10/19/05 d dobbs isu - bcb 444/544x: gene regulation1 10/19/05 gene regulation (formerly gene...
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10/19/05 D Dobbs ISU - BCB 444/544X: Gene Regulation 1
10/19/05
Gene Regulation
(formerly Gene Prediction - 2)
10/19/05 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)
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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.70
02
• Sect 9.1-9.3 DNA binding proteins, Transcription initiation• http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=genomes.section.70
16
* NOTE: Don’t worry about the details!!
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Optional Reading
Reviews:
1) 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
2) Wasserman WW & Sandelin (2004) Applied bioinformatics for the identification of regulatory elements. Nat Rev Genet 5:276-287http://proxy.lib.iastate.edu:2103/nrg/journal/v5/n4/full/nrg1315_fs.html
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Review last lecture: Genes & Genomes
(formerly Gene Prediction - 1)
• Eukaryotes vs prokaryotes • Cells• Genome organization• Gene structure
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Eukaryotes vs Prokaryotes
Eukaryotic cells are characterized by membrane-bound compartments, which are absent in prokaryotes.
“Typical” human & bacterial cells drawn to scale.
BIOS Scientific Publishers Ltd, 1999
Brown Fig 2.1
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Comparison of Gene Structures
BIOS Scientific Publishers Ltd, 1999
Brown Fig 2.2
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Summary: Genes & Genomes(formerly Gene Prediction - 1)
Genes in eukaryotes vs prokaryotesHave 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%)
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• 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”
Summary: Genes & Genomes(formerly Gene Prediction - 1)
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Gene regulation in eukaryotes vs prokaryotesPrimary 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
Summary: Genes & Genomes(formerly Gene Prediction - 1)
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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”
Summary: Genes & Genomes(formerly Gene Prediction - 1)
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http://www.dnai.org/c/index.html
DNA Interactive: "Genomes"
A tutorial on genomic sequencing, gene structure,
genes prediction
Howard Hughes Medical Institute (HHMI)Cold Spring Harbor Laboratory (CSHL)
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But first: a few more words about cDNA & ESTs
Promoters & enhancers
Gene prediction programs (?)
Today: Gene Regulation(formerly Gene Prediction - 2)
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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 [email protected]
10/19/05 15D Dobbs ISU - BCB 444/544X: Gene RegulationPevsner p161
exon 1 exon 2 exon 3intron intron
Transcription
RNA splicing (remove introns)
Capping & polyadenylation
Export to cytoplasm
AAAAA 3’5’
5’
5’
5’ 3’5’3’
3’
3’
7MeG
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DNA RNA
cDNA
Phenotypeprotein
[1] Transcription[2] RNA processing (splicing)[3] RNA export[4] RNA surveillance
Pevsner p160
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Relationship of mRNA to genomic DNA (for RBP4)
Pevsner p162
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Analysis of gene expression in cDNA libraries
A fundamental approach to studying gene expressionis 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
vector
insert
Pevsner p162-163
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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
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Cluster sizes in UniGene
This is a gene with1 EST associated;the cluster size is 1
Pevsner p164
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Cluster sizes in UniGene
This is a gene with10 ESTs associated;the cluster size is 10
Pevsner p164
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Cluster sizes in UniGene - (in 2002)
Cluster size Number of clusters1 34,0002 14,0003-4 15,0005-8 10,0009-16 6,00017-32 4,000500-1000 5002000-4000 508000-16,000 3>16,000 1
Pevsner p164
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Other Resources
Current Protocols in Bioinformaticshttp://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
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Gene Regulation
Promoters & enhancers
What does an RNA polymerase "see"?Eukaryotes vs prokaryotes
• Regulatory regions• Prokaryotic operons & promoters• Eukaryotic promoters & enhancers• Eukaryotic transcription factors
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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
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Promoters for prokaryotic RNA polymerases (e.g., bacterium, E. coli)
BIOS Scientific Publishers Ltd, 1999
Brown Fig 9.17
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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
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Prokaryotic promoters
• RNA polymerase complex recognizes promoter sequences located very close to & on 5’ 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
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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
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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
BIOS Scientific Publishers Ltd, 1999
Brown Fig 9.26
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Eukaryotic genes are transcribed by 3 different RNA polymerases
BIOS Scientific Publishers Ltd, 1999
Brown Fig 9.18
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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
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Assembly of an initiation complex for eukaryotic RNA polymerase II
http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=genomes.figgrp.7095
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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
BIOS Scientific Publishers Ltd, 1999
Brown Fig 9.27
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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
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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
BIOS Scientific Publishers Ltd, 1999
Brown Fig 9.12
37D Dobbs ISU - BCB 444/544X: Gene Regulation
Building “Designer” Zinc Finger DNA-binding Proteins J Sander, Fengli Fu, J Townsend, R Winfrey D Wright, K Joung, D Dobbs, D Voytas (ISU)