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[Music]

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[Applause]

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absolutely fantastic system and it

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really lets us get some clues about

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evolution before Luca but I'd just like

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to let you know it's not the only game

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in town there are there RNAs that we can

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look at that were present in Luca not

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very many but one of them is is a

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nucleus and for those of you who know a

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lot about RNA you priority guess that

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I'm talking about ribonuclease P

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ribonuclease P is a nearly universal

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ribozyme as say there are a few

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organisms scattered here and there

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around the tree of life that have

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dispensed with the RNA component of RNA

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SP but by and large pretty much

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everything uses an rnase p RNA to

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process transfer RNAs so the main thing

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RNA HP does is it takes a pre T RNA cuts

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off sequence from the five prime end and

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gives you a maturity RNA they can go on

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and do its job with the ribosome and

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make proteins okay alright so that's

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what it does as I said it is present in

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all three domains of life here are

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examples from bacteria archaea and

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eukaryotes and as you can see they all

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have they have different secondary

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structures but there are certain

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elements that are shared between all

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three of them and so they have certain

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sequence elements in common they have

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certain structural elements in common

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and they're they're widely dispersed

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across all three domains so it's quite

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clear that this was present in Luca and

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in fact one other interest thinking

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about RNA SP is that it's a ribozyme

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which there are you know not that many

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of and it's it's true in biology RNA SP

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has always has at least one protein

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component but it's not necessary for

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catalysis so this is a ribozyme and

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because it doesn't mean it's protein and

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it's universally conserved and it was in

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Luke I like to point out that I wouldn't

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say that RNA SP is older than ribosome

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but it is something that could be older

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than the ribosome and it's not possibly

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the only other system where we could

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have a to follow a continuous

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evolutionary track from before the

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ribosome so now okay so it's pretty cool

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we should learn more about it so they're

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a bunch of

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and last session about the accretion

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model so I'm going to tune in details

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but some model developed by my largely

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by my collaborators at Georgia Tech and

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it explains ribosome evolution and I

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just there are sort of three key

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features as far as I can tell from from

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you know talking to them and reading

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their papers and that is that one of the

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key features of this model is that

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structural RNA is if you look at an RNA

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structure you can see a record of where

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elements were inserted into it that if

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you look at a three-dimensional

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structure you can also get information

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about the order in which elements were

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added and that these RNAs generally grow

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from a common core and so that's the

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model with the ribosome so why not apply

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to RNA SP the this model relies on

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three-dimensional structures from

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different domains of life and now for

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RNA SP as of about a week ago we have

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bacterial eukaryote and this just came

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out our kale rnase p structure and so we

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have a high-resolution three-dimensional

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structural information for all three

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domains of life and so I just want to go

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through a couple of examples of some of

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the the features the accretion model as

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they show up in RNA speak so this is P

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18 and it's one of the one of the HeLa

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C's in RNA SP from from bacterial

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structure and what I'm showing is what

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we consider an insertion site that is a

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site where it looks like you could have

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put an element into the RNA structure

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without disrupting the rest of the

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structure and that is to say this this

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part that's shown in cyan we think was

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dropped into the part that's shown in

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blue and and you might think okay yeah

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well the backbone it just it comes

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together just because the backbone is

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coming close together doesn't mean that

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it used to be connected but if we

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overlay it with the RK lar an ASP

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structure then we see that you know you

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basically have the same structure - this

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p 18 and of course the other thing about

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that it there are other reasons we kind

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of know this element is probably a later

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edition it's not in eukaryotes or

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archaea and it's not even known all in

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all bacteria so

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but at any rate we can see some of those

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features in RNA SP like we see in the

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ribosome another thing that we've seen

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the ribosome a lot of are these a minor

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interactions they're also present in RNA

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SP and so what's going on there is that

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if you look you can see in blue we have

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a double-stranded RNA that

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double-stranded RNA can form on its own

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it doesn't need the part that's shown in

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cyan and in green okay the part that's

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shown in green these are a couple of

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Dennis teens that are looped out and

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they're interacting with the minor

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groove of the double-stranded RNA that

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structure will not form if the

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double-stranded RNA is not there and so

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the thinking is that the independent

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structure is older than the dependent

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structure okay so we have those two

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elements we can find insertion sites and

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we can you know establish chronology

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based on three-dimensional structure so

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then if we go on from there and we kind

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of build up the structure from a single

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core we can kind of have a model of how

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our NASPE vault and so what I'm showing

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here is RNAs P substrate

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it's a tRNA alright so basically this

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part right here kind of shown in gray is

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the piece that's going to get cut off

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there are a couple of metals at the

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active site where the cleavage happens

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and and we'll just kind of walk through

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the evolution of RNA SP so we start to

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have a you know different chunks get

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added on after the first four pieces are

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added we have basically a step we

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basically have everything you you you

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should need for catalysis and then as

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you continue on in the model with you

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know through time you add on additional

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elements that recognize larger and

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larger portions of the tRNA and also

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help to stabilize some of the structure

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and then we can go beyond Luca with this

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particular structure there are a couple

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of additional elements that are you know

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specific to this lineage okay

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so that's the fun movie here's a couple

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of the views how am I on time okay okay

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oh great please I'm okay so here's a

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couple of different views of the of the

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model and broken up in different pieces

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so what do we do with this now that we

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have this model well there a couple

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things we can do and one of the first

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things I wanted to do is sort of track

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how does the interface between RNA SP

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and its substrate change over time

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so we start here on the left yes

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very early on and so some of the first

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things that they are contacted over the

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cleavage site that's not surprising and

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then there's also part of the the tRNA

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that's one helical turn away from the

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cleavage site and then as we go forward

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in history we start to contact more of

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the leader sequence the part that we cut

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off and then even more of the acceptor

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stem then the T stem T loop and then the

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last thing that gets contacted is the D

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loop and what's kind of interesting

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there is that it's not really until you

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get to Luca that you start interacting

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with parts of the tRNA they're sort of

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unique to tRNA I mean really I mean so

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so obviously the acceptor stem is at the

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ends is unique it has a CCA tail and all

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that but it's essentially interacting

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with what you might call mini helix up

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until Luca potentially so that suggests

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that you know maybe the full tier and I

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didn't come till later okay so and just

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to I don't really have well this is my

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last slide and and I'll just say that

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we're kind of right in the thick of it

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and like figuring out this model and and

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what it all means and but one of the

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things that occurred to me is that by

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tracking the way this interacts with

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tRNA we might be able to synchronize the

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evolution of RNA SP with the evolution

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of the ribosome with the idea that you

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know some of the same when that the

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ribosome and RNA SP might be contacting

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parts of the tRNA at the same time and

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so like I said before RNA SP we don't

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contact the D loop until very late and

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and the ribosome doesn't start to really

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interact with the D loop either until

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fairly late so that something along

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there we've got a lot of work to do to

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really flesh out this model and make

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sure it's well synchronized with the

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ribosome I this is definitely one of

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those projects where you know I'm

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talking about it as I'm thinking about

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it and so we don't have a nicely tied up

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package for what it all means but I

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think this is a very important system it

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has a lot of potential I haven't heard a

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lot of talk about it at origin of life

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or astrobiology conferences in a while

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you know and so I think we need to

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revisit it especially now that we have

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all the structural information and with

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that I just like to thank my

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collaborators at Georgia Tech especially

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Anton who

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I think it was last night or night

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before we spent several hours looking at

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structures having a lot of fun and this

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work was was done I'm at NASA there at

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Georgia Tech and I think you for your

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attention and I hope we have a little

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time for questions is the protein

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ancient or does it what can you say

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about the protein well you can you can

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say that it's it's totally dispensable

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it looks pretty it's I mean it's really

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supposed to care it's got it's a it's

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mixed alpha-helix you some beta sheets I

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mean doesn't really match what some of

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you know the accretion models or the

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Williams Labs model on early proteins

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and it it seems to be a lot of what's

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involved is is recognizing the the Phi

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Prime leader

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more than it does help catalysis but

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it's primarily sort of a recognition

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thing and and I should point out that

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the other artis so that's the bacterial

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RTSP just has this one protein that's

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shown in brown here over the left but

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our kale and eukaryotic Arnie's peas

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have several more proteins eukaryotes

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have up to ten or so and and an half a

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dozen for archaea so and you can and

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what's kind of interesting is in some

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cases you can kind of see deletions that

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happen in eukaryotes archaea where you

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kind of have a protein that seems to be

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coming and and and compensating for or

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you know taking the place of an RNA

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element anyway cool stuff but I was

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taken when he showed the three

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ribonuclease Peas two of them one from

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Ithaca

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called a caucus you know CIN and the

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other are thermophilic are is there any

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implication from what you're doing that

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perhaps the first ribonuclease P was

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high-temperature and that whether or not

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that might actually fit with some of the

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models that indicate that Luca was also

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a high-temperature growing well I'll

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just say that the examples I showed

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where the example were the examples for

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which we have three-dimensional

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structures and so it really just speaks

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the fact that the thermo file

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are easier to get structural information

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on some of the the RNA is associated

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thermophiles tend to be yeah but you

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don't have a model that indicates what

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the first ribonuclease p would have

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looked like well we do I mean as the the

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movie we kind of march through and we

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haven't done I mean what what's nice

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about the system is its fairly small and

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we can go and characterize the activity

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of some of these contracts so some of

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those all those different steps we plan

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to make in the lab and they won't be

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that difficult to make it's not a very

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large RNA and it we know that a lot of

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truncated forms of RNA SP are cadelec

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the active you can cut off tremendous

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amounts of RNA SP and still have

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activity so we're very optimistic that

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as we build constructs that correspond

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to the different steps in the secretion

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model that we'll be able to test them

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and characterize you know what

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temperature do they work best at what pH

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what ions how they respond to iron you

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know things like that I was really taken

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by the fact that do you have

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ribonuclease be functional above 90

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degrees and very interested in how that