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10/19/05 Gene Regulation (formerly Gene Prediction - 2)

10/19/05 Gene Regulation (formerly Gene Prediction - 2). Gene Prediction & Regulation. Mon - Overview & Gene structure review: Eukaryotes vs prokaryotes Wed - Regulatory regions: Promoters & enhancers - Predicting genes Fri - Predicting genes

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10/19/05 Gene Regulation (formerly Gene Prediction - 2)

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  1. 10/19/05Gene Regulation(formerly Gene Prediction - 2) D Dobbs ISU - BCB 444/544X: Gene Regulation

  2. Gene Prediction & Regulation Mon - Overview & Gene structure review: Eukaryotes vs prokaryotes Wed - Regulatory regions: Promoters & enhancers - Predicting genes Fri - Predicting genes - Predicting regulatory regions Next week: Predicting RNA structure (miRNAs, too) D Dobbs ISU - BCB 444/544X: Gene Regulation

  3. Reading Assignment (for Wed) • Mount Bioinformatics • Chp 9 Gene Prediction & Regulation • pp 361-385 Predicting Promoters • Ck Errata:http://www.bioinformaticsonline.org/help/errata2.html • * Brown Genomes 2 (NCBI textbooks online) • Sect 9 Overview: Assembly of Transcription Initiation Complex • http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=genomes.chapter.7002 • Sect 9.1-9.3 DNA binding proteins, Transcription initiation • http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=genomes.section.7016 * NOTE: Don’t worry about the details!! D Dobbs ISU - BCB 444/544X: Gene Regulation

  4. Optional Reading • Reviews: • Zhang MQ (2002) Computational prediction of eukaryotic protein-coding genes. Nat Rev Genet 3:698-709 http://proxy.lib.iastate.edu:2103/nrg/journal/v3/n9/full/nrg890_fs.html • Wasserman WW & Sandelin (2004) Applied bioinformatics for the identification of regulatory elements. Nat Rev Genet 5:276-287 http://proxy.lib.iastate.edu:2103/nrg/journal/v5/n4/full/nrg1315_fs.html D Dobbs ISU - BCB 444/544X: Gene Regulation

  5. Review last lecture: Genes & Genomes(formerly Gene Prediction - 1) Eukaryotes vs prokaryotes Cells Genome organization Gene structure D Dobbs ISU - BCB 444/544X: Gene Regulation

  6. Eukaryotes vs Prokaryotes • Eukaryotic cells are characterized by membrane-bound compartments, which are absent in prokaryotes. • “Typical” human & bacterial cells drawn to scale. Brown Fig 2.1 D Dobbs ISU - BCB 444/544X: Gene Regulation BIOS Scientific Publishers Ltd, 1999

  7. Comparison of Gene Structures Brown Fig 2.2 D Dobbs ISU - BCB 444/544X: Gene Regulation BIOS Scientific Publishers Ltd, 1999

  8. Summary: Genes & Genomes(formerly Gene Prediction - 1) • Genes in eukaryotes vs prokaryotes Have different structuresand regulatory signals • Eukaryotic genomes • Are packaged in chromatin and sequestered in a nucleus • Are larger and have multiple chromosomes • Contain mostly non-protein coding DNA (98-99%) D Dobbs ISU - BCB 444/544X: Gene Regulation

  9. Summary: Genes & Genomes(formerly Gene Prediction - 1) • Eukaryotic genes • Are larger and more complex • * Contain introns that are “spliced” to generate mature mRNA • * Undergo alternative splicing, giving rise to multiple RNAs • Are transcribed by 3 different RNA polymerases * In biology, statements such as this include an implicit “usually” or “often” D Dobbs ISU - BCB 444/544X: Gene Regulation

  10. Summary: Genes & Genomes(formerly Gene Prediction - 1) • Gene regulation in eukaryotes vs prokaryotes • Primary level of control? • Prokaryotes: Transcription • Eukaryotes: Transcription is important, but • Expression is regulated at multiple levels e.g., RNA processing, transport, stability, protein processing, post-translational modification, localization, stability Recent discoveries: small RNAs (miRNA, siRNA) may play very important regulatory roles, often at post-transcriptional levels D Dobbs ISU - BCB 444/544X: Gene Regulation

  11. Summary: Genes & Genomes(formerly Gene Prediction - 1) • Gene prediction? • Prokaryotes: relatively “easy” • Eukaryotes: harder • Genomic organization and gene structures differ in different organisms • Best results obtained with “customized” software for a particular species • In general: • Methods are “good” at locating genes • Have trouble with “details” D Dobbs ISU - BCB 444/544X: Gene Regulation

  12. DNA Interactive: "Genomes" A tutorial on genomic sequencing, gene structure, genes prediction Howard Hughes Medical Institute (HHMI) Cold Spring Harbor Laboratory (CSHL) http://www.dnai.org/c/index.html D Dobbs ISU - BCB 444/544X: Gene Regulation

  13. Today: Gene Regulation(formerly Gene Prediction - 2) But first: a few more words about cDNA & ESTs Promoters & enhancers Gene prediction programs (?) D Dobbs ISU - BCB 444/544X: Gene Regulation

  14. Thanks to Jonathan Pevsner for following Figs & Slides Slightly modified from: "Introduction to Bioinformatics" based on Chp 6 in Pevsner's text: Bioinformatics & Functional Genomics http://pevsnerlab.kennedykrieger.org/wiley J. Pevsner pevsner@jhmi.edu D Dobbs ISU - BCB 444/544X: Gene Regulation

  15. 5’ 3’ exon 1 intron exon 2 intron exon 3 3’ 5’ Transcription 5’ 3’ RNA splicing (remove introns) 3’ 5’ Capping & polyadenylation 5’ 7MeG AAAAA 3’ Export to cytoplasm Pevsner p161 D Dobbs ISU - BCB 444/544X: Gene Regulation

  16. DNA RNA protein Phenotype cDNA [1] Transcription [2] RNA processing (splicing) [3] RNA export [4] RNA surveillance Pevsner p160 D Dobbs ISU - BCB 444/544X: Gene Regulation

  17. Relationship of mRNA to genomic DNA(for RBP4) Pevsner p162 D Dobbs ISU - BCB 444/544X: Gene Regulation

  18. insert vector Analysis of gene expression in cDNA libraries • A fundamental approach to studying gene expression • is through cDNA libraries • Isolate RNA (always from a specific • organism, region, and time point) • Convert RNA to complementary DNA • (with reverse transcriptase) • Subclone into a vector • Sequence the cDNA inserts • These are ESTs or • Expressed Sequence Tags Pevsner p162-163 D Dobbs ISU - BCB 444/544X: Gene Regulation

  19. UniGene: unique genes via ESTs • • Find UniGene at NCBI: • www.ncbi.nlm.nih.gov/UniGene • UniGene clusters contain many ESTs • • UniGene data come from many cDNA libraries. • Thus, when you look up a gene in UniGene • you get information on its abundance • and its regional distribution Pevsner p164 D Dobbs ISU - BCB 444/544X: Gene Regulation

  20. Cluster sizes in UniGene This is a gene with 1 EST associated; the cluster size is 1 Pevsner p164 D Dobbs ISU - BCB 444/544X: Gene Regulation

  21. Cluster sizes in UniGene This is a gene with 10 ESTs associated; the cluster size is 10 Pevsner p164 D Dobbs ISU - BCB 444/544X: Gene Regulation

  22. Cluster sizes in UniGene - (in 2002) Cluster size Number of clusters 1 34,000 2 14,000 3-4 15,000 5-8 10,000 9-16 6,000 17-32 4,000 500-1000 500 2000-4000 50 8000-16,000 3 >16,000 1 Pevsner p164 D Dobbs ISU - BCB 444/544X: Gene Regulation

  23. Other Resources • Current Protocols in Bioinformatics • http://www.4ulr.com/products/currentprotocols/bioinformatics.html • Finding Genes • 4.1 An Overview of Gene Identification: Approaches, Strategies, and Considerations • 4.2 Using MZEF To Find Internal Coding Exons • 4.3 Using GENEID to Identify Genes • 4.4 Using GlimmerM to Find Genes in Eukaryotic Genomes • 4.5 Prokaryotic Gene Prediction Using GeneMark and GeneMark.hmm • 4.6 Eukaryotic Gene Prediction Using GeneMark.hmm • 4.7 Application of FirstEF to Find Promoters and First Exons in the Human Genome • 4.8 Using TWINSCAN to Predict Gene Structures in Genomic DNA Sequences • 4.9 GrailEXP and Genome Analysis Pipeline for Genome Annotation • 4.10 Using RepeatMasker to Identify Repetitive Elements in Genomic Sequences D Dobbs ISU - BCB 444/544X: Gene Regulation

  24. Gene Regulation • Promoters & enhancers • What does an RNA polymerase "see"? • Eukaryotes vs prokaryotes • Regulatory regions • Prokaryotic operons & promoters • Eukaryotic promoters & enhancers • Eukaryotic transcription factors D Dobbs ISU - BCB 444/544X: Gene Regulation

  25. What does an RNA polymerase (or a transcription factor) “see” ? http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=genomes.figgrp.5273 http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=genomes.figgrp.5268 http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=genomes.figgrp.7061 D Dobbs ISU - BCB 444/544X: Gene Regulation

  26. Promoters for prokaryotic RNA polymerases (e.g., bacterium, E. coli) Brown Fig 9.17 D Dobbs ISU - BCB 444/544X: Gene Regulation BIOS Scientific Publishers Ltd, 1999

  27. Prokaryotic genes & operons • Genes with related functions are often clustered in operons (e.g., lac operon) • Operons are transcriptionally regulated as a single unit - one promoter controls several proteins • mRNAs produced are “polycistronic” - one mRNA encodes several proteins; i.e., there are multiple ORFs, each with AUG (START) & STOP codons D Dobbs ISU - BCB 444/544X: Gene Regulation

  28. Prokaryotic promoters • RNA polymerase complex recognizes promoter sequences located very close to & on 5’ side (“upstream”) of initiation site • RNA polymerase complexbinds directly to these. with no requirement for “transcription factors” • Prokaryotic promoter sequences are highly conserved • -10 region • -35 region D Dobbs ISU - BCB 444/544X: Gene Regulation

  29. Eukaryotic genes • Genes with related functions are not clustered, but share common regulatory regions (promoters, enhancers, etc.) • Chromatin structure must be in “right” configuration for transcription D Dobbs ISU - BCB 444/544X: Gene Regulation

  30. Eukaryotic genes have large & complex regulatory regions Cis-acting regulatory elements include: Promoters,enhancers, silencers Trans-acting regulatory factors include: Transcription factors, chromatin remodeling enzymes, small RNAs Brown Fig 9.26 D Dobbs ISU - BCB 444/544X: Gene Regulation BIOS Scientific Publishers Ltd, 1999

  31. Eukaryotic genes are transcribed by 3 different RNA polymerases Brown Fig 9.18 D Dobbs ISU - BCB 444/544X: Gene Regulation BIOS Scientific Publishers Ltd, 1999

  32. Eukaryotic promoters & enhancers • Promoters located “relatively” close to initiation site (but can be located within gene, rather than upstream!) • Enhancers also required for regulated transcription (these control expression in specific cell types, developmental stages, in response to environment) • RNA polymerase complexes do not specifically recognize promoter sequences directly • Transcription factors bind first and serve as “landmarks” for recognition by RNA polymerase complexes D Dobbs ISU - BCB 444/544X: Gene Regulation

  33. Assembly of an initiation complex for eukaryotic RNA polymerase II http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=genomes.figgrp.7095 D Dobbs ISU - BCB 444/544X: Gene Regulation

  34. But, it’s actually more complicated: “Activator & Mediator protein” actually represent a large complex of transcription factors (connected via DNA-protein & protein-protein interactions) that are usually associated with clusters of TF binding sites Brown Fig 9.27 D Dobbs ISU - BCB 444/544X: Gene Regulation BIOS Scientific Publishers Ltd, 1999

  35. Eukaryotic transcription factors • Transcription factors (TFs) are DNA binding proteins that also interact with RNA polymerase complex to activate or repress transcription • TFs contain characteristic “DNA binding motifs” http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=genomes.table.7039 • TFs recognize specific short DNA sequence motifs “transcription factor binding sites” • Several databases for these, e.g.TRANSFAC http://www.generegulation.com/cgibin/pub/databases/transfac D Dobbs ISU - BCB 444/544X: Gene Regulation

  36. Zinc finger-containing transcription factors • Common in eukaryotic proteins • Estimated 1% of mammalian genes encode zinc-finger proteins • In C. elegans, there are 500! • Can be used as highly specific DNA binding modules • Potentially valuable tools for directed genome modification (esp. in plants) & human gene therapy Brown Fig 9.12 D Dobbs ISU - BCB 444/544X: Gene Regulation BIOS Scientific Publishers Ltd, 1999

  37. Building “Designer” Zinc Finger DNA-binding Proteins J Sander, Fengli Fu, J Townsend, R Winfrey D Wright, K Joung, D Dobbs, D Voytas (ISU)

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