Hands-on: Reviewing BLAST What is the appropriate annotation for UniProt entry U4LTX6? U4LTX6 UniProt entry Use “Advanced” option, select UniProtKB and 1000 hits My notes: Lecturer notes: The first thing one notices in the BLAST results is that there seems to be many entries labeled dihydrofolate reductase (DHFR). However, very few of these are from the reviewed section of UniProtKB (UniProtKB/SwissProt). It is clear that the top-hit reviewed entry, DYR_SCHPO, is quite a bit longer than the query. The alignment indicates that the query is similar only to the N-terminal portion of DYR_SCHPO. If one looks at the Pfam graphical view for DYR_SCHPO, it is obvious that the N-terminal portion of DYR_SCHPO corresponds to serine hydrolase (PF03959) rather than DHFR (PF00186). In fact, PF03959 is the same domain observed within the query entry itself. Therefore, one can conclude that the DYR_SCHPO protein is a fusion protein, and the annotation from this fusion created the mass of confusion seen in the sequence databases. Hint: DYR_SCHPO

Hands-on: Finding Literature Starting at the UniProt website, try to find a PUBLICATION describing the FUNCTION of the following: Q1R9Q7 My notes: Lecturer notes: There are numerous ways to find literature associated with a sequence, even if the entry has nothing but “Large Scale” papers cited within (these can be recognized in UniProtKB by looking at the information in the “Cited for” line for each reference; note that some papers might not be labeled as such, but could nonetheless still be non-specific to the entry in question with respect to functional information). For this exercise, try to find a literature citation that addresses the function for the protein given in the entry. The links are to UniProtKB, but consider other databases as well (such as iProClass). It is not necessary for all of you to search all. Just choose one. In five minutes we’ll get your feedback. Search PubMed with Gene Other things to try: Search PubMed with author PDB link Use synonyms for gene/protein Look at organism-specific database

Homework Answer Should the unreviewed (TrEMBL) UniProt entry Q23890--currently named ORF425--be renamed to mitochondrial ribosomal protein L11 (yes or no)? My notes: Lecturer notes: The paper in question did indeed provide the sequence of ORF425, but nothing more. Various methods can be used to determine that the paper refers to a completely different sequence with respect to functional annotation. Answer: No. Getting there (a subset of ways): Hint: Run BLAST against UniProtKB (default parameters). Examine annotations. Nothing about mrpL11. Proof: Go to UniProt website and search for entry. Click Publications, then click on PubMed link. Under Related Information -> Protein (RefSeq). Click on the one labeled ribosomal protein L11. Grab accession number and ID map to UniProtKB. Will find it maps to a different entry (Q23884), which is the actual mrpL11 in UniProtKB (same result if use BLAST instead).

Hands-on: Reasons for Rules Action slide! Hands-on: Reasons for Rules 1 - Three highly similar activities are represented in this group: thymidine phosphorylase (TP, gene deoA, EC 2.4.2.4), pyrimidine-nucleoside phosphorylase (PyNP, gene pdp, EC 2.4.2.2), and AMP phosphorylase (AMPpase, EC 2.4.2.-). 2 - PyNP is typically the only pyrimidine nucleoside phosphorylase encoded by Gram positive bacteria, while eukaryotes and proteobacteria encode two: TP, and the unrelated uridine phosphorylase. AMPpase is found in archaea. 3 - Sequence comparison between the active site residues for TP and PyNP reveals only one difference, which has been proposed to partially mediate substrate specificity. In TP, position 111 is a methionine, while the analogous position in PyNP is lysine. It should be noted that the archaeal members of this family differ in a number of respects from either of these characterized activities. The simplest rule: If Bac/Firmicute, name it “Pyrimidine-nucleoside phosphorylase”, otherwise name it “Thymidine phosphorylase” if length is under 500aa and “Putative thymidine phosphorylase” if length is over 500aa. How to determine it: 1) Read the description 2) Sort by Protein Name then by Taxon Group 3) Sort by Length then by Taxon Group