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BCB 444/544

BCB 444/544. Lecture 15 More Review : RNA, Proteins, Promoters, TFs Next time: Profiles & Hidden Markov Models (HMMs) #15_Sept26. Required Reading ( before lecture). Mon Sept 24 - Lecture 14 Review: Nucleus, Chromosomes, Genes, RNAs, Proteins

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BCB 444/544

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  1. BCB 444/544 Lecture 15 More Review: RNA, Proteins, Promoters, TFs Next time: Profiles & Hidden Markov Models (HMMs) #15_Sept26 BCB 444/544 F07 ISU Dobbs #15 - RNA, Proteins, Promoters, TFs

  2. Required Reading (before lecture) Mon Sept 24 - Lecture 14 Review: Nucleus, Chromosomes, Genes, RNAs, Proteins Surprise lecture: No assigned reading Wed Sept 26 - Lecture 15 Profiles & Hidden Markov Models • Chp 6 - pp 79-84 • Eddy: What is a hidden Markov Model? 2004 Nature Biotechnol 22:1315 http://www.nature.com/nbt/journal/v22/n10/abs/nbt1004-1315.html Thurs Sept 27 - Lab 4 &Fri Sept 28 - Lecture 16 Protein Families, Domains, and Motifs • Chp 7 - pp 85-96 BCB 444/544 F07 ISU Dobbs #15 - RNA, Proteins, Promoters, TFs

  3. Assignments & Announcements Wed Sept 26 • Exam 1 - Graded & returned in class • HW#2 - Graded & returned in class • Answer KEYs posted on website • Grades posted on WebCT • HomeWork #3 - posted online Due: Mon Oct 8 by 5 PM • HW544Extra #1- posted online Due: Task 1.1 - Mon Oct 1 by noon Task 1.2 & Task 2 - Mon Oct 8 by 5 PM BCB 444/544 F07 ISU Dobbs #15 - RNA, Proteins, Promoters, TFs

  4. BCB 544 - Extra Required Reading Mon Sept 24 BCB 544 Extra Required Reading Assignment: • Pollard KS, Salama SR, Lambert N, Lambot MA, Coppens S, Pedersen JS, Katzman S, King B, Onodera C, Siepel A, Kern AD, Dehay C, Igel H, Ares M Jr, Vanderhaeghen P, Haussler D. (2006) An RNA gene expressed during cortical development evolved rapidly in humans. Nature443: 167-172. • http://www.nature.com/nature/journal/v443/n7108/abs/nature05113.html • doi:10.1038/nature05113 • PDF available on class website - under Required Reading Link BCB 444/544 F07 ISU Dobbs #15 - RNA, Proteins, Promoters, TFs

  5. Cell & Molecular Biology: the Basics Slide Credits: Terribilini, 06; & some adapted from Erin Garland BCB 444/544 F07 ISU Dobbs #15 - RNA, Proteins, Promoters, TFs

  6. Eukaryotic Cell • Enclosed & subdivided by membranes • Several compartments called organelles • Multiple linear chromosomes in nucleus BCB 444/544 F07 ISU Dobbs #15 - RNA, Proteins, Promoters, TFs

  7. Prokaryotic Cell • Enclosed by membrane & cell wall • No real organelles • Single circular chromosome (usually) • Has nucleoid but no true nucleus Wrong! DNA is never naked (inside cells)! X BCB 444/544 F07 ISU Dobbs #15 - RNA, Proteins, Promoters, TFs

  8. Translation: mRNA to protein, by ribosomes protein tRNA Amino acids ribosome tRNA mRNA = messenger RNA Codon = 3 nucleotides encode an amino acid BCB 444/544 F07 ISU Dobbs #15 - RNA, Proteins, Promoters, TFs

  9. Genetic Code: Universal (almost!) Stop Codons Start Codon BCB 444/544 F07 ISU Dobbs #15 - RNA, Proteins, Promoters, TFs

  10. Mutations • Nonsense = STOP codon in wrong place! • Missense = mutation that results in an amino acid change in the protein • Synonymous = mutation in DNA that does notresult in an amino acid change in protein • Non-synonymous = mutation in DNA that does result in an amino acid change in protein Question: Can a "synonymous" mutation alter expression of a protein - even though DNA change is "silent" (because it does not change encoded amino acid)? YES! How? This was last Slide covered in Class on Mon 9/24 BCB 444/544 F07 ISU Dobbs #15 - RNA, Proteins, Promoters, TFs

  11. Extra Credit Questions #2-6: • What is the size of the dystrophin gene(in kb)? (Is it still the largest known human protein?) • What is the largest protein encoded in human genome (i.e., longest single polypeptide chain)? • What is the largest protein complex for which a structure is known (for any organism)? • What is the most abundant protein (naturally occurring) on earth? • Which state in the US has the largest number of mobile genetic elements (transposons) in its living (plant and animal) population? • For 1 pt total (0.2 pt each): Answer all questions correctly • & submit to terrible@iastate.edu • For 2 pts total: Prepare a PPT slide with all correct answers • & submit to ddobbs@iastate.edubefore 9 AM on Mon Oct 1 • Choose one option - you can't earn 3 pts! • Partial credit for incorrect answers? only if they are truly amusing! BCB 444/544 F07 ISU Dobbs #15 - RNA, Proteins, Promoters, TFs

  12. Extra Credit Questions #7 & #8: Given that each male attending our BCB 444/544 class on a typical day is healthy (let's assume MH=7), and is generating sperm at a rate equal to the average normal rate for reproductively competent males (dSp/dT = ? per minute): 7a. How many rounds of meiosis will occur during our 50 minute class period? 7b. How many total sperm will be produced by our BCB 444/544 class during that class period? 8. How many rounds of meiosis will occur in the reproductively competent females in our class? (assume FH=5) • For 0.6 pts total (0.2 pt each): Answer all questions correctly • & submit to terrible@iastate.edu • For 1 pts total: Prepare a PPT slide with all correct answers • & submit to ddobbs@iastate.edu before 9 AM on Mon Oct 1 • Choose one option - you can't earn more than 1 pt for this! • Partial credit for incorrect answers? only if they are truly amusing! BCB 444/544 F07 ISU Dobbs #15 - RNA, Proteins, Promoters, TFs

  13. Protein Function Proteins are the workhorses of the cell • Proteins are primary molecules responsible • for carrying out cellular functions • Most "enzymes" that catalyze chemical reactions are proteins (but some are RNAs!) • Proteins have complex structures that are critical for their functions Protein structure for dystrophin: encoded by the largest known gene in humans (but, dystropin is notthe largest known protein in humans) BCB 444/544 F07 ISU Dobbs #15 - RNA, Proteins, Promoters, TFs

  14. Protein Structure: 4 levels of organization BCB 444/544 F07 ISU Dobbs #15 - RNA, Proteins, Promoters, TFs

  15. Key Aspects of Protein Function: Localization & Interactions Protein localization- function depends on proteins being in right place at right time! Protein interactions- function depends on proteins interacting with correct partners inside cells! Both of these are "hot" areas of Bioinformatics research: later, you will use machine learning to "predict" these! BCB 444/544 F07 ISU Dobbs #15 - RNA, Proteins, Promoters, TFs

  16. Protein Sequence-Structure-Function • Amino acid sequence determines protein structure • But some proteins need help folding ("chaperones") in vivo • Proteins fold to a single "native" structure (under a specific set of conditions) • Protein structure determines function • But level, timing & location of expression are important • Interactions with other proteins, DNA, RNA, & small ligands are also very important!! PROBLEMS: • We don't know the "folding code" that determines how proteins fold! • We don't know the "recognition code" that determines how proteins find and bind their correct partners! These are "hot" areas of Computational Biology research: soon, you will try to predict protein structures & protein binding sites! BCB 444/544 F07 ISU Dobbs #15 - RNA, Proteins, Promoters, TFs

  17. Modeling Protein Interaction Networks Is this an engineering problem? This is a "hot" area of Systems Biology research: later, we will try out "Retinal Workbench" for analyzing networks involved in retinal development BCB 444/544 F07 ISU Dobbs #15 - RNA, Proteins, Promoters, TFs

  18. Modeling Metabolic Pathways? seeMetNethttp://metnet.vrac.iastate.edu/MetNet_overview.htm BCB 444/544 F07 ISU Dobbs #15 - RNA, Proteins, Promoters, TFs

  19. Genes Genes in chromatin are not just “beads on a string” they have complex structures that we don't yet fully understand BCB 444/544 F07 ISU Dobbs #15 - RNA, Proteins, Promoters, TFs

  20. Eukaryotic gene structure • In human, on average genes that encode proteins include ~2000-3000 bp coding sequences, but can have >10,000 bp between exons!! • Gene sizes can vary by up to 4 orders of magnitude! • Recall:Eukaryotic genes are fragmented, containing introns between functional exons BCB 444/544 F07 ISU Dobbs #15 - RNA, Proteins, Promoters, TFs

  21. RNA Processing - Splicing One gene One protein Introns are removed to generate a mature mRNA DNA Transcribed RNA Introns removed by splicing Different combinations of exons can be used to make different proteins (alternative splicing) mRNA BCB 444/544 F07 ISU Dobbs #15 - RNA, Proteins, Promoters, TFs

  22. Gene regulation • Transcriptional regulation is primarily mediated by proteins that interact with cis-acting DNA elements associated with genes: • DNA level (sequence-specific) regulatory signals • Promoters, terminators • Enhancers, repressors, silencers • Chromatin level (global) regulation • Heterochromatin (inactive) • e.g., X-inactivation in female mammals • In eukaryotes, genes are often regulated at other levels: • Post-transcriptional (RNA transport, splicing, stability) • Post-translational (protein localization, folding, stability) BCB 444/544 F07 ISU Dobbs #15 - RNA, Proteins, Promoters, TFs

  23. Promoter = DNA sequences required for initiation of transcription; contain TF binding sites, usually "close" to start site • Transcription factors (TFs) - proteins that regulate transcription • (In eukaryotes) RNA polymerase binds by recognizing a complex of • TFs bound at promotor First, TFs must bind TF binding sites (TFBSs) within promoters; then RNA polymerase can bind and initiate transcription of RNA ~200 bp Pre-mRNA BCB 444/544 F07 ISU Dobbs #15 - RNA, Proteins, Promoters, TFs

  24. Enhancers & repressors = DNA sequences that regulate initiation of transcription; contain TF binding sites,can be far from start site! Enhancers "enhance" transcription Repressors or silencers "repress" transcription RNAP = RNA polymerase II Promoter Enhancer Gene 10-50,000 bp Repressor Enhancer binding proteins (TFs) interact with RNAP Repressor binding proteins (TFs) block transcription BCB 444/544 F07 ISU Dobbs #15 - RNA, Proteins, Promoters, TFs

  25. Transcription factors (TFs) & their binding sites (TFBSs) • Transcription factors - proteins that either activate or repress transcription, usually by binding DNA (via a DNA binding domain) & interacting with RNA polymerase (via a "trans-activating domain) to affect rate of transcription initiation • Promotors, enhancers, and repressors - all contain binding sites for transcription factors • Promoters - usually located close to start site; • Enhancers/Silencers/Repressor sequences - can be close or very far away: located upstream, downstream or even within the coding sequence of genes !! BCB 444/544 F07 ISU Dobbs #15 - RNA, Proteins, Promoters, TFs

  26. "Non-coding" DNA? Many genes encode RNA that is not translated 4 Major Classes of RNA: • mRNA = messenger RNA • tRNA = transfer RNA • rRNA = ribosomal RNA • "Other" -Lots of these, diverse structures & functions: • "Natural" RNAs: • siRNA, miRNA, piRNA, snRNA, snoRNA, … • ribozymes • Artificial RNAs: • RNAi • antisense RNA BCB 444/544 F07 ISU Dobbs #15 - RNA, Proteins, Promoters, TFs

  27. Web Resources for more information: • BioTech’s Life Science Dictionary • Online textbooks – NCBI bookshelf BCB 444/544 F07 ISU Dobbs #15 - RNA, Proteins, Promoters, TFs

  28. Algorithms & Software for MSA? #3 (NOT covered on Exam1) Heuristic Methods - continued • Progressive alignments (Star Alignment, Clustal) • Others: T-Coffee, DbClustal -see text: can be better than Clustal • Match closely-related sequences first using a guide tree • Partial order alignments (POA) • Doesn't rely on guide tree; adds sequences in order given • PRALINE • Preprocesses input sequences by building profiles for each • Iterative methods • Idea: optimal solution can be found by repeatedly modifying existing suboptimal solutions(eg: PRRN) • Block-based Alignment • Multiple re-building attempts to find best alignment (eg:DIALIGN2 & Match-Box) • Local alignments • Profiles, Blocks, Patterns - more on these soon! BCB 444/544 F07 ISU Dobbs #15 - RNA, Proteins, Promoters, TFs

  29. Chp 6 - Profiles & Hidden Markov Models SECTION II SEQUENCE ALIGNMENT Xiong: Chp 6 Profiles & HMMs • √Position Specific Scoring Matrices (PSSMs) • √PSI-BLAST Thurs & Fri: • Profiles • Markov Models & Hidden Markov Models BCB 444/544 F07 ISU Dobbs #15 - RNA, Proteins, Promoters, TFs

  30. Chp 7 - Protein Motifs & Domain Prediction SECTION II SEQUENCE ALIGNMENT Xiong: Chp 7 Protein Motifs and Domain Prediction • Identification of Motifs & Domains in Multple Sequence Alignment • Motif & Domain Databases Using Regular Expressions • Motif & Domain Databases Using Statistical Models • Protein Family Databases • Motif Discovery in Unaligned Sequences • Sequence Logos BCB 444/544 F07 ISU Dobbs #15 - RNA, Proteins, Promoters, TFs

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