1. Prosite and UCSC Genome Browser Exercise 3

2. Protein motifs and Prosite

3. Turning information into knowledge  The outcome of a sequencing project is masses of raw data  The challenge is to turn this raw data into biological knowledge  A valuable tool for this challenge is an automated diagnostic pipe through which newly determined sequences can be streamlined

4. From sequence to function  Nature tends to innovate rather than invent  Proteins are composed of functional elements: domains and motifs Domains are structural units that carry out a certain function Domains are structural units that carry out a certain function The same domains are The same domains are shared between different proteins Motifs are shorter Motifs are shorter sequences with certain biological activity

5. What is a motif?  A sequence motif = a certain sequence that is widespread and conjectured to have biological significance  Examples: KDEL – ER-lumen retention signal PKKKRKV – an NLS (nuclear localization signal)

6. More loosely defined motifs  KDEL (usually) +  HDEL (rarely) =  [HK]-D-E-L: H or K at the first position  This is called a pattern (in Biology), or a regular expression (in computer science)

7. Syntax of a pattern  Example: W-x(9,11)-[FYV]-[FYW]-x(6,7)-[GSTNE]

8. Patterns  W-x(9,11)-[FYV]-[FYW]-x(6,7)-[GSTNE] Any amino-acid, between 9-11 times F or Y or V WOPLASDFGYVWPPPLAWS ROPLASDFGYVWPPPLAWS WOPLASDFGYVWPPPLSQQQ 

9. Patterns - syntax  The standard IUPAC one-letter codes.  ‘x’ : any amino acid.  ‘[]’ : residues allowed at the position.  ‘{}’ : residues forbidden at the position.  ‘()’ : repetition of a pattern element are indicated in parenthesis. X(n) or X(n,m) to indicate the number or range of repetition.  ‘-’ : separates each pattern element.  ‘‹’ : indicated a N-terminal restriction of the pattern.  ‘›’ : indicated a C-terminal restriction of the pattern.  ‘.’ : the period ends the pattern.

10. Profile-pattern-consensus GTTCAA GCTGAA CTTCAC 54321.0010.66A.1000T.00.6600.33C.00.3300G GTTCAA [AC]-A-[GC]-T-[TC]-[GC] multiple alignment consensus pattern profileNNTNAN

11. http://www.expasy.ch/prosite/

12. Prosite  A method for determining the function of uncharacterized translated protein sequences  Database of annotated protein families and functional sites as well as associated patterns and profiles to identify them

13. Prosite  Entries are represented with patterns or profiles pattern 54321.0010.66A.1000T.00.6600.33C.00.3300G profile [AC]-A-[GC]-T-[TC]-[GC] Profiles are used in Prosite when the motif is relatively divergent and it is difficult to represent as a pattern

14. Scanning Prosite Query: sequence Query: pattern Result: all patterns found in sequence Result: all sequences which adhere to this pattern

15. prosite sequence query

17. Prosite profile

18. Prosite profile  sequence logo

19. Sequence logo

20. WebLogo http://weblogo.berkeley.edu/logo.cgi

21. Searching Prosite with a sequence

22. Patterns with a high probability of occurrence  Entries describing commonly found post- translational modifications or compositionally biased regions.  Found in the majority of known protein sequences  High probability of occurrence

23. Searching Prosite with a pattern

24. prosite pattern query

27. Searching Prosite with a Prosite AC

28. UCSC Genome Browser

31. Reset all settings of previous user UCSC Genome Browser - Gateway

34. UCSC Genome Browser query results

35. UCSC Genome Browser Annotation tracks Vertebrate conservation mRNA (GenBank) RefSeq UCSC Genes Base position Single species compared SNPs Repeats Direction of transcription (<) CDS Intron UTR

36. USCS Gene

37. UCSC Genome Browser - movement Zoom x3 + Center

38. UCSC Genome Browser – Base view

39. Annotation track options dense squish full pack

40. Annotation track options Another option to toggle between ‘pack’ and ‘dense’ view is to click on the track title Sickle-cell anemia distr. Malaria distr.

41. BLAT  BLAT = Blast-Like Alignment Tool  BLAT is designed to find similarity of >95% on DNA, >80% for protein  Rapid search by indexing entire genome. Good for: 1. Finding genomic coordinates of cDNA 2. Determining exons/introns 3. Finding human (or chimp, dog, cow…) homologs of another vertebrate sequence 4. Find upstream regulatory regions

42. BLAT on UCSC Genome Browser

44. BLAT Results

45. Match Non-Match (mismatch/indel) Indel boundaries

46. BLAT Results

47. BLAT Results on the browser

48. Getting DNA sequence of region