It’s a bird, It’s a plane … Nope it’s a tRNA!

DNA annotation is the final step of successfully capturing a phage and really gets into the knitty gritty. Through the use of PECAAN [1] , Phamarator [2] and DNA master [3] software I was able to get to the bottom of which genes coded for particular functions in Phage Bazzle [4]!

Bazzle EM Picture

Phage Bazzle (https://phagesdb.org/phages/Bazzle/)

After feeling like an annotation expert, I was blind sided by a bizarre large gap between genes, which can be seen when looking at the start sites of genes of Bazzle in PECAAN. When further investigating, DNA master revealed these to be tRNA’s. TRNA’s are transfer adaptor molecules that serve as the link between mRNA and the amino acid sequence during translation [5]. Within the sequence tRNA’s don’t appear as normal coding in the genome, therefore the software identifies that these are rogue. They are often easy to spot by a signature gene gap as which puzzled me since they appear in clusters, multiple tRNA’s in the same region. The can also be seen on Pham Maps of PECAAN represented as +’s. Bazzle hoarded 11 cheeky tRNA’s that all required special annotation.

PECAAN showing tRNA of Bazzle as +

TRNA annotation is making sure these molecules are the correct structure, containing the right bases on their acceptor stem loop. This loop must have CCA bases at the 3′ as well as 7bp in total. Errors after transcription can alter these bases so we’ve gotta be prepared to give them a trim (even if it’s a mullet, yikes).

Image result for tRNA loop
tRNA with acceptor loop containing CCA at 3′ end (http://www.bx.psu.edu/~ross/workmg/TranslationCh14.htm)

Thankfully gene annotation is ahead of the game and I simply had to run the scripts of tRNA through Aragorn [6] and tRNAScanSE [7] which develop two options for which they think the tRNA should be trimmed. The best one is which matches the conditions above, this is then confirmed in PECAAN and saved along with the rest of the annotation, BOOM done.

And just like that a year of phaging has finished! I will forever be grateful for the experience and confidence this course has given me, as well as the passion it has sparked for microbiology. The future is looking exciting!

Bibliography

  1. PECAAN annotation software https://seaphages.org/media/docs/PECAAN_User_Guide_Dec7_2016.pdf
  2. Phamerator annotation software https://bmcbioinformatics.biomedcentral.com/articles/10.1186/1471-2105-12-395
  3. DNA Master Software https://phagesdb.org/DNAMaster/
  4. Phage Bazzle https://phagesdb.org/phages/Bazzle/
  5. tRNA definition https://www.nature.com/scitable/definition/trna-transfer-rna-256/
  6. Aragorn Software https://www.ncbi.nlm.nih.gov/pmc/articles/PMC373265/
  7. tRNAScanSE Software https://www.ncbi.nlm.nih.gov/pmc/articles/PMC146525/

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