RNase. RNase. RNase. This is the name for “RNA cutters” and I’m writing it over and over because, even though I think (and worry) about them a lot, capitalize the name correctly, I often do not! I always write RNAse! The other day I saw it written “RNAse” in a paper and felt so happy and vindicated! But, in a journal article is basically the only place I want to find these (at least the generic ones). Because RNases are everywhere – they’re secreted by us and by microbes to degrade viral RNA etc. which is good. But they can also shred up RNA you’re trying to work with, which is not good. In addition to keeping things super clean when working with them (about the only time you’ll see me working with a clean bench…) we can use a chemical called DEPC (DiEthyl PyroCarbonate) to put a permanent safety shield on the RNAse (ugh, I mean RNase) scissor blades.
The beast to rule them all is RNase A (& it’s related chewers). EDTA doesn’t protect against RNaseA because these scissors don’t need the help of metal to cut RNA. How does it pull it off? You can “phile” this case away as a nucleophilic attack! As the “-ase” ending indicates, RNases are enzymes (reaction-speeder-uppers). And what this type of enzyme speeds up is RNA cutting. And, unlike lots of RNA or DNA cutters (nucleases) RNaseA isn’t inhibited if you hide the metals!
Genetic information is written & stored in the form of nucleic acids (DNA & RNA). DNA is used for “permanent storage” whereas RNA is used for “temporary copies” like the messenger RNA (mRNA) copies of protein recipes that allow for a “seasonal assortment” of proteins to be made. More on this here: http://bit.ly/31IwofL & here: http://bit.ly/2FqasfN
But basically there are times when your cells want to destroy RNA – whether it’s foreign RNA from viruses or proteins you’re done making for now. So your cells have proteins called RNases that can catalyze (speed up) the hydrolytic cleavage of RNA (using water (hydro) to cut (lytic) the phosphodiester bond connecting the individual RNA letters (nucleotides). And, in the lab, we often want to keep them from doing this in our samples.
Different RNases have different “Achille’s heels” because the enzymes aren’t really the scissors themselves, they’re more like the scissors holders. They hold the blades in places so they can cut. Lots of nucleases use metals to help do this, so we can use EDTA (a chelator that “bites down” on a metal in multiple places) to “hide their metals” and inactivate them. But this trick doesn’t work for RNaseA because, instead of needing a metal to convince water to attack it, it convinces the RNA to attack itself! (and then uses amino acids (protein letters) sticking out into active site) to convince water to finish the job.
One reason RNA, and not DNA, is used for “temporary” purposes is that it’s easier to degrade. And this is because it has an extra “leg.” The D in DNA stands for “deoxyribose” and it indicates that it has one less oxygen than the ribose sugar you find in RNA. Instead of having a 2’ OH (right leg), DNA just has an H. And H is much more “boring” than OH. And speaking of boring, that O can “get bored” and go looking for excitement if conditions are right (or wrong depending on what you’re wanting….)
The O & H atoms are bound together by sharing a pair of subatomic particles called electrons & oxygen doesn’t share fair! The oxygen hogs the electrons it shares with hydrogen. And since electrons are negatively charged, this makes the oxygen partly negative & the hydrogen partly positive. But that’s not the only effect. Because it’s pulling the electrons away from the hydrogen, it’s kinda like the H has “already lost” so it has less to lose by getting lost!
The more the O pulls away, the easier it is to break the O-H bond. When that bond breaks and the electrons stay with the oxygen, a proton (H⁺ ) is released. (H only had 1 proton & 1 electron to begin with, and if it leaves the electron behind, it’s now just a proton). We say the OH has been deprotonated and has “acted as an acid.”
Once deprotonated, the O now has more electrons than it can neutralize, so it’s negatively charged. (another way to remember this is that you have to have conservation of charge and the H+ is positive so the other part must be negative). O likes having that extra electron but it doesn’t like having that charge. So it wants to find something positive to help it neutralize the charge.
One place you’ll positively find positive charge is a nucleus because that’s where the positively-charged protons live. So we call such positivity-seekers NUCLEOPHILES. Nucleophiles often have a lone pair of electrons, so you can remember nucleophiles by thinking of the u as a smiley face. Sometimes, but not always, nucleophiles have a – charge. note: protons are another subatomic particle – the 3rd type of subatomic particle is the neutron, which also hangs out in the nucleus but is neutral.
The “opposite” of a nucleophile is an electrophile. An electrophile has “too much” positive charge – sometimes this is enough to make it positively charged, though sometimes it’s still neutral.
When you remove the hydrogen from that -OH of ribose (the one that RNA has but DNA doesn’t), the O can “get bored” and when it gets bored it does looking for fun – and for a nucleophile, fun is found with electrophiles. So it looks tor an electrophile and finds one right nearby in the phosphate group.
Wait, isn’t that negative? Overall yes, but not the phosphorus! A phosphate ion has a phosphorus atom hooked up to 4 oxygen atoms. And each of those oxygens is pulling electrons away from the phosphorus. So even though the phosphate is negative overall, the phosphorus at the center is partly positive. And thus it’s electrophilic and attractive to nucleophiles.
Left alone, if the 2’ OH “gets bored” it can attack the phosphate connecting it to the next nucleotide, kicking that nucleotide off and creating a cyclic 2’,3’ phsopshodiester and releasing a 5’ O-nucleoside. Or, instead of kicking off the other nucleotide & thus breaking the chain, it can just swap which leg it’s connected to to get a 2’,5’-phosophodiester instead of the normal 3’-5’.
These are transesterification reactions and you don’t want them to happen when you don’t want them to happen or else you’d get jumbled shredded RNA!But you do want them to happen when you do want them to happen! So we want to “selectively bore” specific oxygens and RNases are great at this.
A reason you can have charge-seeking without “full charges” is that you can have “partial charges” – like in water, the O pulls electrons away from the H’s so the O’s are partly negative & the H’s are partly positive. If an H⁺ leaves, you get those full charges, but they were already “in the works” before you split it – kinda like a frayed cord stretched to the limit. Things can make an -OH more reactive by pulling harder on that cord, making it easier to break. So RNases have active sites that hold RNA in position and pull on that cord, making the oxygen bored!
pH is a measure of how many free H⁺ there are – the more there are, the lower the pH & the more acidic the solution. Under acidic conditions, there are plenty of H⁺ around, so each O can be an OH. But under basic (alkaline conditions) there are fewer H⁺, so the O is more likely to “donate”
But you don’t want to increase the pH everywhere or you’d get shredded RNA. Instead you want to do it in a controlled fashion. The histidine (one of the amino acid building blocks or “letters” that make up proteins) in the active site of RNase A acts as a sort of local pH raiser – it’s kinda like it tricks the RNA in that location into thinking that the H⁺ stock’s running low, so the OH deprotonates and gives that proton to His.
His can do this because it has an N with a lone pair of electrons and that lone pair can act as a “general base” & pull off that proton, turning the 2’ OH into a 2’ O- which is now very nucleophilic & can attack the phosphate.
I thought you said this was a hydrolysis reaction?! Where’s the water come in? Well, when the O attacks the P you get a 2’-3’ cyclic intermediate. Water is used to get that phosphate to uncyclize in a way that the phosphate ends up back on the 3’ leg where it “belongs.” It does this with the help of another histidine, this time coming from the other side. Instead of directly attacking, though, it convinces a water to attack. And this back-forthness of the His-mediated protons regenerates the His, letting it do it again.
A lot of DNA cutters (DNases) as well as some other RNases use metals to help them do this type of reaction. Metal atoms are particularly good for things involving DNA & RNA because metal atoms are usually positively-charged (cationic), whereas DNA & RNA are negatively-charged (anionic). This negative charge is what allows us to separate DNA & RNA pieces by length using electricity to bribe them through a gel mesh using positive charge to bribe them. And it also is what allows positively-charged molecules to grab it.
Nucleases have positively-charged binding pockets, where the positive charge comes from basic amino acids like lysine & arginine. That helps the binding, but when the reaction is actually occurring, when that phosphate is getting attacked, you have a really unstable, highly negatively charged pentacovalent (5 things attached) intermediate that needs some extra charge stabilization in order for the cleavage to occur. Some nucleases use metals for this, holding metal cations such as Mg2+ in the active site to help stabilize that intermediate (and activate the nucleophile). So you can inhibit those nucleases with EDTA or other chelators (things that bind metals in multiple places), which strip the nuclease of the metal they need. http://bit.ly/2SV2156
But RNase A and some other nucleases don’t need metal. Instead, they use positive-charged amino acids like Lys & Arg instead of positively charged metals.
So, how can we stop RNase? One way is using DEPC (DiEthyl PyroCarbonate). Instead of taking away something the RNase needs, it adds something to hide what it needs. It alkylates the catalytic His residues, sticking carbethoxyl groups on those crucial N’s preventing them from doing that proton ping-pong. This is a covalent inhibition (the DEPC part gets added onto the catalytic residue “permanently” instead of just competing with it.
But it’s not specific for *just those* N’s. It can alkylate any Ns. and lysine & cysteines, and tyrosines. So you don’t want to just stick it into your reaction mix. Instead, we usually only use it to neutralize and RNase A in water we want to use when working with RNA. And after we treat the water, we autoclave it, which heats it up really hot under high pressures. https://bit.ly/autoclavessteam
Surprisingly, although that would kill almost any “normal” protein, such autoclaving doesn’t permanently inactivate all the RNase A – this beast can survive autoclaving – at least some of it – autoclaving can kill some of its activity but this protein has a lot of disulfide bonds that can withstand the high heat and keep the protein partly folded so that it can more easily retold when time comes.
Autoclaving might not inactivate all the RNase A, but it does permanently inactivate the DEPC by causing it to fall apart forming CO₂ & ethanol. Which can evaporate away.
Tech note: you can use DEPC-treated, autoclaved, water to make buffers that contain Tris, HEPES, or other amines. But you can’t (or at least you shouldn’t) directly treat amine-containing buffers because DEPC isn’t picky. In addition to just the active site amino acids of RNases, it reacts with “any” amines, thiols, & alcohols. And once it reacts, it’s “useless.” Kinda like a party popper – you can only pop it once. So you want to make sure you start with enough poppers and don’t accidentally set them off before you’re ready. Then, the point of the autoclaving is to pop all the poppers that haven’t gone off before you add it to something where you don’t want it to go off. (and you don’t don’t want it to go off and leave confetti stuck to your reaction!)
ThermoFisher suggests ~0.1% DEPC. If you go lower, you might not inhibit all the RNase. But if you go too high, you might not inactivate all the DEPC before you go use it in your reactions. Or, even if you do inactivate it all, some of the DEPC by-products can inhibit some of those reactions you’re going to use it with (such as in vitro translation) and potentially even modify RNA instead of just RNases.
If you’ve ever worked with DEPC-treated water and noticed a fruity smell and gotten kinda freaked out that it had gone bad or something – don’t worry! That smell comes from esters that are formed when ethanol given off during the RNase-killing reacts with trace amounts of carboxylic acid contaminates.
Perhaps one of the most important things to keep in mind when working with DEPC-treated water is that the water is only as clean as you keep it! Since you’ve inactivated all the DEPC, there’s nothing in there to protect against any RNases that are subsequently introduced.
And there are a lot of RNases a lot of places – There are a lot of different RNases. Some do more specific things in our cells and work in a more controlled fashion. For example, the protein I study, Argonaute cuts RNA but only when that RNA matches an RNA guide that Argonaute is bound to in a process called RNA interference (RNAi). https://bit.ly/rnainterference
So I like that RNase :). But these generic guys are the ones we really worry about. RNase A is the name for the bovine version of one of them. The most similar one in humans is RNase 1. They’re both referred to as “pancreatic-type” cuz they’re secretory RNases made by the pancreas – but that name is kinda misleading because they’re also made by most cells – especially endothelial cells (cells lining body tubing and compartments) – so we can secrete it in our spit & sweat, etc. to add a barrier of protection against viruses before they can even get in. Bacteria & fungi and other tiny life forms also do this – and they’re the main source we worry about in the lab.
In addition to DEPC treatment and cleaning well, we take other precautions when working with RNA including using filtered pipet tips which prevent RNases from getting pushed out of the pipet into the sample when you’re pipetting.
This post might seem kinda random but I was working with RNA today 🙂
source of figure & more info: https://bit.ly/32KTllv
more on topics mentioned (& others) #365DaysOfScience All (with topics listed) 👉 http://bit.ly/2OllAB0