TRNAs & Phamerator

What are tRNA's for?

Protein synthesis!

- They act as an adaptor molecule in translation

- The genetic info in mRNAs get decoded into amino acids

T/F: tRNAs are highly conserved

True

What is the typical size for tRNAs?

70-90 base pairs

What shapes are conserved for tRNAs?

secondary clover shape and tertiary L-shape

Do tRNAs have limited or unliimited sequence similarity?

limited

What's the deal with the 5' end of tRNA?

7bp segment known as the "acceptor stem"

What is the general shape of a tRNA?

3 stem loops with variable length

What are the conserved bases in tRNA

R (for purines), y (for pyrimidines), G,A,C

How are potential tRNAs scored?

The tRNA algorithms score potential tRNAs based on their adherence to the conserved bases and stem-loop lengths.

Why can't we use BLAST to identify tRNAs?

The variability in tRNA sequences is extensive enough that tools like BLAST, which rely on sequence similarity to identify features, cannot be used for sensitively identifying a genome’s tRNAs de novo

What type of tool do we need to identify tRNAs?

However, because the size and structure of tRNAs are well conserved, tools that interrogate a genome for sub-sequences capable of adopting a tRNA-like fold (following RNA base-pairing rules) are very successful in identifying tRNA genes.

What's the deal with Aragon (v1.1)

- run durinig auto-annotation by DNA Master

-Identifies most tRNAs

- fails to predict ends correctly

- creates placeholders for tRNAs so we don't need to do manual entry

What about Aragon (V1.2.38)

- Run during manual annotation

- Strong overlap with Aragon V1.1, but better at getting ends

- Useful for correctly triming tRNAs

Whats the deal with tRna scan-SE (v2.0.6)

- Run during manual annotation

- identifies most basic tRNAs and many non-canonical tRNAs

Which is preferred for tRNA coordinates?

Aragon

What are tRNAs unlikely?

- be in an ORF called by Glimmer and GeneMark with coding potential

- be on the opposite strand as other tRNAs

- be genetically distant from other tRNAs

Average number of tRNAs per genome?

0-1

What are we looking for when checking Aragon ends?

-cca sequnce at the 3' end must be present (for charge purposes)

- Aragon 1.2.38 correctly trims the ends of tRNAs!

What is the best data for tRNAs?

- tRNA scan-SE score > 35

- also found by Aragon

When to use caution for tRNAs?

- longer than 90 bp

- tRNAs in a protein encoding gene

- tRNA s in the middle and opposite of a tRNA cluster

what is unique about terminase (think placement)

it is usually first or close to first

which 4 proteins are significant for packaging (viral structural and assembly genes)

terminase, portal protein, capsid maturation protease, scaffolding protein

which protein can translate itself in two different frames

tail assembly chaperones

true or false: tail assembly chaperones are experimentally shown to have a frame shift in B3 phage but not C1s

false, it’s the opposite

which proteins are involved in endolysis

holin, lysin A, lysin B (note not all of them present in every phage)

what is an example of the proper syntax for synteny describing phamerator results

Holin (Lakes_7) is downstream of Lysin A and upstream of Lysin B just like in D32 and Naji.

what is required for synteny to be used

correct size, adjacent to other structural genes of known verifiable function, the only possible option for that function in that genome

true or false: you must meet all three requirements to use synteny

true

what is the first step in doing synteny

locally blast the phage to identify 2 closely related phages

what does holin do

help put a “hole in” the cell wall of the host

what proteins are applicable for temperate phages that go through the lysogenic cycle

integrase and recombinase

what proteins are involved in DNA replication

helicase, DNA polymerase, etc

in terms of endolysis proteins what do C1s have

lysin A, holin, and lysin B

in terms of endolysis proteins what do B3s have

lysin A and lysin B

in terms of endolysis proteins what do A6s have

lysin A and holin

in terms of endolysis proteins what do B1s have

lysin A and lysin B