10/19/05 d dobbs isu - bcb 444/544x: gene regulation1 10/19/05 gene regulation (formerly gene...

10/19/05 D Dobbs ISU - BCB 444/544X: Gene Regulation 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)


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!!

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=Books



http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=genomes.chapter.7002

http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=genomes.chapter.7002

http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=genomes.section.7016

http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=genomes.section.7016


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


Review last lecture: Genes & Genomes

(formerly Gene Prediction - 1)

• Eukaryotes vs prokaryotes • Cells• Genome organization• Gene structure


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

http://www.bios.co.uk/


Comparison of Gene Structures


Brown Fig 2.2


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%)


• 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 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



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”



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)


But first: a few more words about cDNA & ESTs

Promoters & enhancers

Gene prediction programs (?)

Today: Gene Regulation(formerly Gene Prediction - 2)


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

10/19/05 16D Dobbs ISU - BCB 444/544X: Gene Regulation

DNA RNA

cDNA

Phenotypeprotein

[1] Transcription[2] RNA processing (splicing)[3] RNA export[4] RNA surveillance

Pevsner p160

10/19/05 17D Dobbs ISU - BCB 444/544X: Gene Regulation

Relationship of mRNA to genomic DNA (for RBP4)

Pevsner p162


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


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

http://www.ncbi.nlm.nih.gov/UniGene


Cluster sizes in UniGene

This is a gene with1 EST associated;the cluster size is 1

Pevsner p164


Cluster sizes in UniGene

This is a gene with10 ESTs associated;the cluster size is 10

Pevsner p164


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


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

http://www.4ulr.com/products/currentprotocols/bioinformatics.html

http://www.4ulr.com/products/currentprotocols/bioinformatics.html


Gene Regulation

Promoters & enhancers

What does an RNA polymerase "see"?Eukaryotes vs prokaryotes

• Regulatory regions• Prokaryotic operons & promoters• Eukaryotic promoters & enhancers• Eukaryotic transcription factors


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









Promoters for prokaryotic RNA polymerases (e.g., bacterium, E. coli)


Brown Fig 9.17


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


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


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


Brown Fig 9.26


Eukaryotic genes are transcribed by 3 different RNA polymerases


Brown Fig 9.18


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


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


Brown Fig 9.27


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

http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=genomes.table.7039


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

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)

10/19/05 d dobbs isu - bcb 444/544x: gene regulation1 10/19/05 gene regulation (formerly gene...

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