Annotating a Gene Do I Have the Whole Gene?

1. Annotating a GeneDo I Have the Whole Gene? Planctomyces limnophilus imidazole glycerol phosphate synthase subunit hisF VKGVNFLNLQDAGDPVEVAARYERDGADELVFLDITASHEERGIIHDVVR RTSEVCFMPLTVGGGIRNLDDIRTLLQAGCDKVSINSAAVKDPEFVRAAA LKFGKQCIVVNIDPRRVTQPDGSVKWEVHINGGRKPTGLEAVSWAKEVER LGAGEIVLTSMDADGTKDGYDFEMTRAVADAVEIPVVASGGAGHPEHLRQ ILQEGHASAALAASIFHYGTHPIGETKEYLLKHGVPIRLNQPIP 17 AA’s missing Pl VKGVNFLNLQDAGDPVEVAARYERDGADELVFLDITASHEERG YP_902708.1| MLTKRIIPCLDVKGGRVVKGVQFLELRDAGDPVEIAEIYDRQGADELTFLDITASSDERD NP_954136.1| MLTKRIIPCLDVKGGRVVKGVQFLELRDAGDPVEIAELYDRQGADELTFLDITASSDERS ZP_01855067.1 MLAKRIIPCLDVHAGRVVKGVNFLNLQDAGDPVEVAARYEEQGADELVFLDITASHEERE NP_869248.1| MLSARVIPCLDVHGGRVVKGTNFVNLRDAGDPVEVARRYEAEGADELVFLDITASHEERA ZP_01090903.1| MLAHRVIPCLDVDRGRVVKGTNFVNLRDAGDPVQVASRYEQEGADELVFLDICASHEERA NP_623694.1| MLAKRIIPCLDVKDGRVVKGINFVNLKDAGDPVEIAERYNELGADELVFLDITASYEKRK YP_359936.1| MLTVRIIPCLDVNKGRVVKGVNFLNLVDAGDPVELAAFYDREGADEVVFLDITASFEGRQ

2. Operons as a Model for RegulationTranslational Nuances • Most basic definition of operon = >1 gene that shares the same promoter – this requires that the genes be close to one another and in the same orientation! • Having >1 ORF on a mRNA sets up another problem (potential point of regulation) = how to get ribosomes starting on each ORF solution #1 = separate rbs for each ORF lacZ ORF … 62 nt … lacY ORF … 64 nt … lacA ORF solution #2 = ORFs are so close together that same ribosome can finish one, then start another (often called translational coupling)

3. Finding the Right Start CodonBacteria Domain

4. Finding the Right Start CodonHow Does Planctomyces Do It?

5. Finding the Right Start CodonLeaderless mRNAs

6. TrpR + tryptophan Small ORF in front of trpE ORF = trpL E. coli trp OperonMultiple Levels of Regulation P

7. E. coli trp OperonAttenuation = Regulation of Transcriptional Elongation that Involves Translation RNA Pol stalls somewhat while making polyU tract, giving ribosome a chance to catch up

8. E. coli trp OperonAttenuation = Regulation of Transcriptional Elongation that Involves Translation RNA Pol stalls somewhat while making polyU tract, giving ribosome a chance to catch up

9. trp Operon in Bacillus & Related Gm+Variations on Attenuation

10. trp Operon in Bacillus & Related Gm+How Might Ammonifex Regulate its trp Operon in Ammonifex: ABFCDGE ABFCDGE

11. trp Operon in Bacillus & Related Gm+How Might Ammonifex Regulate its trp Operon Ammonifex has a TRAP homolog (but no AT homolog) Region upstream of 1st gene of trp operon (operon reads right to left; red indicates repeats found in TRAP binding sites; green is rbs) ....trpE ((((((((((((((((...))).))))))))))))) UUUUUGUAggacgaaagcggaggcaagcgaauccccggcccgaacuuuuuuaauucccggucgcgcuucgucgagugcgugaccgggaaguuugaa ((((......))))((((..((((... agagcgguccgagcucgaggggagcggagaugagcagagaagagcagagccagagucgauuuaguauacgucaaaauuucggucucaaacgggcaac .))))..)))).(((((..((((.((((((((.((........))...((.(((.....))).))...)))).....)))).))))...))))) When [tryptophan] is low, TRAP has no trp and does not bind to gag repeats, so the upstream region of the trp operon mRNA can form a strong antiterminator loop (in blue). When [tryptophan] are high, TRAP has trp bound to it and binds to gag repeats, preventing antiterminator loop and allowing terminator loop to form (in red) kicking RNA Polymerase off.

12. Comparative GenomicsSynteny Ortholog Neighborhood Viewer img.jgi.doe.gov

13. Comparative GenomicsSynteny

14. Comparative GenomicsSynteny

15. intragenome transfer

16. 10 kb 0 kb 20 kb Annotation Pipeline • Gene finding & operon prediction • Blast & global sequence alignments • Protein domain prediction • Protein localization prediction • Functional prediction • Functional call, linkage to experimental data, & testable hypotheses (community involvement)

17. Pathway-based AnnotationStudents Identify in the Holes One of the proteins, 3-phosphoserine aminotransferase, was not originally found in the Ammonifex degensii genome by its name or by homology to the typical bacterial SerC protein. However, I was able to find a paper in the research literature showing that some members of the Archaea (e.g., Methanococcus) use a different protein to do the same job (Helgadottir, et al., 2007, J Bacteriology 189:575-82). A. degensii and its related genera Moorella & Thermoanaerobacter all have the archaeal-type SerC. As another example, there is no clear SerB homolog in the Planctomyces limnophilus, indicative of a relative dearth of SerB annotation calls across all sequenced genomes

18. Pathway-based AnnotationStudents Identify the Holes These 2 functions are done by one bifunctional enzyme in E. coli, but not in Agrobacterium

19. Pathway-based AnnotationFilling in the Holes (Transposon Mutagenesis) Have a Tn hit in gene Atu3885: - annotated as myo-inositol monophosphatase family member - mutation leads to auxotrophy - now know it cannot make histidine - TIGR Fam hit to alternative histidinol-P phosphatase These 2 functions are done by one bifunctional enzyme in E. coli, but not in Agrobacterium

20. Pathway-based AnnotationStudents Identify the Holes Looking for 10 genes of glycolysis & 3 additional genes for gluconeogenesis Agrobacterium & Chromohalobacter genomes lack FBPase There are 6 different proteins that have FBPase activity There must be a 7th way (& maybe 8th way) as well

21. Pathway-based AnnotationFilling in the Holes (Functional Complementation) E. coli fbp-

22. Fbp complementing clone from Chromohalobacter salexigens

26. Pathway-based AnnotationPossible Gene Redundancy Looking for genes encoding F0 & F1 components of ATP Synthase in Azotobacter vinelandii Found 2 operons Operons have different gene orders & different evolutionary histories Why 2 different operons?

27. Bd/e1ACBag IACBagbe The two operons have different gene orders & different evolutionary histories