Gene Regulation
10/19/05
Gene Prediction & Regulation
10/19/05
Mon - Overview & Gene structure review:
Eukaryotes vs prokaryotes
Gene Regulation
Wed - Regulatory regions:
Promoters & enhancers
(formerly Gene Prediction - 2)
- Predicting genes
Fri - Predicting genes
- Predicting regulatory regions
• Next week: Predicting RNA structure (miRNAs, too)
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Reading Assignment (for Wed)
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Optional Reading
Reviews:
Mount Bioinformatics
• Chp 9 Gene Prediction & Regulation
1)
• pp 361-385 Predicting Promoters
• Ck Errata: http://www.bioinformaticsonline.org/help/errata2.html
Zhang MQ (2002) Computational prediction of eukaryotic proteincoding genes. Nat Rev Genet 3:698-709
http://proxy.lib.iastate.edu:2103/nrg/journal/v3/n9/full/nrg890_fs.html
* Brown Genomes 2 (NCBI textbooks online)
2)
• Sect 9 Overview: Assembly of Transcription Initiation Complex
• http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=genomes.chapter.7002
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
• 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!!
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Eukaryotes vs Prokaryotes
Review last lecture: Genes & Genomes
(formerly Gene Prediction - 1)
“Typical” human & bacterial
cells drawn to scale.
• Eukaryotes vs prokaryotes
• Cells
Eukaryotic cells are
characterized by
membrane-bound
compartments, which are
absent in prokaryotes.
• Genome organization
• Gene structure
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Brown Fig 2.1
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BIOS Scientific Publishers Ltd, 1999
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1
Gene Regulation
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Summary: Genes & Genomes
Comparison of Gene Structures
(formerly Gene Prediction - 1)
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 2.2
BIOS Scientific Publishers Ltd, 1999
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Summary: Genes & Genomes
(formerly Gene Prediction - 1)
Gene regulation in eukaryotes vs prokaryotes
• Eukaryotic genes
Primary level of control?
• Are larger and more complex
• * Contain introns that are “spliced” to generate
mature mRNA
Prokaryotes: Transcription
•
Eukaryotes: Transcription is important, but
e.g., RNA processing, transport, stability,
protein processing, post-translational
modification, localization, stability
• Are transcribed by 3 different RNA polymerases
Recent discoveries: small RNAs (miRNA, siRNA)
may play very important regulatory roles, often
at post-transcriptional levels
* In biology, statements such as this include an implicit “usually” or “often”
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• Expression is regulated at multiple levels
• * Undergo alternative splicing, giving rise to
multiple RNAs
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Summary: Genes & Genomes
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DNA Interactive: "Genomes"
(formerly Gene Prediction - 1)
Gene prediction?
A tutorial on genomic sequencing, gene structure,
genes prediction
• Prokaryotes: relatively “easy”
•
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Summary: Genes & Genomes
(formerly Gene Prediction - 1)
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D Dobbs ISU - BCB 444/544X: Gene Regulation
Eukaryotes: harder
Howard Hughes Medical Institute (HHMI)
Cold Spring Harbor Laboratory (CSHL)
• Genomic organization and gene structures differ in
different organisms
• Best results obtained with “customized” software for a
particular species
http://www.dnai.org/c/index.html
• In general:
• Methods are “good” at locating genes
• Have trouble with “details”
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2
Gene Regulation
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Today: Gene Regulation
Thanks to Jonathan Pevsner
for following Figs & Slides
(formerly Gene Prediction - 2)
But first:
a few more words about cDNA & ESTs
Slightly modified from:
"Introduction to Bioinformatics"
based on Chp 6 in Pevsner's text:
Bioinformatics & Functional Genomics
Promoters & enhancers
http://pevsnerlab.kennedykrieger.org/wiley
Gene prediction programs (?)
J. Pevsner [email protected]
10/19/05
5’ exon 1
3’
intron
D Dobbs ISU - BCB 444/544X: Gene Regulation
exon 2
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exon 3 3’
5’
intron
Transcription
DNA
5’
RNA
protein
Phenotype
3’
RNA splicing
(remove introns)
3’
5’
cDNA
Capping & polyadenylation
5’ 7MeG
AAAAA 3’
Export to cytoplasm
Pevsner p161
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Relationship of mRNA to genomic DNA (for
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Pevsner p160
[1] Transcription
[2] RNA processing (splicing)
[3] RNA export
[4] RNA surveillance
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Analysis of gene expression in cDNA libraries
RBP4)
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
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Pevsner p162-163
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insert
vector
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3
Gene Regulation
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UniGene: unique genes via ESTs
Cluster sizes in UniGene
This is a gene with
1 EST associated;
the cluster size is 1
• 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
Pevsner p164
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Pevsner p164
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Cluster sizes in UniGene
Cluster sizes in UniGene - (in 2002)
This is a gene with
10 ESTs associated;
the cluster size is 10
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
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Other Resources
Pevsner p164
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Gene Regulation
Current Protocols in Bioinformatics
http://www.4ulr.com/products/currentprotocols/bioinformatics.html
Promoters & enhancers
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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What does an RNA polymerase "see"?
Eukaryotes vs prokaryotes
• Regulatory regions
• Prokaryotic operons & promoters
• Eukaryotic promoters & enhancers
• Eukaryotic transcription factors
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4
Gene Regulation
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Promoters for prokaryotic RNA polymerases
(e.g., bacterium, E. coli)
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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Brown Fig 9.17
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BIOS Scientific Publishers Ltd, 1999
Prokaryotic genes & operons
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Prokaryotic promoters
• Genes with related functions are often clustered in
operons (e.g., lac operon)
• RNA polymerase complex recognizes promoter
sequences located very close to & on 5’ side
(“upstream”) of initiation site
• Operons are transcriptionally regulated as a single
unit - one promoter controls several proteins
• RNA polymerase complex binds directly to these.
with no requirement for “transcription factors”
• mRNAs produced are “polycistronic” - one mRNA
encodes several proteins; i.e., there are multiple
ORFs, each with AUG (START) & STOP codons
• Prokaryotic promoter sequences are highly conserved
• -10 region
• -35 region
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Eukaryotic genes have large & complex
regulatory regions
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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Cis-acting regulatory elements include:
Promoters,enhancers, silencers
Trans-acting regulatory factors include:
Transcription factors, chromatin remodeling
enzymes, small RNAs
Brown Fig 9.26
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BIOS Scientific Publishers Ltd, 1999
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5
Gene Regulation
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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 9.18
BIOS Scientific Publishers Ltd, 1999
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But, it’s actually more complicated:
Assembly of an initiation complex for
eukaryotic RNA polymerase II
http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=genomes.figgrp.7095
“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
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Brown Fig 9.27
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Eukaryotic transcription factors
BIOS Scientific Publishers Ltd, 1999
10/19/05
• Common in eukaryotic proteins
• Estimated 1% of mammalian
genes encode zinc-finger
proteins
• TFs contain characteristic “DNA binding motifs”
http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=genomes.table.7039
• In C. elegans, there are 500!
• TFs recognize specific short DNA sequence motifs
“transcription factor binding sites”
• Can be used as highly specific
DNA binding modules
• Several databases for these, e.g. TRANSFAC
http://www.generegulation.com/cgibin/pub/databases/transfac
• Potentially valuable tools for
directed genome modification
(esp. in plants) & human gene
therapy
Brown Fig 9.12
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Zinc finger-containing transcription factors
• Transcription factors (TFs) are DNA binding proteins
that also interact with RNA polymerase complex to
activate or repress transcription
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BIOS Scientific Publishers Ltd, 1999
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6
Gene Regulation
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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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