1. Gene Structure and Identification
Genes and Genomes
ORFs and more
Consensus Sequences
Gene Finding
Reading: sections 1.3,
BIO520 Bioinformatics Jim Lund

2. 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.

3. 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.

4. Cells recognize genes from DNA sequence.
ACGT to Gene
Cells recognize genes from DNA sequence.

5. Protein Coding Genes RNA genes rRNA tRNA siRNA, miRNA, snRNA, snoRNA…
snRNA -small nuclear RNA (mRNA splicing), U1, U2, U4, U5, and U6
snoRNA -smal nucleolar RNA (functional/catalytic in rRNA maturation)
siRNA bp active element that binds RNA-induced silencing complex (RISC) and degrades complementary RNAs, see 2006 Nobel Prize in biology.
miRNA-noncoding regulatory endogenous hairpin RNA bind to a gene’s 3' UTR and block translation Good ref:

6. 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.
snRNA -small nuclear RNA (mRNA splicing)
snoRNA -smal nucleolar RNA (functional/catalytic in RNA maturation)

7. Genomes
3,100,000,000
2002 Mus musculus ,700,000,000

8. 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.
Yeast, ~1% of genes have ORFs<100 aa

9. Infer Gene Structure “Gene Model”
Promoter
Strength
Regulation
mRNA
Exons
Splicing
Stability
ORF=protein

10. Genomes Gene Content E. coli 4000 genes X 1 kbp/gene=4 Mbp
Genome=4 Mbp!
Gene-rich

11. Regulatory regions=300 Mb?
Genomes Gene Content
Human
27,148 genes X 2 kbp=54 Mb mRNA
Introns=300 Mb?
Regulatory regions=300 Mb?
2,446 Mb = ?

12. Complex Genome DNA Hard!! ~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
Hard!!

13. Easy problem: Bacterial Gene Finding
Dense Genomes
Short intergenic regions
Uninterrupted ORFs
Conserved signals
Abundant comparative information
Complete Genomes

14. E. coli genome 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).
E. coli was the first typical bacteria sequenced, also the first model organism sequenced.

15. Prokaryotic Gene Expression
Promoter
Cistron1
Cistron2
CistronN
Terminator
Transcription
RNA Polymerase
mRNA 5’ 3’ 1 2 N
Translation
Ribosome, tRNAs,
Protein Factors N N C N C C 1 2 3
Polypeptides

16. 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.

17. 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)
Detect consensus sequences using a position probability matrix or a neural network algorithm.

18. P(ORF)=(61/64)n P(20)=(61/64)20=.38 P(100)=0.008 P(200)=10-4 ORFs
Long open reading frames are relatively rare.
P(100)=0.008
P(200)=10-4

19. ORF finding tools Artemis Testcode (Fickett’s) CodonPreference
analyze ORFs
Testcode (Fickett’s)
CodonPreference
ORF Finder (NCBI)
BCM Search Launcher
NCBI ORF finder:

20. ORFs in E. coli
Frame 1 2 3
ORFs are shown in blue. -1 -2 -3

21. 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

22. Codon Bias
Gly GGG
Gly GGA
Gly GGT
Gly GGC

23. Codon Bias Gene Differences
GAL4 ADH1
Gly GGG
Gly GGA
Gly GGT
Gly GGC

24. Nucleotide Bias Useful: DNA sequence Errors?
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
DNA sequence
Errors?
Described by James Fickett in Nucleic Acids Research 10(17); (1982).
Designed to work on a window of 200bp or more.

25. We found ORFs-now what? Work backwards Locate adjacent cistrons
Locate RBS
Locate promoter
Locate terminator
Locate regulatory sites

26. Operon Structure Promoter? Determine based on:
Spacing of ORFs. <50bp, likely not a separate gene.
2)Promoter seq. If a gene has its own promoter, it is not part of the preceding operon.
3) Functional similarity. Operons often contain genes that function together.
Promoter?

27. Translation Ribosome Binding Site, Shine-Dalgarno Site
nnAGGAGGnnnnnATG…
Consensus not always used,
example E. coli gene:
nnAaGAGGnnnnATG
ATG used >90% of time, GTG or TTG used infrequently, fMet (formylmethionine) still incorporated as 1st aa.
(Better represented as a PSSM or a HMM)

28. Alternate sigma factors
Bacterial Promoter
-35
T82T84G78A65C54A45…
(16-18 bp)…
T80A95T45A60A50T96…(A,G)
Prokaryotic sigma 70 promoters usually used, but there are other sigma factors with different site preferences.
Alternate sigma factors
CCCTTGAA….CCCGATNT

29. Terminators Stem/loop 3’-U tail Rho-independent C-rich G-poor
structural only
3’-U tail
Rho-independent
C-rich
G-poor
“loose” consensus
Rho-dependent

30. Difficulties in gene prediction
Frame shifts
sequencing errors
Overlapping ORFs
Rare (a few percent)
Short ORFs
Unusual genes
bp composition
signal sequences

31. Programs for prokaryotic gene prediction
Glimmer
ORPHEUS
GeneMark
90%+ sensitivity and specificity
GENSCAN
Links to many gene prediction programs at