Gene Structure and Identification III

BIO520 Bioinformatics Jim Lund

Previous reading: 1.3, 9.1-9.6 10.2, 10.4, 10.6-8

For real prediction we need…

• Solve the protein folding problem• Solve the molecular docking/binding

problem• Develop realistic simulations of

molecules in cells• Simulate multicellular systems

Promoter/Enhancer analysis

• Regulatory Sequences– Known Consensus Sequences

– Consensus Sequence Generation• Using functional (experimental) Data

• HBB as an example

Gene Regulatory Sequences

• Functional sites–Consensus

–Experimental tests

• Inferred sites–Transcriptome analysis

Sequence Logos

• http://weblogo.berkeley.edu/

Position Weight Matrix:

PO A C G T01 6 4 4 6 N02 4 9 3 4 N03 12 4 3 1 A04 6 1 11 2 R05 3 2 11 4 G06 3 3 4 10 N07 3 10 3 4 N08 11 2 4 3 A09 4 9 3 4 N10 3 6 3 8 N

EUKARYOTES

• More complex signals– Basal/core promoter– Promoter– Enhancers

• More genes• More dispersed signals

– Larger promoters, distant enhancers, regulatory sites in introns.

• Combinatoric regulation common

Basal Promoter Analysis

Myers and Maniatis, Genes VI, 831

• TATA-box -25 to -30 TBP• CCAAT-box -212 to -57 CTF/NF1• GC-box -164 to +1 SP1• K C W K Y Y Y Y +1 to +5 cap signal

TATA CAATGC

+1

Finding PolII sites (transcription start

site)• Promoter Scan• TSSG/TSSW (TSSP for plants)

• Core-Promoter• FPROM

• BCM Search Launcher

Enhancer Elements

• Octamer OCT1, OCT2B NF B• ATF ATF• AP1… AP1• ……..

Consensus Sequence Databases

• TRANSFAC

• TFD (transcription factor database)

Consensus Sequence Databases

• Finding sites in promoter regions:– TESS

• http://www.cbil.upenn.edu/cgi-bin/tess/tess

– TFSEARCH• http://www.cbrc.jp/research/db/TFSEARCH.html

– BCM Search Launcher• http://searchlauncher.bcm.tmc.edu/seq-search/gene-

search.html

HBB promoter (TESS)

Sequence-based algorithms for identifying enhancer binding sites

• Genes from: – Microarray transcription analysis

– ChIP::chip experiments

– Orthologous sequences

– Experimental/other

• Programs for finding consensus sites:– MEME analysis of clusters

– AlignAce

– BioProspector/CompareProspector

Practical Gene Finding

• Use ALL tools– Predictive: Stitch together a consensus

• ORF finders

• Find patterns (and WWW pattern searches)

• HMM: GRAIL, Genscan…

– Comparative• BLASTN, BLASTX

• Compare genomes (human:mouse)

– cDNA, protein, genetic evidence

ORFs-aldolase gene

Genomic DNA-cDNA alignment

DNA sequencing

cDNAAlign (GAP)

Infer Promoter, EnhancerTest in cis

P

Comparative Genomics

• Conservation of coding regions• Identification of transcription signals

– “words” in common

• Example-yeast comparisons

Ensembl prediction pipeline

RepeatMasker

Genscan

Blast genscan peptides vProtein,unigene,est,vert mrna

Pmatch all human Proteins and cdnas

MiniGenewiseMiniEst2genome

Genes

DNA

Genscan features

• Model both strands at once• Each state may output a string of symbols

(according to some probability distribution).• Explicit intron/exon length modeling• Advanced splice site modeling• Complete intron/exon annotation for sequence• Able to predict multiple genes and partial/whole

genes• Parameters learned from annotated genes• Separate parameter training for different CpG

content groups (< 43%, 43-51%, 51-57%,>57% CG content)

GENSCAN predictions

Gn.Ex Type S .Begin ...End .Len Fr Ph I/Ac Do/T CodRg P.... Tscr..----- ---- - ------ ------ ---- -- -- ---- ---- ----- ----- ------

7.00 Prom + 63096 63135 40 -2.75 7.01 Init + 63183 63274 92 2 2 103 77 142 0.997 14.61 7.02 Intr + 63403 63625 223 1 1 83 96 181 0.999 15.61 7.03 Term + 64524 64652 129 2 0 101 50 83 0.373 3.00 7.04 PlyA + 64758 64763 6 1.05

8.00 Prom + 70508 70547 40 -4.75 8.01 Init + 70595 70686 92 1 2 103 77 133 0.990 13.71 8.02 Intr + 70817 71039 223 2 1 100 96 217 0.999 20.91 8.03 Term + 71890 72018 129 0 0 116 43 119 0.827 7.40 8.04 PlyA + 72126 72131 6 1.05

9.00 Prom + 74399 74438 40 -8.25 9.01 Sngl + 76602 76847 246 2 0 71 50 218 0.886 11.13 9.02 PlyA + 76928 76933 6 1.05

GENSCAN predicted exons

Annotated predicted exons

HBB gene

• HBB exons 1-3• 70545..70686• 70817..71039• 71890..72150

• GENSCAN• 70595 70686• 70817 71039• 71890 72018