1. Recitation 7 2/4/09 PSSMs+Gene finding
Comp. Genomics
Recitation 7
2/4/09
PSSMs+Gene finding
Partially based on slides by Irit Gat-Viks and Metsada Pasmanik-Chor

2. Biological Motifs
Biological units with common functions frequently exhibit similarities at the sequence level. These include very short “motifs”, such as:
Gene splice sites
DNA regulatory binding sites (bound by transcription factors)
Often it is desirable to model such motifs, to enable searching for new ones. Probabilistic models are very useful. Today we deal with PSSM - the simplest.

3. E. Coli Promoters

4. Regulation of Genes Transcription Factor (Protein) RNA polymerase
DNA
Gene
Regulatory Element

5. Regulation of Genes Transcription Factor (Protein) RNA polymerase DNA
Regulatory Element
Gene

6. Regulation of Genes New protein RNA polymerase Transcription Factor
DNA
Regulatory Element
Gene

7. Motif Logo Motifs can mutate on less important bases.
Position:
TGGGGGA TGAGAGA TGAGGGA
Motifs can mutate on less important bases.
The five motifs at top right have mutations in position 3 and 5.
Representations called motif logos illustrate the conserved regions of a motif.

8. Example: Calmodulin-Binding Motif
(calcium-binding proteins)

9. PSSM Starting Point
A gap-less MSA of known instances of a given motif. Representing the motif by either:
Consensus.
Position Specific Scoring Matrix (PSSM).

10. Usage of a PSSM
For a putative k-mer GTGC– multiply the probabilities: p1(G)·p2(T)·p3(G)·p4(C)
This gives the likelihood of the motif given the PSSM model
TATA box motif

11. Gene finding Only part of the genome encodes proteins
80-90% in bacteria, ab. 2% in humans
Goal: Given a genome sequence, identify gene boundaries

12. The genetic code
A protein-coding gene, an open reading frame (ORF) begins with an ATG and ends with one of three stop codons

13. Prokaryotic genes The ‘easy’ problem
Difficulty – not all possible ORFs are actually genes
In E.Coli: 6500 ORFs while there are 4290 genes.
Additional “handles” are needed

14. Handle #1: Long ORFs
In random DNA, one stop codon every 64/3=21 codons on average.
Average protein is ~300 codons long.
=> search long ORFs.
Problems:
Short genes
Overlapping long ORFs on opposite strands

15. Handle #2: Codon frequencies
Coding DNA is not random:
In random DNA, expect Leu : Ala : Trp ratio of 6 : 4 : 1
In real proteins, 6.9 : 6.5 : 1
Different frequencies for different species.

16. Using Codon Frequencies/Usage
Assume each codon is independent.
For codon abc calculate frequency f(abc) in coding region.
Given coding sequence a1b1c1,…, an+1bn+1cn+1
Calculate
The probability that the ith reading frame is the coding region:

17. Handle #3: G+C content
C+G content (“isochore”) has strong effect on gene density, gene length etc.
< 43% C+G : 62% of genome, 34% of genes
>57% C+G : 3-5% of genome, 28% of genes
Gene density in C+G rich regions is 5 times higher than moderate C+G regions and 10 times higher than rich A+T regions
Amount of intronic DNA is 3 times higher for A+T rich regions. (Both intron length and number).
Etc…

18. Handle #4: Promoter motifs
Transcription depends on regulatory regions.
Common regulatory region – the promoter
RNA polymerase binds tightly to a specific DNA sequence in the promoter

19. Gene prediction programs
Scan the sequence in all 6 reading frames:
Start and stop codons
Long ORF
Codon usage
GC content
Gene features: promotor, terminator, poly A sites, exons and introns, …
Frame +1
Frame +2
Frame +3

20. Moving to eukaryotes
Less of the genome is protein coding + introns are a (very) serious headache

21. Eukaryote gene structure
Gene length: 30kb, coding region: 1-2kb
Binding site: ~6bp; ~30bp upstream of TSS
Average of 6 exons, 150bp long
Huge variance: - dystrophin: 2.4Mb long
Blood coagulation factor: 26 exons, 69bp to 3106bp; intron 22 contains another unrelated gene

22. Splicing Splicing: the removal of the introns.
Performed by complexes called spliceosomes, containing both proteins and snRNA.
The snRNA recognizes the splice sites through RNA-RNA base-pairing
Recognition must be precise: a 1nt error can shift the reading frame making nonsense of its message.
Many genes have alternative splicing which changes the protein created.

23. Splice Sites