Gene Structure and Identification

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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
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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.

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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.

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ACGT to Gene

• Cells recognize genes from DNA sequence.

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Genes

• Protein Coding
• RNA genes
• rRNA
• tRNA
• siRNA, miRNA, snRNA, snoRNA

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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.

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Genomes
2002 Mus musculus 2,500,000,000
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Characteristics of Protein Coding Genes

• ORF
• long (usually gt100 aa)
• known proteins?likely
• Basal signals
• Transcription, splicing, translation
• Regulatory signals
• Depend on organism
• Prokaryotes vs Eukaryotes
• Verterbrate vs fungi, eg.

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Infer Gene StructureGene Model

• Promoter
• Strength
• Regulation

• mRNA
• Exons
• Splicing
• Stability
• ORFprotein

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GenomesGene Content
E. coli 4000 genes X 1 kbp/gene4 Mbp Genome4
Mbp!
Gene-rich
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GenomesGene Content
Human 26,755 genes X 2 kbp54 Mbp mRNA
Introns300 Mbp? Regulatory regions300 Mbp?
2344 Mbp???
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Complex Genome DNA

• 10 highly repetitive (300 Mbp)
• NOT GENES
• 25 moderate repetitive (750 Mbp)
• Some genes
• 10 exons and introns (340 Mbp)
• 55 ?
• Regulatory regions
• Intergenic regions

Hard!!
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Easy problemBacterial Gene Finding

• Dense Genomes
• Short intergenic regions
• Uninterrupted ORFs
• Conserved signals
• Abundant comparative information
• Complete Genomes

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E. coli genome

• 4415 genes
• Ave. distance between genes 118bp
• 318 aa, average protein length
• 57 proteins longer than 1000 aa.
• 318 shorter than 100 aa.
• 2584 operons, 70 contain one gene.
• 1.5 repetitive DNA (mostly viral fragments).

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Prokaryotic Gene Expression
Promoter
Cistron1
Cistron2
CistronN
Terminator
Transcription
RNA Polymerase
mRNA 5
3
1
2
N
N
N
C
N
C
C
1
2
3
Polypeptides
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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
  N-terminal protein sequences.

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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 8bps)

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ORFs
P(ORF)(61/64)n
P(20)(61/64)20.38
P(100)0.008
P(200)10-4
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ORF finding tools

• VectorNTI
• Analyze/ORF
• Testcode (Ficketts)
• CodonPreference
• WWW tools
• ORF Finder (NCBI)
• BCM Search Launcher...

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ORFs in E. coli
Frame
1
2
3
-1
-2
-3
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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

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Codon Bias
Gly GGG 6 0.21 Gly GGA 6 0.17 Gly GGT 6 0.38 Gly G
GC 6 0.24
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Codon Bias Gene Differences
GAL4 ADH1 Gly GGG 0.21 0 Gly GGA 0.17 0 Gly
GGT 0.38 0.93 Gly GGC 0.24 0.07
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Nucleotide Bias

• Coding DNA vs non-Coding DNA
• often GC content higher than bulk
• Empirical statistics (Ficketts TESTCODE)
• Useful
• ORF matches typical
• organism, bias
• ORF obscured by STOP codons

DNA sequence Errors?
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We found ORFs-now what?

• Work backwards
• Locate adjacent cistrons
• Locate RBS
• Locate promoter
• Locate terminator
• Locate regulatory sites

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Operon Structure
Promoter?
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TranslationRibosome Binding Site, Shine-Dalgarno
Site
nnAGGAGGnnnnnATG Consensus not always used,
example E. coli gene nnAaGAGGnnnnATG
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Bacterial Promoter
-35 T82T84G78A65C54A45 (16-18
bp) T80A95T45A60A50T96(A,G) -10 1
Alternate sigma factors CCCTTGAA.CCCGATNT
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Terminators

• Stem/loop
• structural only
• 3-U tail
• Rho-independent

• C-rich
• G-poor
• loose consensus
• Rho-dependent

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Difficulties in gene prediction

• Frame shifts
• sequencing errors
• Overlapping ORFs
• Rare (a few percent)
• Short ORFs
• Unusual genes
• bp composition
• signal sequences

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Programs for prokaryotic gene prediction

• Glimmer
• ORPHEUS
• GeneMark
• 90 sensitivity and specificity
• GENSCAN
• Vector NTI (ORF analysis)