Download 8:Genes

Gene Structure and
Identification
Genes and Genomes
ORFs and more
Consensus Sequences
Gene Finding
Reading: sections 1.3, 9.1-9.6
BIO520 Bioinformatics
Jim Lund
Gene
The functional and physical unit of
heredity passed from parent to
offspring. Genes are pieces of
DNA, and most genes contain
the information for making a
specific protein.
Gene-Informatics
Genes are character strings
embedded in much larger strings
called the genome. A gene usually
encodes a protein. Genes are
composed of ordered elements
associated with the fundamental
genetic processes including
transcription, splicing, and
translation.
ACGT to Gene
Cells recognize genes
from DNA sequence.
Genes
• Protein Coding
• RNA genes
– rRNA
– tRNA
– siRNA, miRNA, snRNA,
snoRNA…
Genomes
• Genome seq. has only limited use by itself
– Markers, SNPs, etc.
• Functional annotation
– Identify proteins and their functions.
– And regulatory regions, etc.
• Parts list: a source for understanding all
biology--and ushers in the post-genomic age
of biology.
Genomes
2002 Mus musculus
3,100,000,00
0
2,700,000,000
Characteristics of Protein
Coding Genes
• ORF
– long (usually >100 aa)
– “known” proteinslikely
• Basal signals
– Transcription, splicing, translation
• Regulatory signals
– Depend on organism
• Prokaryotes vs Eukaryotes
• Verterbrate vs fungi, eg.
Infer Gene Structure
“Gene Model”
Promoter
•Strength
•Regulation
mRNA
•Exons
•Splicing
•Stability
•ORF=protein
Genomes
Gene Content
E. coli
4000 genes X 1 kbp/gene=4 Mbp
Genome=4 Mbp!
Genomes
Gene Content
Human
27,148 genes X 2 kbp=54 Mb mRNA
Introns=300 Mb?
Regulatory regions=300 Mb?
2,446 Mb = ?
Complex
Genome DNA
• ~10% highly repetitive (300 Mb)
– NOT GENES
• ~25% moderate repetitive (750 Mb)
– Some genes
• ~10% exons and introns (354 Mb)
• 55% = ?
– Regulatory regions
– Intergenic regions
Easy problem:
Bacterial Gene Finding
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Dense Genomes
Short intergenic regions
Uninterrupted ORFs
Conserved signals
Abundant comparative information
Complete Genomes
E. coli genome
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4,415 genes
Ave. distance between genes: 118 bp
318 aa, average protein length
57 proteins longer than 1000 aa.
318 shorter than 100 aa.
2,584 operons, 70% contain one gene.
1.5% repetitive DNA (mostly viral fragments).
Prokaryotic Gene
Expression
Promoter Cistron1 Cistron2 CistronN Terminator
Transcription
RNA Polymerase
mRNA 5’
3’
1
2
Translation
C
N
N
1
N
Ribosome, tRNAs,
Protein Factors
C
N
C
2
3
Polypeptides
Prokaryotic gene prediction
•ORFs
•Biased nucleotide distribution
–Periodicity of 3
–Codon bias (codon usage statistics)
–Also called Codon Adaptation Index (CAI).
•Signal sequences
•Homology
•Other biological info: for E. coli, partial Nterminal protein sequences.
Prokaryotic signal sequences
•Ribosome binding site (RBS)/Shine-Delgarno
element
•3-9 purines complementary to sequence at
3’ end of the 16S rRNA in the small subunit
of the ribosome.
•Located: 4-7 bps 5’ of the AUG.
•Promoter
•-35 consensus site (TTGACA)
•-10 consensus site (TATAAT)
•Signal peptides
•Regulatory protein binding sites (4 to 8 bps)
ORFs
n
P(ORF)=(61/64)
20
P(20)=(61/64) =.38
P(100)=0.008
-4
P(200)=10
ORF finding tools
• Artemis
– analyze ORFs
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Testcode (Fickett’s)
CodonPreference
ORF Finder (NCBI)
BCM Search Launcher
ORFs in E. coli
Frame
1
2
3
-1
-2
-3
Codon Bias
• Genetic code degenerate
• Codon usage varies
– Organism to organism
– Gene to gene
• High bias correlates with high level
expression
• Bias correlates with tRNA isoacceptors
• Change bias or tRNAs, change
expression
Codon Bias
Gly
Gly
Gly
Gly
GGG
GGA
GGT
GGC
6
6
6
6
0.21
0.17
0.38
0.24
Codon Bias
Gene Differences
Gly
Gly
Gly
Gly
GGG
GGA
GGT
GGC
GAL4
0.21
0.17
0.38
0.24
ADH1
0
0
0.93
0.07
Nucleotide Bias
• Coding DNA vs non-Coding DNA
– often G+C content higher than bulk
• Empirical statistics (Fickett’s
TESTCODE)
Useful:
• ORF matches “typical”
– organism, bias
• ORF obscured by STOP codons
We found ORFs-now what?
• Work backwards
–Locate adjacent cistrons
–Locate RBS
–Locate promoter
–Locate terminator
–Locate regulatory sites
Operon Structure
Promoter?
Translation
Ribosome Binding Site, ShineDalgarno Site
nnAGGAGGnnnnnATG…
Consensus not always used,
example E. coli gene:
nnAaGAGGnnnnATG
(Better represented as a PSSM or a HMM)
Bacterial Promoter
-35
T82T84G78A65C54A45…
(16-18 bp)…
T80A95T45A60A50T96…(A,G)
-10
+1
Alternate sigma factors
CCCTTGAA….CCCGATNT
Terminators
• Stem/loop
• C-rich
– structural only • G-poor
• 3’-U tail
• “loose”
consensus
Rhoindependent
Rho-dependent
Difficulties in gene prediction
• Frame shifts
– sequencing errors
• Overlapping ORFs
– Rare (a few percent)
• Short ORFs
• Unusual genes
– bp composition
– signal sequences
Programs for prokaryotic gene
prediction
•Glimmer
•ORPHEUS
•GeneMark
•90%+ sensitivity and specificity
•GENSCAN