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[Music]

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[Applause]

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today I'm going to talk to you guys

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about some work I did looking at the

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roll iron as a cofactor for the ribosome

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in vivo and what that what that means

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for the origin of life okay so the

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ribosome is very ancient and rare

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conserved so much so in fact that it

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likely predates the last Universal

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common ancestor and even cellular life

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itself so we think about the origin of

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the ribosome that really sort of puts it

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at around four billion years ago here at

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the beginning of the Archaean and the

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ribosome evolved an environment we're on

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an earth where the atmosphere was devoid

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of oxygen and because of that you had

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abundant stable soluble ferrous iron in

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the environments on the early Earth and

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not only did the ribosome evolve in this

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environment it was encapsulated by cells

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and used in early life forms for at

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least an additional 1 to 2 billion years

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before oxygen started to rise during

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what's known as the great oxidation

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event and precipitate out this iron so

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thinking about why the iron content or

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the ferrous iron content of the

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environment would be important for the

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origin of the ribosome is is clear when

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we consider that developmental cations

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are absolutely essential to the

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structure and function of the ribosome

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and actually the whole translation

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system and historically magnesium has

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really been implicated as the sole

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divalent cation in this process however

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we've recently shown that iron can

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actually near totally replace magnesium

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in in the entire translation system and

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mediate the translation of functional

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protein so in these experiments you can

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see that just on in the lower line here

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with with iron we very much took the

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translation system in and transported it

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to the environment of its ancestors and

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were able to show that it retains

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function in such an environment in vitro

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of course and after showing that in

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vitro we we then wanted to go and see if

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there's anything going on in in vivo

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with with iron in the ribosome and more

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specifically are there environmental

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conditions or growth conditions under

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which a bacterial cell we're under you

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know sort of canonical normal laboratory

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conditions might have most

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magnesium associated with its ribosomes

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can we impart conditions under which

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there are there's it can sort of swap

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out either some or all that magnesium in

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its ribosomes and so to do that we grew

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coli cells under four different growth

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conditions we grew them aerobic Li

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either in the presence or absence of

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added ferrous ion or anaerobically again

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in the presence or absence of add added

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ferrous iron and in thinking was it was

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really this culture that had both no

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oxygen because of that allowing for this

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ferrous iron high amounts of ferrous

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iron to remain stable and solution what

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we're going to call our pre great

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oxidation event culture pre Joey culture

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ribosomes from those cells would we

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thought would have the most iron

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associated with them so we grew we grew

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the cells under those growth conditions

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and then in Aleksic chamber to keep

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everything nice to know to free I puree

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lysis cells purified out the ribosomes

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using chromatography based method it

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concentrated the ribosomes using ultra

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centrifugation and then was able to do

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some downstream analysis and so I'll

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first look at the iron content analysis

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we we measured using a total x-ray

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fluorescence spectroscopy and so from

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that method we you just get an iron

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concentration in in your sample you know

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the ribosomal concentration of your

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sample and from that we just calculate

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an iron per ribosome ratio and that's

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what we're showing on the y-axis here

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and on the x-axis we have our four

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growth conditions so our two aerobic

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conditions and then our two anaerobic

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conditions and again our pre goe

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anaerobic 1 millimolar ferrous chloride

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condition and what we can see is that

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indeed in the pre juhi conditions we

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have significantly more iron associated

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with those ribosomes compared to the

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other four growth conditions so about

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about 10 versus about one for the other

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three growth conditions when we looked

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at the ribosomal RNAs of these of these

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guys so irregardless of growth condition

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the 23's of the 16s ribosomal RNA czar

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intact and therefore these ribosomes are

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also functional in in in in vitro

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translation assays regardless of the

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iron content or growth condition so then

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when we thought sort of more about the

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ribosomes and the way we were purifying

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them we realized something really

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interesting

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it if if any of you purified ribosomes

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before familiar with this field really

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all canonical ribozyme purification

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you're just bathing these things in

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millimolar amounts of magnesium during

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the purification process and and all the

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buffers and so what we're thinking

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what's happened was happening is that

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you know there may be some iron pool

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that's associated in vivo that during

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the purification process is getting

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spontaneously exchanged for with

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magnesium from the buffers and what we

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might actually be seeing is magnesium's

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that are preferentially associated at

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least in vitro with magnesium and you

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know thinking about if that's happening

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well we should be able to do the

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opposite we should be able to purify

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this stuff and oxic aliy in buffers

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containing normal amounts of ferrous

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iron and swap in irons and ending up

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with with ribosomes that are

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preferentially enriched for iron and

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that's what we did and so that is the

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duct these dots on the top of the graph

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here and we can see is that you know

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regardless regardless of growth

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condition these ribosomes are all able

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to soak up about 500 to 600 irons per

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ribosome during the purification process

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from that buffer and so that was really

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interesting and it tells us that there's

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a huge capacity at least in vitro for

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the ribosome to to associate extensively

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with ferrous iron molecules it also

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tells us something kind of really cool

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about about these data from the ions

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that are left over left over if you will

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after your purification with magnesium

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these are the these are ions basically

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we couldn't wash out and sort of suggest

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at their sort of binding nature with the

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ribosome being either tightly very

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tightly associated with the ribosomal

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RNA and or tightly that are deeply

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buried within the ribosomal structure

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and really thinking about the behavior

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of these these ions and the fact that

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we're seeing around ten sort of also

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possibly hints at the the exact

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mechanism of association with the

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ribosome so Lauren Williams and his

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group have previously identified these

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these interactions called Dyne nuclear

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micro clusters these are interactions

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that happen in the ribosome RNA where

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you have two divalent cations that are

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bridged by a common phosphate oxygen on

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the backbone of the ribosomal RNA and so

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these divalent cations are very tightly

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associated and highly coordinated with

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with the fought with phosphate

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they're also most of them are very

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deeply buried within the ribosomal

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structure actually many many of them

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being very important for framing the

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prep the pebble transfer a center and

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then when you go to you know see how

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many of these things are in the ribosome

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we find that there are four on the large

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subunit and one in the small subunit and

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there should be two divalent cations in

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each of these dining clear micro

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clusters and so we would expect around

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ten divalent cations to be participating

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in these sorts of interactions per

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ribosome and in fact we've actually

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recently got some support for this this

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hypothesis a paper came out showing that

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these elisa in vitro the RNA responsible

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for making these micro clusters looks

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like it can fold in the presence of iron

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in vitro and so right now we're sort of

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thinking about the the next experiment

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or good experiments to do to to to

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really see you know a where this iron is

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and what its binding to in the ribosome

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okay so then we went back and we looked

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at the the ribosomal RNA of both the

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ribosomes that were purified in

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magnesium and compared that to the

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ribosomal RNA of the ribosomes that were

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purified in iron and so again this is

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the gel i already showed you so these

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these are the intact ribosomal RNAs of

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the of the ribosomes that were purified

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in buffers containing magnesium and

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these are the ribosomal RNAs of the

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buffers purified in the presence of iron

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and what you can see here is that these

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RNAs really seem to be heavily heavily

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degraded right after getting them off

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the purification process already have

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heavily degraded and you know we really

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took a lot of great care to not do not

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introduce too much oxygen during this

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purification process so it's very

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unlikely that it's significant amount of

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this cleavage is due to any any sort of

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iron mediated oxidative damage and so

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thinking about other mechanisms of

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cleavage

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one of the ones we thought about was

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in-line cleavage and so this is a

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spontaneous cleavage mechanism of RNA

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molecules where you basically get

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hydrolysis of the phosphate backbone

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this this this cleavage mechanism is

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also accelerated by the association of

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divalent

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cations with the phosphate backbone of

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the RNA molecule and actually uh Becca

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good-good Metzler I another grad student

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in Lauren Williams lab and has just

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really recently shown that iron can not

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only mediate in line cleavage suggesting

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that associates with the the backbone of

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the RNA but media added orders of

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magnitude higher rate than another

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divalent cation like magnesium

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essentially essentially basically

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showing a new pathway of iron mediated

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cleavage of ribosomal RNA which is an

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extremely exciting result in its own

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right and actually she gave a poster

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last night but I think it's still up so

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I urge any of you haven't already seen

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it to to go and look at it but basically

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that's what we think is going on here

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during the purification in the in the an

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toxic chamber over you know multiple our

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purification in a anoxic chamber where

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it's difficult to keep things very cold

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we think the the iron that is soaking

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into those ribosomes from the buffer is

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is associating with the ribosomal RNA

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and therefore mediating this in-line

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cleavage so like I just mentioned in the

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early part of the talk growing you coli

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under these pre jewy conditions these

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these conditions conducive to the

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Archaean earth leads to elevated levels

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of iron within the ribosomes and the

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fact these cells retain this is very

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suggestive of iron possibly being you

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know at least one in may possibly in

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tannin with magnesium and others

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divalent cofactors for early translation

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and that these ribosomes are intact and

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functional the in vivo iron we're seeing

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is because it can't be washed out is

281
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likely bound to RNA spots where it's

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very tightly associated or deeply buried

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like those daniel a micro clusters and

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also we're seeing iron associated in

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vitro extensively with the ribosomal RNA

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and mediate that in ninth cleavage and

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so given those two points that that

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really suggests that possibly the iron

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00:10:59,870 --> 00:10:58,300
that we're seeing you know here is very

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much a muted signal and that

291
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purification under buffers of magnesium

292
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is is washing out possibly significantly

293
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more in vivo associated iron and so

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thinking about you know experiments to

295
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to really get at the the true number of

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Virant associated

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with the ribosome and with that I just

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like to thank my advisor dr. Jen glass

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my co advisor dr. Laura Williams

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but with members of both labs and NASA

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for funding all this and thank you guys

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for listening we have time for exactly

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do questions okay the ones who anybody

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thank you for the nice paper

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there's an interesting calculation that

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00:11:53,790 --> 00:11:51,610
you might want to make I'll just call

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this to your attention and that is that

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we have a pretty good knowledge of how

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much iron ore there is in the Earth's

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crust if you dissolve all of that back

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into the ocean you only get about five

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micromolar iron in the seawater and

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right now of course iron has rusted out

314
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and it's way down below it's down the

315
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nano molar range so just keep in mind

316
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that iron might have been very dilute

317
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much more than much less than the

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millimolar concentrations yeah yeah yeah

319
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and yeah that's always something that

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comes up thinking about sort of the

321
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actual environmental relevance of iron

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versus other other dye valence

323
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definitely the the environments on the

324
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earlier earth weren't totally

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homogeneous and there could have been

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environments where or iron magnesium

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00:12:42,330 --> 00:12:39,520
could have been possibly competing

328
00:12:43,830 --> 00:12:42,340
levels of concentration yeah I didn't

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show this data but even manganese looks

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to be able to to to mediate trend

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translation function I think really the

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00:12:54,120 --> 00:12:51,970
most true answer is probably it was some

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00:12:56,370 --> 00:12:54,130
combination of those three and possibly

334
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even more dive ants especially at the

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early origins of the ribosome before you

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had other data dedicated systems to go

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out and sequester and compartmentalize

338
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and concentrate one divalent over

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another but yeah I think it's always

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important to very much keep the

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environmental relevance of all these

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things in mind thank you yeah two

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00:13:17,040 --> 00:13:15,010
question you showed a plot of the goe in

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the higher availability of ferrous iron

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00:13:21,060 --> 00:13:18,970
ore before the goe what was the

346
00:13:23,190 --> 00:13:21,070
availability it does magnesium available

347
00:13:23,460 --> 00:13:23,200
yeah that's one quick the other question

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is

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have you looked at anaerobic iron-rich

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conditions today for bacteria that lived

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00:13:32,340 --> 00:13:30,010
there to see if they have two irons in

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these dot in these places yeah so I'll

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cover the first question first and that

354
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goes back to the previous answer it

355
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part of it was definitely sort of where

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you were on the earth of the time

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00:13:43,500 --> 00:13:41,890
magnesium was definitely lower due to

358
00:13:44,700 --> 00:13:43,510
the increased hydrothermal activity of

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the earth especially because

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hydrothermal activity actually

361
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sequester's magnesium in crustal

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00:13:54,420 --> 00:13:52,270
minerals so definitely more towards the

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00:13:56,700 --> 00:13:54,430
ocean and the closer you got to any type

364
00:13:58,620 --> 00:13:56,710
of hydrothermal activity actually your

365
00:14:00,270 --> 00:13:58,630
ratio of iron to magnesium would

366
00:14:01,950 --> 00:14:00,280
increase drastically but overall

367
00:14:04,160 --> 00:14:01,960
magnesium was what's probably lower than

368
00:14:06,720 --> 00:14:04,170
it significant lower than it is today

369
00:14:10,110 --> 00:14:06,730
and then part of the second question is

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00:14:12,270 --> 00:14:10,120
yeah that's sort of that's sort of the

371
00:14:13,710 --> 00:14:12,280
logical as I see the logical next step

372
00:14:15,930 --> 00:14:13,720
of all this but really the biggest

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00:14:17,760 --> 00:14:15,940
barrier to that is just that nothing

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00:14:19,620 --> 00:14:17,770
really grows that well that's it that's

375
00:14:22,890 --> 00:14:19,630
an anaerobic now look at anaerobe

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00:14:25,200 --> 00:14:22,900
possibly to to the the Odie's that you

377
00:14:27,510 --> 00:14:25,210
can get e.coli and just this purifies so

378
00:14:28,890 --> 00:14:27,520
I definitely think it's possible and I

379
00:14:30,360 --> 00:14:28,900
think it's an experiment that needs to

380
00:14:32,640 --> 00:14:30,370
be done because you can think about

381
00:14:34,800 --> 00:14:32,650
obligate anaerobes that possibly haven't

382
00:14:36,480 --> 00:14:34,810
seen oxygen for millions of years or

383
00:14:38,730 --> 00:14:36,490
possibly never in their in their

384
00:14:42,410 --> 00:14:38,740
evolutionary history and and postulate

385
00:14:45,000 --> 00:14:42,420
about you know not not only the

386
00:14:47,370 --> 00:14:45,010
biochemistry of iron versus magnesium

387
00:14:48,990 --> 00:14:47,380
the translation system but but possibly

388
00:14:51,030 --> 00:14:49,000
they don't use magnesium for anything if

389
00:14:52,200 --> 00:14:51,040
if they're in an iron abundant

390
00:14:55,440 --> 00:14:52,210
environment don't have to worry about

391
00:14:56,520 --> 00:14:55,450
oxygen oxidative immediate toxicity but

392
00:14:59,220 --> 00:14:56,530
I think there's a lot of method

393
00:15:01,620 --> 00:14:59,230
methodological challenges to that that

394
00:15:03,270 --> 00:15:01,630
finding that out I have I don't know

395
00:15:07,260 --> 00:15:03,280
billion questions to ask but later or