SEA-PHAGES | How close can one pack protein and tRNA's genes

Link to this post \| posted 18 Feb, 2016 16:58
cdshaffer	OK, general question. We have a new phage with lots of tRNA genes packed interdigitated with glimmer/Gene protein predictions. There are long spaces between the tRNA genes so there is certainly room for a protein gene but some of the glimmer and genemark predictions come pretty close to the aragorn hits. How much space should there be between these features? Do we need to leave room for a promoter anytime we switch from a tRNA gene to a protein gene or vice versa? Should I tell the students to leave at least 25-50 bp needed for the promoter? I will have students look at the protein vs. tRNA distribution in the host bacteria but wanted to check if there is some kind of general rule to maintain consistency across all SEA phages annotations. Edited 18 Feb, 2016 17:34

Link to this post | posted 18 Feb, 2016 16:58

OK, general question. We have a new phage with lots of tRNA genes packed interdigitated with glimmer/Gene protein predictions. There are long spaces between the tRNA genes so there is certainly room for a protein gene but some of the glimmer and genemark predictions come pretty close to the aragorn hits. How much space should there be between these features? Do we need to leave room for a promoter anytime we switch from a tRNA gene to a protein gene or vice versa?

Should I tell the students to leave at least 25-50 bp needed for the promoter? I will have students look at the protein vs. tRNA distribution in the host bacteria but wanted to check if there is some kind of general rule to maintain consistency across all SEA phages annotations.

Edited 18 Feb, 2016 17:34

Link to this post \| posted 24 Feb, 2016 15:03
welkin	Nope, the tRNA cassettes are weird, and no you don't have to have a promoter for each of them or for each protein encoding gene interspersed within the cassette. We tend to steer clear of a tRNA and a protein occupying the same space, but there are definitely genomes where they get pretty close (take a look at the Ms). The only promoter rule is still about switching directions– presumably, switching transcriptional directions would still require two promoters even for tRNAs.

Link to this post | posted 24 Feb, 2016 15:03

welkin

Nope, the tRNA cassettes are weird, and no you don't have to have a promoter for each of them or for each protein encoding gene interspersed within the cassette. We tend to steer clear of a tRNA and a protein occupying the same space, but there are definitely genomes where they get pretty close (take a look at the Ms). The only promoter rule is still about switching directions– presumably, switching transcriptional directions would still require two promoters even for tRNAs.

Link to this post \| posted 24 Feb, 2016 17:46
cdshaffer	OK thanks. Looking more closely we have now found at least several tRNA's where there are NO bases between the end of the tRNA and the first base of the auto-annotation. Splicing of the RNA to excise the tRNA would thus produce a leaderless message. I have been talking to our local faculty who works almost exclusively with Streptomyces (the phage host in this case) and apparently in some of these strains leaderless messages are not that uncommon, so I think we will go ahead and annotate both no matter how close as along as they don't overlap. I tend to agree with the general annotation policy that it is better to annotate a false positive than a false negative; so when things are truly ambiguous over-predict don't under-predict. He also raised the possibility that this is an intentional organization that could be used as some sort of regulation of expression, by splicing out an overlapping tRNA you effectively kill the protein encoded gene, resulting in less protein production than if the tRNA wasn't there. I don't know if this has ever been described before but would be very cool if true.

Link to this post | posted 24 Feb, 2016 17:46

cdshaffer

OK thanks.
Looking more closely we have now found at least several tRNA's where there are NO bases between the end of the tRNA and the first base of the auto-annotation. Splicing of the RNA to excise the tRNA would thus produce a leaderless message. I have been talking to our local faculty who works almost exclusively with Streptomyces (the phage host in this case) and apparently in some of these strains leaderless messages are not that uncommon, so I think we will go ahead and annotate both no matter how close as along as they don't overlap. I tend to agree with the general annotation policy that it is better to annotate a false positive than a false negative; so when things are truly ambiguous over-predict don't under-predict.

He also raised the possibility that this is an intentional organization that could be used as some sort of regulation of expression, by splicing out an overlapping tRNA you effectively kill the protein encoded gene, resulting in less protein production than if the tRNA wasn't there. I don't know if this has ever been described before but would be very cool if true.

Link to this post \| posted 02 Feb, 2017 19:41
GregFrederick@letu.edu	To follow up with this discussion, we have a tRNA gene identified by Aragorn on the +3 frame and a clear ORF on the -2 frame. In other words, they are in opposite orientation on the genome. However, the both look very "REAL". The Frames output is below. https://drive.google.com/open?id=0B_7KYneUM8kdVWRKWjJrQlFBajQ We are planning to call both unless we hear otherwise here. Let me know your thoughts. Thank you. GF Edited 16 Feb, 2017 15:20

Link to this post | posted 02 Feb, 2017 19:41

GregFrederick@letu.edu

To follow up with this discussion, we have a tRNA gene identified by Aragorn on the +3 frame and a clear ORF on the -2 frame. In other words, they are in opposite orientation on the genome.

However, the both look very "REAL". The Frames output is below.

https://drive.google.com/open?id=0B_7KYneUM8kdVWRKWjJrQlFBajQ

We are planning to call both unless we hear otherwise here.

Let me know your thoughts. Thank you. GF

Edited 16 Feb, 2017 15:20

Link to this post \| posted 16 Feb, 2017 15:17
GregFrederick@letu.edu	GregFrederick@letu.edu To follow up with this discussion, we have a tRNA gene identified by Aragorn on the +3 frame and a clear ORF on the -2 frame. In other words, they are in opposite orientation on the genome. However, the both look very "REAL". The Frames output is below. https://drive.google.com/open?id=0B_7KYneUM8kdVWRKWjJrQlFBajQ We are planning to call both unless we hear otherwise here. Let me know your thoughts. Thank you. GF To clarify: The ORF and the tRNA gene in question are overlapping, and on the opposite strands. See the picture of the region in the image at the link. I did not see a reply to this question. We are about finished with the annotation portion of the course. However, we are uncertain how to deal with this region. Thoughts? Edited 16 Feb, 2017 15:22

Link to this post | posted 16 Feb, 2017 15:17

GregFrederick@letu.edu

GregFrederick@letu.edu
To follow up with this discussion, we have a tRNA gene identified by Aragorn on the +3 frame and a clear ORF on the -2 frame. In other words, they are in opposite orientation on the genome.

However, the both look very "REAL". The Frames output is below.

https://drive.google.com/open?id=0B_7KYneUM8kdVWRKWjJrQlFBajQ

We are planning to call both unless we hear otherwise here.

Let me know your thoughts. Thank you. GF

To clarify: The ORF and the tRNA gene in question are overlapping, and on the opposite strands. See the picture of the region in the image at the link.

I did not see a reply to this question. We are about finished with the annotation portion of the course. However, we are uncertain how to deal with this region.

Thoughts?

Edited 16 Feb, 2017 15:22

Link to this post \| posted 16 Feb, 2017 15:25
GregFrederick@letu.edu	Welkin Pope Nope, the tRNA cassettes are weird, and no you don't have to have a promoter for each of them or for each protein encoding gene interspersed within the cassette. We tend to steer clear of a tRNA and a protein occupying the same space, but there are definitely genomes where they get pretty close (take a look at the Ms). The only promoter rule is still about switching directions– presumably, switching transcriptional directions would still require two promoters even for tRNAs. Welkin: Based on this statement above ("We tend to steer clear of a tRNA and a protein occupying the same space, but there are definitely genomes where they get pretty close" ) ….It seems we should delete the tRNA gene. Could you take a look at the image linked in my post above and let me know your thoughts on how to deal with this specific situation? Thank you. G Edited 16 Feb, 2017 15:27

Link to this post | posted 16 Feb, 2017 15:25

GregFrederick@letu.edu

Welkin Pope
Nope, the tRNA cassettes are weird, and no you don't have to have a promoter for each of them or for each protein encoding gene interspersed within the cassette. We tend to steer clear of a tRNA and a protein occupying the same space, but there are definitely genomes where they get pretty close (take a look at the Ms). The only promoter rule is still about switching directions– presumably, switching transcriptional directions would still require two promoters even for tRNAs.

Welkin: Based on this statement above ("We tend to steer clear of a tRNA and a protein occupying the same space, but there are definitely genomes where they get pretty close" )

….It seems we should delete the tRNA gene.

Could you take a look at the image linked in my post above and let me know your thoughts on how to deal with this specific situation? Thank you. G

Edited 16 Feb, 2017 15:27

Link to this post \| posted 09 Mar, 2017 15:11
megan6	I'm a student in the bioinformatics class and we would really like to finish this genome, as well as my lab partner and I would like to do a presentation concerning what appears to be this anomaly. If anyone does have information, please report this to us!

Link to this post \| posted 13 Mar, 2017 20:32
welkin	Yes, Greg, from this picture I think you need to delete the tRNA. I can't tell what cluster this is from the picture, but I am guessing there are many other cluster relatives that likely have the same prediction, and the tRNA was deleted. With no bench evidence one way or the other, I am inclined to go for the protein encoding gene that fits into the operon nicely, and to stay consistent with other cluster relatives. Best, Welkin

Link to this post | posted 13 Mar, 2017 20:32

welkin

Yes, Greg, from this picture I think you need to delete the tRNA. I can't tell what cluster this is from the picture, but I am guessing there are many other cluster relatives that likely have the same prediction, and the tRNA was deleted. With no bench evidence one way or the other, I am inclined to go for the protein encoding gene that fits into the operon nicely, and to stay consistent with other cluster relatives.

Best,
Welkin

Link to this post \| posted 15 Mar, 2017 22:36
GregFrederick@letu.edu	Thanks Welkin. We will delete the tRNA. It really seems real. So is there any empirical evidence that there are not tRNAs overlapping with structural genes? Since these are in the opposite orientation, if seems feasible that both could be real.

Link to this post \| posted 20 Mar, 2017 17:54
welkin	Our RNA seq and mass spec data from various clusters supports the guiding principle that the vast majority phage genes and genomes are only transcribed in one direction for any given region. So while I don't have data for this particular instance (and there are always exceptions to every principle), the data that we do have says that you need to choose one or the other when it comes to two genes occupying the same piece of DNA in different frames.

Link to this post | posted 20 Mar, 2017 17:54

welkin

Our RNA seq and mass spec data from various clusters supports the guiding principle that the vast majority phage genes and genomes are only transcribed in one direction for any given region. So while I don't have data for this particular instance (and there are always exceptions to every principle), the data that we do have says that you need to choose one or the other when it comes to two genes occupying the same piece of DNA in different frames.

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How close can one pack protein and tRNA's genes