. I will make additional articles available. Themes throughout the course: gene/protein families The course will use retinol-binding protein 4 (RBP4) as a model gene/protein. RBP4 is a member of the lipocalin family. It is a small, abundant carrier protein. We will study it in a variety of contexts including --sequence alignment --gene expression --protein structure --phylogeny --homologs in various species We will also use the Pol protein of IV-1 as an example.

6 The IV-1 pol gene encodes three proteins Aspartyl protease Reverse transcriptase Integrase PR RT IN

7 Themes throughout the course: lab activities We will try to schedule a lab for the class as a whole. You can do each assigned lab on your own if you wish. owever, during the group lab you can get help on problems, and in some cases the computers will have specialized software. Grading 30% final exam 40% discovery of a novel gene (by April 30) and phylogenetic tree (by May 13) 20% portfolio/notebook 10% presentation extra credit: find a mistake in a database

8 What is bioinformatics? Interface of biology and computers Analysis of proteins, genes and genomes using computer algorithms and computer databases Genomics is the analysis of genomes. The tools of bioinformatics are used to make sense of the billions of base pairs of DNA that are sequenced by genomics projects. Top ten challenges for bioinformatics Precise models of where and when transcription will occur in a genome (initiation and termination) Precise, predictive models of alternative RNA splicing Precise models of signal transduction pathways; ability to predict cellular responses to external stimuli Determining protein:dna, protein:rna, protein:protein recognition codes Accurate ab initio protein structure prediction

9 Top ten challenges for bioinformatics Rational design of small molecule inhibitors of proteins Mechanistic understanding of protein evolution Mechanistic understanding of speciation Development of effective gene ontologies: systematic ways to describe gene and protein function Education: development of bioinformatics curricula Source: Ewan Birney, Chris Burge, Jim Fickett Three perspectives on bioinformatics The tree of life The organism The cell

10 Body region, physiology, pharmacology, pathology Time of development

11 DNA RNA protein phenotype

12 Small Molecules Thousands of different ones in many cells Some are burned as fuel (sugars, fatty acids) Others transfer energy generated by oxidation to the organelles Others are intermediates in metabolism

13 Large Molecules Small molecules strung together Instead of synthesizing large biological molecules like small ones (by adding C, O, and N here and there) Nature decided to make big molecules by simply linking together smaller ones in long linear chains Large molecules are polymers made up of many small molecules called monomers Proteins, DNA and RNA are the major classes of large molecules (Macromolecules is the technical term) Proteins Composed of assemblages of small molecules called amino acids joined in a long unbranched chain Amino acids are composed of 4 parts Central carbon atom Amino group Carboxyl group Side group (One of 20 amino acids)

14 Protein: Central Carbon Atom C Protein: Amino Group + N Properties similar to ammonia Basic can neutralize an acid

15 Protein: Carboxyl Group O C O - Found in familiar substances like acetic acid (vinegar) Responsible for acidic properties of amino acids Negatively charged at neutral p Protein: Side Group C 3 One of 20: some are oily (repel water) some attract water some are charged In every amino acid this is (basically) the only change Their symbols are GLY, ALA, VAL, LEU, ILE, SET, CYS, MET, TYR, PE TRP, IS, ARG, LYS, ASP, GLU, ASN, GLN, PRO, TR

16 Protein Structure 1 C Protein Structure 2 N C

17 Protein Structure 3 O N C C O Protein Structure 4 C 3 O N C C O

18 Protein Structure: note charges C 3 O + N C C O - Protein Formation C 3 O C 3 O + N C C O - + N C C O - Loss of water

19 Protein Formation C 3 C 3 O N C C O N C C O - 2 O Protein Formation: A Peptide Bond C 3 C 3 O N C C N C C O O In this case we get a dipeptide with two units of alanine Note amino terminal end does not change growth is at carboxyl end

20 Critical Point The character of a protein (a polypeptide) is determined by the sequence of its amino acids Not the number of kinds of amino acids It is the sequence the order from one end to the other that distinguishes one protein from another Correct Definitions Polypeptide consists of a single unbranched chain of amino acids linked together by peptide bonds Proteins consists of one or more polypeptides in very close association with each other. These associations do NOT occur by the formation of interchain peptide bonds. Instead they most often interact through and are joined together by a series of many weak bonds

21 Sickle Cell Anemia emoglobin consists of 4 chains of amino acids, four polypeptides 2 identical chains of 141 amino acids called α-globin 2 identical chains of 146 amino acids called β-globin Individuals with sickle cell anemia are born with β-globin chains that contain a valine at amino acid #6 (numbering from the amino terminal end) instead of the glutamin acid that normally occurs there. This mutation affect the structure and function of the hemoglobin. The red blood cell itself become distorted taking on the familiar sickle shape that characterizes the disease. These cells get stuck in capillaries and eventually impede blood flow causing further complications. Without transfusions and despite of them people with this condition often die before becoming adults. Characteristics of Polypeptides Vary considerably in size 10s to 1,000s of amino acids Average is 350 Titin, a polypetide which has some 25,000 amino acids, is found in vertebrate striated muscle May group together (to form proteins like hemoglobin) through weak associations and bonds Are irregularly shaped Not straight rods The different amino acids interact with each other so that the typical polypeptide bends back and forth forming a complicated three-dimensional structure

22 retinol-binding protein (NP_006735) β-lactoglobulin (P02754) Page 42 DNA base Deoxyribonucleic acid (DNA) is a polymer The monomers from which it is formed are called nucleotides (deoxyribonucleotides) There are four kinds of these nucleotides deoxyadenosine-5 -phospage (A) deoxycytidine-5 -phospage (C) deoxyguanosine-5 -phospage (G) deoxythymidine-5 -phospage (T)

23 DNA base 2 These nucleotides consist of three parts A five carbon sugar (deoxyribose) A ring-shaped nitrogen containing a structure called a base (A, C, G, or T) A phosphate group (negatively charge imposing a negative charge on the nucleotide and this on the DNA) Figure from Sofer On oage 8 showing DNA

24 DNA The deoxyribonucleotides are linked together via their phosphate groups by strn bonds like the peptide bond by the elimination of water The resultant polymer which may contain hundreds of millions of nucleotides is DNA These consist of intertwining chains or strands that take on the shape of a helix DNA figure

25 RNA Ribonucleic acid (RNA) resembles DNA It is composed of chains of nucleotides These nucleotides are ribonucleotides Ribose sugar instead of deoxyribose sugar Ribose has an O group instead of an The base thymine (T) of DNA is replaced by uracil (U) in RNA RNA is most often single stranded whereas DNA is double stranded Figure of RNA

26 Proteins Major constituents of cells Involved in virtually all cellular processes Actin one of the most abundant proteins in higher organisms Actin is associated with movement Actin forms filaments that help cells move The contraction of our heart, the movement of our limbs are mediated by muscle cells, each of which contains a molecular motor composed of actin in combination with other muscle proteins Proteins 2 Other proteins act as molecular police offices recognizing or fastening onto undesirable elements like bacteria and viruses so these can be eliminated from the organisms Antibodies of vertebrates are examples Some proteins transport molecules in and out of cells Like molecular pumps, they transfer foodstuffs and desirables salts into the cell and force out waste products and poisons

27 Enzymes as Proteins Catalysts Proteins most often serve as specific catalysts, directing and accelerating the multitude of chemical reactions that occur in cells These are called enzymes They help break down complex substances into simpler ones They aid in piecing together a variety of small molecules from parts of smaller (or little) ones. They play a role in the synthesis of other proteins as well as DNA and RNA Enzymes = Protein Catalysts Direct chemical reactions in all living things by acting as biological catalysts Catalysts speed up the rate of a chemical reaction without being used up in the process Without catalysts most reactions in living things would proceed far too slowly (several orders of magnitude) to support life These differ from chemical catalysts (like platinum and palladium) in their extraordinary specificity Most enzymes only catalyze a single chemical reaction

28 ow much is the speedup? Extraordinary In some cases they accelerate the rate of a chemical step more than hundreds of trillions of times that of an uncatalyzed reaction ow much catalysis occurs? There are hundreds of different kinds of small molecules in a cell There are different proteins that may be present The vast majority are enzymes which affect the rate of a single chemical step This means that there are 100s or even 1000s of reactions being catalyzed at any given time within the cell A phenomenal machine to coordinate the result is an orderly series of reactions with one product often serving as a reactant for another Each enzyme seems to play a unique role in the complicated pathways of chemical synthesis and breakdown going on allowing the cell to function as an efficient factory

29 Enzymes speed up reactions in 3 ways Proximity They bring chemical reactants (substrates) very close together so they have an increased opportunity to interact (molecular collision increase interaction) They increase local concentration of reactants by binding to them and dragging them into proximity Orientation They bind to substrates only in certain orientations thereby aligning reactants so they can interact more efficiently Reaction They chemically react (reversibly) with one or more substances forming intermediates that are more readily converted into the required products ow do they do that? Enzymes take on particular 3D structures Enzyme catalysis requires that certain amino acids in the enzyme be positioned so that they can interact properly with the reactants If this positioning is faulty or disrupted the enzymes will no longer work Key to medicinal properties of drugs

30 The amino ac 3D Structures 3D Structures The amino acids of proteins can rotate around their peptide bond allowing polypeptides to assume many different configurations This confirmation of the protein is determined primarily by its amino acid sequence which will naturally and of its own accord fold in space to take on a specific shape Apparently many weak interactions occur among the different side chains of the amino acids and one particular conformation of the protein forms a uniquely stable structure

31 Classic Experiment 1 Researchers made active enzymes by chemically synthesizing a given amino acid sequence outside the cell They used a series of chemical reactions to knit together amino acids into a polymer with the same sequenced as that produced by living cells In most cases these artificial enzymes took on a specific shape, without benefit of help from any elements in the living cell And, they worked properly in catalysis Classic Experiment 2 An enzyme s native structure was disrupted (denatured) by agents (called denaturants) such as acids, high temperature, and detergents A variety of tests showed that the protein had lost its normal shape It had also lost all detectible enzyme activity The denaturing agents were slowly removed After a while, the protein regained its original shape And, its enzyme activity was restored

32 Conclusions The formation of a specific shape is inherent in a protein s amino acid sequence Proper folding does not necessarily require the intervention of other molecules such as other enzymes Protein Synthesis ow do organisms build proteins with the correct amino acids at each position? The structure IS determined by its amino acid sequence Issues Yield: if a machine adds amino acids one at a time with 90% accuracy, after 10 or 20 amino acids, the efficiency will be considerably below 1% (.9 n ) but cells synthesize thousands of different proteins with an efficiency very close to 100% Program: what tells the protein-synthesis machinery of the cell to insert the right amino acid at the right position at the right time?

33 DNA: the instruction carrier DNA is simply like a program for making 1000s of enzymes It is the sequence of nucleotides htat is responsible for informing the cell where each amino acid will be inserted in a protein The sequence is translated into a protein sequence The Genetic Code DNA Protein Mapping DNA and proteins are linear polymers DNA consists of 4 nucleotides Proteins consist of 20 different amino acids Possible mappings of DNA to Proteins 1 nucleotide yields 4 possible choices 2 nucleotides yield 16 choices So need 3 nucleotides to get to 20 amino acids Since there are 64 possibilities this is not a 1-1 mapping

34 The Triplet Code Picture of code page 19 As well as Ben Fry s version Examples TTT PE 3 Thymines tells machinery to insert the amino acid Phenylalanine AAA LYS 3 Adenines says insert Lysine 3 triplets code for nothing (actually code as terminators) TAA, TAG, TGA Some triplets code for the same amino acid TTA, TTG, CTT, CTC, CTA, CTG all code for LEU

35 Protein Synthesis DNA does not participate directly in protein synthesis It acts through an intermediary called RNA DNA makes a copy of one of its chains in the form of RNA (called messenger RNA or mrna) This process is called transcription mrna enters a ribosome, a large and complex subcellular molecular machine which translates its sequence of nucleotides into the protein sequence translation is used because the languages are different (one has 4 words, the other 20) As the ribosomes move along the mran small adaptor RNAs called transfer or trnas read the sequence on the mrna and add the appropriate amino acid to the next position on the growing peptide chain There are many trnas Each has a look at one end that bears a sequence of nucleotides complementary to a triplet on the mrna On the other end, the trna bears an amino acid The particular amino acid that the trna carries corresponds to the sequence of the complementary loop near its other end The protein is synthesized starting at its amino acid terminal end (the N terminus) Double Stranded DNA A phosphate group connects carbon 5 of the sugar to carbon 3 of the sugar of the next nucleotide This imparts an asymmetry to the moelcule Two strands with the bases pointing inward representing the ladder rungs (paired A/T and C/G, called base pairs) and the sugar phosphates linkages being the sides of the ladder See figure page 23

36 Base Pairing experiments Complementary strands at high temperatures will denature If temperatures are moderated and a solution of denatured DNA is allowed sufficient time each strand will tend to finds its complement and reform the original double stranded molecule (renaturation or nucleic acid hybridization) The same is true for complementary strands of RNA) Thus complementary strands of DNA have an affinity for each other which nature (and engineers) will use DNA replication Complex Semi-conservative (one strand from parent, the other newly synthesized) Proceeds by adding to the 3 end of the growing chain Mediated by an enzyme called DNA polymerase Uses one strand as a template base pairing the other DNA polymerase needs a preexisting chain (a primer) to add nucleotides to: a single strand complementary to part of the template In the presence of this primer and template DNA polymerase adds nucleotide units to the primer in a sequence dictated by the template

37 Transcription The two strands of DNA separate temporarily (so one can be used as a template for the RNA synthesis) The synthesis of the RNA goes from its 5 end toward its 3 end. Each nucleotide added is the complement of the base on the DNA (where the DNA has an A, the RNA adds a U, where DNA has a T the RNA has an A, and so on) The synthesis is directed by an enzyme called RNA polymerase The enzyme starts the synthesis at specific points on the DNA and ends at specific sites Note that only one strand is produced (a ribonucleotide chain) No primer is necessary Thousands of different RNA molecules can be synthesized from the DNA

38 Chromosomes DNA is found in the nucleus of cells in the form of a few extremely long molecules (chromosomes) each containing the information for constructing many different proteins Each chromosome come in pairs (usually one from the father and one from the mother) Each usually carry the same information and thus are called homologs (redundancy is good) Usually one correct copy of a sequence helps remedy the mistake on the other (that is the organism will appear normal even though one of its genes is defective) Chromosomes 2 Drosophila melanogaster the fruit fly has four pairs of chromosomes and each has sufficient DNA to code for thousands of proteins umans have 23 pairs of chromosomes The nucleotide sequence of DNA in each of the chromosomes in any organism carries all the information required to specify the amino acids sequences of all proteins made by that organism in its lifetime

39 Genes In humans there are 3 billion base pairs of DNA in the 23 chromosomes Only a portion is transcribed into RNA These transcribed areas are scattered throughout all the chromosomes Each RNA molecule that is manufactured represents therefore only a small fraction of the total DNA In general, each RNA molecule contains information for making a single specific protein Genes 2 A gene is a section of a chromosome that contains the information to make a specific protein through the production of a specific RNA A gene is more than a transcribed portion of DNA. It also contains additional instructions, data encoded in the DNA sequence outside the transcribed areas, that are responsible for telling the cell how much RNA to make and in which tissues and under what circumstances to transcribe a given segment of DNA These controlling or regulatory regions of DNA may act over long distances to modulate the generic activity of the DNA The unsolved question still is: how the non-transcribed regions of the DNA actually control gene expression

40 DNA RNA protein phenotype genomic DNA databases cdna ESTs UniGene protein sequence databases

41 There are three major public DNA databases EMBL GenBank DDBJ The underlying raw DNA sequences are identical There are three major public DNA databases EMBL oused at EBI European Bioinformatics Institute GenBank oused at NCBI National Center for Biotechnology Information DDBJ oused in Japan

42 >100,000 species are represented in GenBank all species 128,941 viruses 6,137 bacteria 31,262 archaea 2,100 eukaryota 87,147 The most sequenced organisms in GenBank omo sapiens (6.9 million entries) Mus musculus (5.0 million) Zea mays (896,000) Rattus norvegicus (819,000) Gallus gallus (567,000) Arabidopsis thaliana (519,000) Danio rerio (492,000) Drosophila melanogaster (350,000) Oryza sativa (221,000)

43 National Center for Biotechnology Information (NCBI)

44 PubMed is National Library of Medicine's search service 11 million citations in MEDLINE links to participating online journals PubMed tutorial (via Education on side bar)

45 Entrez integrates the scientific literature; DNA and protein sequence databases; 3D protein structure data; population study data sets; assemblies of complete genomes Entrez is a search and retrieval system that integrates NCBI databases

46 BLAST is Basic Local Alignment Search Tool NCBI's sequence similarity search tool supports analysis of DNA and protein databases 80,000 searches per day OMIM is Online Mendelian Inheritance in Man catalog of human genes and genetic disorders edited by Dr. Victor McKusick, others at JU

47 Books is searchable resource of on-line books TaxBrowser is browser for the major divisions of living organisms (archaea, bacteria, eukaryota, viruses) taxonomy information such as genetic codes molecular data on extinct organisms

48 Structure site includes Molecular Modelling Database (MMDB) biopolymer structures obtained from the Protein Data Bank (PDB) Cn3D (a 3D-structure viewer) vector alignment search tool (VAST) Four questions we can answer at NCBI (and elsewhere): [1] ow can I do a literature search using PubMed? [2] ow can WelchWeb help? [3] ow can I use Entrez to find information about a particular gene or protein? (What is an accession number?) [4] ow can I find information about a particular disease?

49 Question #1: ow can I use PubMed at NCBI to find literature information? PubMed is the NCBI gateway to MEDLINE. MEDLINE contains bibliographic citations and author abstracts from over 4,000 journals published in the United States and in 70 foreign countries. It has 12 million records dating back to 1966.

50 MeS is the acronym for "Medical Subject eadings." MeS is the list of the vocabulary terms used for subject analysis of biomedical literature at NLM. MeS vocabulary is used for indexing journal articles for MEDLINE. The MeS controlled vocabulary imposes uniformity and consistency to the indexing of biomedical literature.

51 PubMed search strategies Try the tutorial ( education on the left sidebar) Use boolean queries lipocalin AND disease Try using limits Try LinkOut to find external resources Obtain articles on-line via Welch Medical Library (and download pdf files):

52 1 AND lipocalin AND disease (35 results) 1 OR lipocalin OR disease (1,300,000 results) 1 NOT lipocalin NOT disease (350 results) Question #2: ow can I use WelchWeb (from the Welch Medical Library) to do literature (and other) searches? WelchWeb is available at

53 WelchWeb is available at gateway

54 PubMed gateway Library catalog

55 Remote access to Welch services Request literature

56 Browse journals Browse databases

57 WelchWeb URLs of interest Basic Sciences Subject Guide RAUL (remote access) Weldoc (Inter Library Loan, and electronic delivery of articles) MyWelch (personal library portal) Welch E-Learning page (online tutorials and hand-outs) Johns opkins Author Publishing Tool Browse Welch E-Resources by Subject Liaison Librarian Program (every dept has a liaison librarian) Thanks to Brian Brown (bbrown20@jhmi.edu), the Welch Medical Library liason to the basic sciences Visit the Basic Sciences Subject guide for a long list of bioinformaticsrelated sites...

58 This lecture continues in part 2 with a discussion of more NCBI resources