Gene Regulation 10/19/05

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1 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 - Predicting regulatory regions Next week: Predicting RNA structure (mirnas, too) 10/19/05 D Dobbs ISU - BCB 444/544X: Gene Regulation 1 10/19/05 D Dobbs ISU - BCB 444/544X: Gene Regulation 2 Reading Assignment (for Wed) Mount Bioinformatics Chp 9 Gene Prediction & Regulation pp Predicting Promoters Ck Errata: * Brown Genomes 2 (NCBI textbooks online) Sect 9 Overview: Assembly of Transcription Initiation Complex Reviews: Optional Reading 1) Zhang MQ (2002) Computational prediction of eukaryotic proteincoding genes. Nat Rev Genet 3: ) Wasserman WW & Sandelin (2004) Applied bioinformatics for the identification of regulatory elements. Nat Rev Genet 5: Sect DNA binding proteins, Transcription initiation * NOTE: Don t worry about the details!! 10/19/05 D Dobbs ISU - BCB 444/544X: Gene Regulation 3 10/19/05 D Dobbs ISU - BCB 444/544X: Gene Regulation 4 Review last lecture: Genes & Genomes Eukaryotes vs prokaryotes Cells Genome organization Gene structure Eukaryotes vs Prokaryotes Typical human & bacterial cells drawn to scale. Eukaryotic cells are characterized by membrane-bound compartments, which are absent in prokaryotes. 10/19/05 D Dobbs ISU - BCB 444/544X: Gene Regulation 5 Brown Fig /19/05 D Dobbs ISU - BCB 444/544X: Gene Regulation 6 D Dobbs ISU - BCB 444/544X 1

2 Comparison of Gene Structures Genes in eukaryotes vs prokaryotes Have different structures and 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%) Brown Fig /19/05 D Dobbs ISU - BCB 444/544X: Gene Regulation 7 10/19/05 D Dobbs ISU - BCB 444/544X: Gene Regulation 8 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 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 10/19/05 D Dobbs ISU - BCB 444/544X: Gene Regulation 9 10/19/05 D Dobbs ISU - BCB 444/544X: Gene Regulation 10 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 DNA Interactive: "Genomes" A tutorial on genomic sequencing, gene structure, genes prediction Howard Hughes Medical Institute (HHMI) Cold Spring Harbor Laboratory (CSHL) 10/19/05 D Dobbs ISU - BCB 444/544X: Gene Regulation 11 10/19/05 D Dobbs ISU - BCB 444/544X: Gene Regulation 12 D Dobbs ISU - BCB 444/544X 2

3 Today: Gene Regulation (formerly Gene Prediction - 2) But first: a few more words about cdna & ESTs Promoters & enhancers Gene prediction programs (?) 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 J. Pevsner pevsner@jhmi.edu 10/19/05 D Dobbs ISU - BCB 444/544X: Gene Regulation 13 10/19/05 D Dobbs ISU - BCB 444/544X: Gene Regulation 14 exon 1 intron exon 2 intron exon 3 Transcription RNA splicing (remove introns) 7Me G Capping & polyadenylation AAAAA Export to cytoplasm Pevsner p161 10/19/05 D Dobbs ISU - BCB 444/544X: Gene Regulation 15 Pevsner p160 DNA RNA cdna protein [1] Transcription [2] RNA processing (splicing) [3] RNA export [4] RNA surveillance Phenotype 10/19/05 D Dobbs ISU - BCB 444/544X: Gene Regulation 16 Relationship of mrna to genomic DNA (for RBP4) 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 insert vector Pevsner p162 10/19/05 D Dobbs ISU - BCB 444/544X: Gene Regulation 17 Pevsner p /19/05 D Dobbs ISU - BCB 444/544X: Gene Regulation 18 D Dobbs ISU - BCB 444/544X 3

4 UniGene: unique genes via ESTs Cluster sizes in UniGene Find UniGene at NCBI: UniGene clusters contain many ESTs This is a gene with 1 EST associated; the cluster size is 1 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 10/19/05 D Dobbs ISU - BCB 444/544X: Gene Regulation 19 Pevsner p164 10/19/05 D Dobbs ISU - BCB 444/544X: Gene Regulation 20 Cluster sizes in UniGene This is a gene with 10 ESTs associated; the cluster size is 10 Cluster sizes in UniGene - (in 2002) Cluster size Number of clusters 1 34, , , , , , ,000 3 >16,000 1 Pevsner p164 10/19/05 D Dobbs ISU - BCB 444/544X: Gene Regulation 21 Pevsner p164 10/19/05 D Dobbs ISU - BCB 444/544X: Gene Regulation 22 Other Resources Current Protocols in Bioinformatics 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 Gene Regulation Promoters & enhancers What does an RNA polymerase "see"? Eukaryotes vs prokaryotes Regulatory regions Prokaryotic operons & promoters Eukaryotic promoters & enhancers Eukaryotic transcription factors 10/19/05 D Dobbs ISU - BCB 444/544X: Gene Regulation 23 10/19/05 D Dobbs ISU - BCB 444/544X: Gene Regulation 24 D Dobbs ISU - BCB 444/544X 4

5 What does an RNA polymerase (or a transcription factor) see? Promoters for prokaryotic RNA polymerases (e.g., bacterium, E. coli) /19/05 D Dobbs ISU - BCB 444/544X: Gene Regulation 25 Brown Fig /19/05 D Dobbs ISU - BCB 444/544X: Gene Regulation 26 Prokaryotic genes & operons Prokaryotic promoters 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 RNA polymerase complex recognizes promoter sequences located very close to & on side ( upstream ) of initiation site RNA polymerase complex binds directly to these. with no requirement for transcription factors Prokaryotic promoter sequences are highly conserved -10 region -35 region 10/19/05 D Dobbs ISU - BCB 444/544X: Gene Regulation 27 10/19/05 D Dobbs ISU - BCB 444/544X: Gene Regulation 28 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 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 10/19/05 D Dobbs ISU - BCB 444/544X: Gene Regulation 29 Brown Fig /19/05 D Dobbs ISU - BCB 444/544X: Gene Regulation 30 D Dobbs ISU - BCB 444/544X 5

6 Eukaryotic genes are transcribed by 3 different RNA polymerases 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 Brown Fig /19/05 D Dobbs ISU - BCB 444/544X: Gene Regulation 31 10/19/05 D Dobbs ISU - BCB 444/544X: Gene Regulation 32 Assembly of an initiation complex for eukaryotic RNA polymerase II 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 10/19/05 D Dobbs ISU - BCB 444/544X: Gene Regulation 33 Brown Fig /19/05 D Dobbs ISU - BCB 444/544X: Gene Regulation 34 Eukaryotic transcription factors Zinc finger-containing 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 TFs recognize specific short DNA sequence motifs transcription factor binding sites Several databases for these, e.g. TRANSFAC 10/19/05 D Dobbs ISU - BCB 444/544X: Gene Regulation 35 Brown Fig 9.12 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 10/19/05 D Dobbs ISU - BCB 444/544X: Gene Regulation 36 D Dobbs ISU - BCB 444/544X 6

7 Building Designer Zinc Finger DNA-binding Proteins J Sander, Fengli Fu, J Townsend, R Winfrey D Wright, K Joung, D Dobbs, D Voytas (ISU) D Dobbs ISU - BCB 444/544X 7