10/19/05

Gene Regulation

(formerly Gene Prediction - 2)

10/19/05
 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
 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!!

10/19/05
 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 5:276-287
http://proxy.lib.iastate.edu:2103/nrg/journal/v5/n4/full/nrg1315_fs.html

10/19/05
Review last lecture: Genes & Genomes
(formerly Gene Prediction - 1)

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

10/19/05
 Eukaryotes vs Prokaryotes

“Typical” human & bacterial

cells drawn to scale.

Eukaryotic cells are

characterized by
membrane-bound
compartments, which are
absent in prokaryotes.

Brown Fig 2.1

BIOS Scientific Publishers Ltd, 1999 10/19/05
 Comparison of Gene Structures

Brown Fig 2.2

BIOS Scientific Publishers Ltd, 1999 10/19/05
 Summary: Genes & Genomes
(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%)

10/19/05
 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”

10/19/05
 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

10/19/05
 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”

10/19/05
 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

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

But first:
a few more words about cDNA & ESTs

Promoters & enhancers

Gene prediction programs (?)

10/19/05
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
 5’ exon 1 3’
intron exon 2 intron exon 3 5’
3’
Transcription
5’ 3’
RNA splicing (remove introns)

5’ 3’
Capping & polyadenylation

5’ 7MeG AAAAA 3’

Export to cytoplasm
Pevsner p161 10/19/05 D Dobbs ISU - BCB 444/544X: Gene Regulation 15
 DNA RNA protein Phenotype

cDNA

[1] Transcription
[2] RNA processing (splicing)
[3] RNA export
[4] RNA surveillance
Pevsner p160 10/19/05 D Dobbs ISU - BCB 444/544X: Gene Regulation 16
 Relationship of mRNA to genomic DNA (for RBP4)

Pevsner p162 10/19/05 D Dobbs ISU - BCB 444/544X: Gene Regulation 17

 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) insert
• Subclone into a vector
• Sequence the cDNA inserts
These are ESTs or
Expressed Sequence Tags
vector

Pevsner p162-163 10/19/05

 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 10/19/05

 Cluster sizes in UniGene

This is a gene with

1 EST associated;
the cluster size is 1

Pevsner p164 10/19/05

 Cluster sizes in UniGene

This is a gene with

10 ESTs associated;
the cluster size is 10

Pevsner p164
10/19/05
 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
10/19/05
 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

10/19/05
 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
 What does an RNA polymerase
(or a transcription factor) “see” ?
http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=genomes.figgrp.527
3

http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=genomes.figgrp.526
8

http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=genomes.figgrp.7061

10/19/05
 Promoters for prokaryotic RNA polymerases
(e.g., bacterium, E. coli)

Brown Fig 9.17

BIOS Scientific Publishers Ltd, 1999 10/19/05
 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

10/19/05
 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

10/19/05
 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

10/19/05
 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

BIOS Scientific Publishers Ltd, 1999 10/19/05
 Eukaryotic genes are transcribed by
3 different RNA polymerases

Brown Fig 9.18

BIOS Scientific Publishers Ltd, 1999 10/19/05
 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

10/19/05
 Assembly of an initiation complex for
eukaryotic RNA polymerase II

http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=genomes.figgrp.7095

10/19/05
 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

BIOS Scientific Publishers Ltd, 1999 10/19/05
 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

10/19/05
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

BIOS Scientific Publishers Ltd, 1999 10/19/05
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: Gene Regulation 37