Genome of the week Bacillus subtilis Grampositive soil

Genome of the week Bacillus subtilis Gram-positive soil bacterium Genetically tractable, well-studied Developmental pathways (sporulation, genetic competence) Industrial and agricultural importance 4. 2 Mb genome (sequence completed 1997) Close relative of Bacillus anthracis (Anthrax)

B. subtilis genome features • 4, 106 protein coding genes • 10 r. RNA operons • Nearly 50% of the genome consists of paralogous genes. – 77 ABC transporter binding proteins • 10 phage like regions - horizontal transfer. Low GC regions in the genome. • 18 sigma factors - initiate transcription. • 34 two-component regulatory systems.

Annotating genes • How to assign preliminary functions to genes. • Automated programs. • Similarity searches – BLAST and PSI-BLAST – COGs, Pfam, CDD, other databases – Only 50 -75% of genes will have a predicted function. Some have no known homologs in any other genome. • Functional characterization (individual genes) – Gene knockouts – Overexpression

• In many cases computer annotation will only be able to predict function - NOT assign function! – The biological function of many genes have not been determined, even in model systems. – As genomic characterization of gene function continues - more and more computer generated annotations will be correct.

• Molecular function - activity of a protein at the molecular level. – Examples would be ATPase, metal binding, converting glucose-6 -phosphate to fructose-6 phosphate. • Biological function - cellular role of the protein. – Examples would be translation initiation, DNA replication, glycolysis.

Homologs, orthologs, and paralogs. • Homologous genes are genes that share a common evolutionary ancestor. – Orthologs are genes found in different organisms that arose from a common ancestor – Paralogs are genes found in the same organism that arose from a common ancestor. Duplication could have occurred in the species or earlier.

Using BLAST to predict gene function. • BLAST predicted protein sequence against the non-redundant database. • Determine best hits • Automated annotation programs will often assign the best hit function to the gene being searched. • Must manually confirm automated annotations. (Final project).

Basic Local Alignment Search Tool § § § Calculates similarity for biological sequences Finds best local alignments Heuristic approach based on Smith-Waterman algorithm Searches for matching “words” rather than individual residues Uses statistical theory to determine if a match might have occurred by chance NCBI Field Guide

Nucleotide Words Query: GTACTGGACATGGACCCTACAGGAA Minimum word size = 7 Word Size = 11 GTACTGGACAT blastn default = 11 TACTGGACATG megablast default = 28 Make a lookup ACTGGACATGG table of words CTGGACATGGAC GGACATGGACCC ACATGGACCCT NCBI Field Guide. . .

Protein Words Query: GTQITVEDLFYNIATRRKALKN GTQ Word Size can be 2 or 3 (default = 3) TQI Make a lookup Neighborhood Words QIT table of words LTV, MTV, ISV, LSV, etc. ITV TVE VED EDL DLF NCBI Field Guide. . . Word Size = 3

Minimum Requirements for a Hit ATCGCCATGCTTAATTGGGCTT CATGCTTAATT exact word match one match • Nucleotide BLAST requires one exact match • Protein BLAST requires two neighboring matches within 40 aa GTQITVEDLFYNI YYN neighborhood words SEI two matches NCBI Field Guide

Scoring Systems - Nucleotides Identity matrix A G C T A +1 – 3 – 3 G – 3 +1 – 3 CAGGTAGCAAGCTTGCATGTCA || |||||| CACGTAGCAAGCTTG-GTGTCA C – 3 +1 – 3 T -3 -3 -3 +1 raw score = 19 -9 = 10 NCBI Field Guide

Scoring Systems - Proteins Position Independent Matrices PAM Matrices (Percent Accepted Mutation) • • Derived from observation; small dataset of alignments Implicit model of evolution All calculated from PAM 1 PAM 250 widely used BLOSUM Matrices (BLOck SUbstitution Matrices) • Derived from observation; large dataset of highly conserved blocks • Each matrix derived separately from blocks with a defined percent identity cutoff • BLOSUM 62 - default matrix for BLAST Position Specific Score Matrices (PSSMs) PSI- and RPS-BLAST NCBI Field Guide

BLOSUM 62 NCBI Field Guide A 4 R -1 5 N -2 0 6 D -2 -2 1 6 Common amino acids have low weights C 0 -3 -3 -3 9 Q -1 1 0 0 -3 5 E -1 0 0 2 -4 2 5 G 0 -2 0 -1 -3 -2 -2 6 H -2 0 1 -1 -3 0 0 -2 8 I -1 -3 -3 -3 -1 -3 -3 -4 -3 4 L -1 -2 -3 -4 -3 2 4 Rare amino acids have high weights K -1 2 0 -1 -3 1 1 -2 -1 -3 -2 5 M -1 -1 -2 -3 -1 0 -2 -3 -2 1 2 -1 5 F -2 -3 -3 -3 -1 0 0 -3 0 6 P -1 -2 -2 -1 -3 -1 -1 -2 -2 -3 -3 -1 -2 -4 7 S 1 -1 1 0 -1 0 0 0 -1 -2 -2 0 -1 -2 -1 4 T 0 -1 -1 -2 -2 -1 -1 -2 -1 1 5 Negative for less likely substitutions W -3 -3 -4 -4 -2 -2 -3 -1 1 -4 -3 -2 11 Y -2 -2 -2 -3 -2 -1 -2 -3 2 -1 -1 -2 -1 3 -3 -2 -2 2 7 V 0 -3 -3 -3 -1 -2 -2 -3 -3 3 1 -2 1 -1 -2 -2 0 -3 -1 4 X 0 -1 -1 -1 for -2 -1 -1 -1 -2 0 0 -2 -1 -1 -1 Positive more likely substitutions A R N D C Q E G H I L K M F P S T W Y V X

Scores Simply add the scores for each pair of aligned residues V V BLOSUM 62 +4 PAM 30 +7 D S – C Y E T L C F +2 +1 -12 +9 +3 +2 0 -10 +2 7 11 Different matrices produce different scores! NCBI Field Guide

Local Alignment Statistics High scores of local alignments between two random sequences follow the Extreme Value Distribution Expect Value E = number of database hits you expect to find by chance Alignments size of database your score expected number of random hits At low E values E approximates a P value Score NCBI Field Guide

BLAST Databases for Proteins nr (non-redundant protein sequences) – Gen. Bank CDS translations – NP_ Ref. Seqs – PIR, Swiss-Prot, PRF – PDB (sequences from structures) swissprot NCBI Field Guide pat - patents pdb – sequences with 3 D structures month – sequences updated within 30 days

Assessment of BLAST output • What is the level of identity and similarity of the best hits? – More identity - more likely the proteins may have similar functions. • Does the area of similarity occur over the entire protein? Or just part of the protein? (fig. 2. 19) – Often you will find hits to only part of your protein. A GTP-binding domain for example. • Have any of the best hits been characterized experimentally? – With so many microbial genomes sequenced chances are you will have to search extensively to find a hit that has been characterized experimentally. NCBI Field Guide

BLAST Formatting Page NCBI Field Guide

BLAST Output: Graphic Overview SH 3 PX NCBI Field Guide

BLAST Output: Descriptions 4 X 10 -68 links to entrez default e value cutoff = 10

Tax. BLAST: Taxonomy Reports

BLAST Output: Alignments >gi|12643956|sp|Q 9 Y 5 X 1|SNX 9_HUMAN Sorting nexin 9 (SH 3 and PX domaincontaining protein 1) (SDP 1 protein) Length = 595 Score = 255 bits (652), Expect = 4 e-68 Identities = 140/322 (43%), Positives = 185/322 (56%), Gaps = 7/322 (2%) Query: 221 SSATVSRNLNRFSTFVKSGGEAFVLGEASGFVKDGDKLCVVLGPYGPEWQENPYPFQCTI 280 SS+++ LN+F F K G E ++L A K +K+ +++G YGP W F C + Sbjct: 197 SSSSMKIPLNKFPGFAKPGTEQYLL--AKQLAKPKEKIPIIVGDYGPMWVYPTSTFDCVV 254 Query: 281 DDPTKQTKFKGMKSYISYKLVPTHTQVPVHRRYKHFDWLYARLAEKF-PVISVPHLPEKQ 339 DP K +K G+KSYI Y+L PT+T V+ RYKHFDWLY RL KF I +P LP+KQ Sbjct: 255 ADPRKGSKMYGLKSYIEYQLTPTNTNRSVNHRYKHFDWLYERLLVKFGSAIPIPSLPDKQ 314 Query: 340 ATGRFEEDFISKRRKGLIWWMNHMASHPVLAQCDVFQHFLTCPSSTDEKAWKQGKRKAEK 399 TGRFEE+FI R + L WM M HPV+++ +VFQ FL + DEK WK GKRKAE+ Sbjct: 315 VTGRFEEEFIKMRMERLQAWMTRMCRHPVISESEVFQQFL---NFRDEKEWKTGKRKAER 371 NCBI Field Guide

Blink – Protein BLAST Alignments • Lists only 200 hits • List is nonredundant NCBI Field Guide

Nucleotide vs. Protein BLAST Comparing ADSS from H. sapiens and A. thaliana aaccgggtgacggtggtgctcggtgcgcagtggggcgacgaaggc Human: N R V T V V L G A Q W G D E G + + V L G Q W G D E G A. th. : S Q V S G V L G C Q W G D E G agtcaagtatctggtgtactcggttgccaatggggagatgaaggt BLASTp finds three matching words BLASTn finds no match, because there are no 7 bp words Protein searches are generally more sensitive than nucleotide searches. NCBI Field Guide

N ucleotide Translated BLAST P rotein Particularly useful for nucleotide sequences without protein annotations, such as ESTs or genomic DNA tblastn P N PPP tblastx PPP P N N PPP PPP N Database PPP blastx Query PPP Program

Linking Protein Sequence, Structure, and Function Protein Domains Protein sequences CDD: Conserved functional domains in proteins represented by a PSSM PSI-BLAST, RPS-BLAST, CDART 3 D Domains NCBI Field Guide

Position Specific Substitution Rates Weakly conserved serine Active site serine

Position Specific Score Matrix (PSSM) 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 D G V I S S C N G D S G G P L N C Q A A 0 -2 -1 -3 -2 4 -4 -2 -2 -5 -2 -3 -3 -2 -4 -1 0 0 -1 R N D C Q E G H I L K M F -2 0 2 -4 2 4 -4 -3 -5 -4 0 -2 -6 -1 0 -2 -4 -3 -3 6 -4 -5 -5 0 -2 -3 1 -3 -3 -5 -1 -2 6 -1 -4 -5 1 -5 -6 3 -3 -4 -6 0 -1 -4 -1 2 -4 6 -2 -5 -5 0 8 -5 -3 -2 -1 -4 -7 -6 -4 -6 -7 -4 -4 -1 -4 -2 -3 -3 -5 -4 -4 -5 -7 -6 -7 12 -7 -7 -5 -6 -5 -5 -7 -5 0 Serine scored 0 2 -1 -6 7 0 is-2 0 -6 differently -4 2 0 -2 -3 -3 -4 -4 -5 two 7 -4 positions -7 -7 -5 -4 -4 in-4 these -5 -2 9 -7 -4 -1 -5 -5 -7 -7 -4 -2 -4 -4 -3 -3 -3 -4 -6 -6 -3 -5 -6 -6 -4 -5 -6 8 -6 -8 -7 -5 -6 -7 -6 -4 -5 -6 8 -6 -7 -7 -5 -6 -7 Active -6 -6 -5 site -6 nucleophile -5 -5 -6 -6 -6 -7 -4 -6 -7 -7 -5 -5 -6 -7 0 -1 6 -6 1 0 -6 -4 -4 -6 -6 -1 3 0 -5 4 -3 -4 -5 -5 10 -2 -5 -5 1 -1 -1 -5 0 -1 1 4 2 -5 2 0 0 0 -4 -2 1 0 0 -1 1 3 -4 -1 1 4 -3 -1 -2 -2 P 1 -2 -4 -5 -5 -1 -7 -5 -6 -5 -4 -6 -6 9 -6 -6 -4 0 -3 S 0 -2 0 -3 1 4 -4 -1 -3 -4 7 -4 -2 -4 -6 -2 -1 -1 0 T -1 -1 -2 0 -3 3 -4 -3 -5 -4 -2 -5 -4 -4 -5 -1 0 -1 -2 W -6 0 -6 -1 -7 -6 -5 -3 -6 -8 -6 -6 -6 -7 -5 -6 -5 -3 -2 Y -4 -6 -4 -4 -5 -5 0 -4 -6 -7 -5 -7 -7 -7 -4 -1 0 -3 -2 V -1 -5 -2 0 -6 -3 -4 -3 -6 -7 -5 -7 -7 -6 0 -4 -3

PSIBLAST NCBI Field Guide Create your own PSSM: Confirming relationships of purine nucleotide metabolism proteins query PSSM BLOSUM 62 Alignment

PSI BLAST >gi|113340|sp|P 03958|ADA_MOUSE ADENOSINE DEAMINASE (ADENOSINE AMINOH MAQTPAFNKPKVELHVHLDGAIKPETILYFGKKRGIALPADTVEELRNIIGMDKPLSLPGFLAKFDYY VIAGCREAIKRIAYEFVEMKAKEGVVYVEVRYSPHLLANSKVDPMPWNQTEGDVTPDDVVDLVNQGLQ EQAFGIKVRSILCCMRHQPSWSLEVLELCKKYNQKTVVAMDLAGDETIEGSSLFPGHVEAYEGAVKNG RTVHAGEVGSPEVVREAVDILKTERVGHGYHTIEDEALYNRLLKENMHFEVCPWSSYLTGAWDPKTTH VRFKNDKANYSLNTDDPLIFKSTLDTDYQMTKKDMGFTEEEFKRLNINAAKSSFLPEEEKKELLERLY e value cutoff for PSSM NCBI Field Guide

PSI Results: Initial BLAST Run NCBI Field Guide

First PSSM Search Other purine nucleotide metabolizing enzymes not found by ordinary BLAST NCBI Field Guide

Third PSSM Search: Convergence Just below threshold, another nucleotide metabolism enzyme NCBI Field Guide

Domains Entrez Domains (CDD) 16, 482 records A Database of Position Specific Score Matrices SMART 4% CDD 2% • EMBL • HMM based models originally concentrating on eukaryotic signaling domains, now expanding • NCBI • Eukaryotic COGs NCBI Curated Alignments LOAD 0. 3% • NCBI • Library of Ancient Domains KOG 29% • NCBI • BLAST based alignments derived from complete proteomes of unicelluar organisms Pfam 35% COG 30% • Sanger Center • Pfam-A seeds: HMM based models representing a wide variety of functional domains derived from SWISS-PROT NCBI Field Guide