I started watching this, and at first I was thinking "yeah, yeah, whatever; this isn't really helping." But then I kept watching, and it became the clearest explanation of this concept that I have ever seen. Thank you very much.
Same here! , The expanation is really plane but it has what it should. 1 The exothermal character of the polimerization is mentioned and linked to making that oxygen on the first phosphate of the nucleotide's 5' position a good leaving group. 2 The nucleophilic character of the oxygen on the primer's 3' position is shown to be the cause for 3' in the primer to bind to the oxygen of the phosphate in 5' position of the nucleotide. 3.It is shown how the reaction will not procede trying to bind 3' in the nucleotide with 5' in the primer because the primer does not carry the triphosphate moiety ...hope I got it right ☺
4 minutes in and im thinking the same thing, "Let me peek at the comments and see if I should finish this video or he is just yapping" Lol alright I'll finish thanks man
Mad praise for this guy. He actually clearly explained using the displayed skeletal formula of each of the molecules what all my Year 1 lecturers so far have just dictated to me. Books aren't as easy to learn from I feel, so this video is greatly appreciated. Will share with anyone else who is looking to understand this :D
This is the best explanation I've found on the biochemical reasons for the 5' reaction direction. Super helpful to my own students. Thanks for posting.
You're right, if the additional phosphates were retained on the 5' end, you could add a nucleotide there. However, they're lost by spontaneous hydrolysis, so there typically is only a single phosphate on the 5' end of any nucleotide strand. dNTPs are constantly being synthesized. I suppose you could have an enzyme that would go around adding extra phosphates on 5' ends of strands where they'd been lost, but it would be rather a waste of energy, as lots of strands aren't going to be extended.
Hi There! 😁 Thanks a ton for your explanation. Am curious - What’s stopping the 5’ end from binding to an oxygen from water surrounding? Seems if water binds before 3’ oxygen - no space for reaction to continue. Thoughts?
I've always wondered WHY DNA has a leading and lagging strand but none of my professors ever get specific when the 5'-->3' synthesis question comes up. Thanks so much. This is so helpful! Best, TL
This was very very helpful doctor ...thank you so much. Can you please make another video as to why RNA primee is really required for starting dna replication.
this video was made when i was four years old and now im using it 13 years later to help me get into medical school, really hope the man that made this is well, he has explained it better than any teacher ive had :)))))))
The way Prof. Eric Lander explained in his Intro Bio class is that, indeed, 5' end on the growing DNA strand can have three phosphates attached PPP (as cd4600 pointed out) and the reaction can go from 3' to 5'. But here is the problem. This PPP end is highly reactive. So DNA went to all this trouble replicating only to have its 5' PPP-end react with something and halting replication. While if PPP on a free nucleotide reacts - not a big deal - just one nucleotide was lost.
Thank you so much! This explanation cleared everything up for me! I never understood why DNA polymerase only made bonds at the 3' end and just had to go with it. Now I get it! Thank you so much!
I have seen a few very ridiculous videos but this one is really amazing and although it is beyond the depth of basic biology I needed this to understand something that is important to my studies, the comments are helpful as well. thank you from Houston.
also, if you would take a look at my other conclusion regarding the ribosomes, it would be great too. here goes, Ribosomes read the mRNA from 5' to 3'. They do this by attaching to AUG on the 5' end of the mRNA molecule. They then read all the way till a stop codon is found. The Anticodon (on the tRNA) must be complementary to this, so it must be read from 3' to 5'. That is the anticodon for 5' AUG 3' is 3' UAC 5'.
@Platypusti - In principle, it could work. The main problem is the inherent instability of the triphosphate. That's why it's reactive, right? If you're going to put that triphosphate on the 5' end of the primer, you're going to have to use that primer very soon, before the extra phosphates come off spontaneously. As far as I know, there's no example in nature of an organism doing it that way, but I'm not saying there couldn't be.
@agathman oh thats true, I think I just got very excited and wanted to type it all out fast! So yea, the template strand is read from the 3' to the 5' direction. the dna polymerase (or rna polymerase) adds makes 5' to 3'. It does this by adding to the free 3' OH group.
@basantologist Actually, this could be either one; the process of replication works the same on both strands. I have some illustrations that show the whole process of replication in E. coli, with both lagging and leading strands, which might be helpful to @AM loki. I'll put a link in the video description.
@AM loki You see, originally, the DNA has two strands which gets unzipped so the DNA polymerase can come and add the new nucleotides. In this video, you were shown only one of the template strands.. The other template strand, because it is anti parallel and the DNA polymerase can only go 5' to 3', it has to do it in segments which are called the Okazaki fragments. This occurs on the lagging strand. The strand shown here as the template was the leading strand.
This is really great, i found a lot of explanations regurgitating the same ideas that everyone teaches but wasnt getting at the key problem i had with why you couldnt flip the strand upside down and label it 5' at the top by mistake
+QuickStart Language Because triphosphates are unstable. The existing end wouldn't have them unless it had just been put there. Furthermore, in many situations, synthesis starts at a nick in existing DNA, and there wouldn't be three phosphates there in the first place.
So it's not hard to prove that with a : simple white board and a black marker you can give the clearest explanations of the concepts without leaving slightest of doubts in the minds of the audience/viewers/seekers. This professor for sure knows what is meant by "to understand" and that is precisely what he has tried make us do. Compare it with all those flashy animations with loads of blabber going no where... Thank you professor for this...
@GJEViLakaTHEKING -- I think you're misstating the conclusion. DNA polymerase (or RNA polymerase) moves along the template strand in the 3' to 5' direction (as seen from the template strand). The newly synthesized nucleic acid strand is antiparallel to that template, and is made by adding nucleoside triphophates to the 3' end of that new strand. Thus the new strand grows in the 5' to 3' direction. The 5' end of, for instance, an RNA molecule is made first; the 3' end is made last.
@GJEViLakaTHEKING Yes, absolutely. Anticodons are the one exception to the rule that nucleic acid sequences are given 5' to 3' -- generally we state anticodon sequences 3' to 5', so we'd say the methionine anticodon (pairs with AUG) is UAC. Normally you'd say CAU, since that's 5' to 3', but it would be confusing to say it that way.
Actually cd4600, that's THE more plausible explanation, because there is no reason to think that the receiving nucleotide is in the monophospate form, it should be in the triphosphate form, but then if something promotes the hydrolisis of this "last" nucleotide it would be imposible to continue adding more nucleotides to this chain in the 3' to 5' direction.
This was awesome review for my MCAT studying, thank you so much for going into detail about the concepts behind how AND why dna is replicated in such a manner!
Summary of lecture" when 5>3 occurs 5 prime has to 2 extra phosphate attached to it which which pulls off one oxygen from 5' phosphate hence it can now bond with 3' OH. Other way 3>5, 3' can bring same OH group but there at 5' it's stable it has no extra 2 phosphate which can pulls off oxygen from 5' phosphate and leave space for 3' OH. I hope you got it
When he explains why there can't be a nucleophilic attack from 3' to 5' - why is the assumption that the template nucleotide only has one phosphate? If it had 3 phosphates that would make it viable for nucleophilic attack from the dNTP being added. In one his replies to an earlier comment, he states that the reason this can't happen is because the triphosphate is too reactive to be on the 5' end because the pp gets lost by spontaneous hydrolysis. However, why would this not be the case for the 3' end?
You Explain things like an amazing Boss. It would be great if my university hired you as a prof! I always have a hard time understand with the directions, so THANKS! in video's conclusion, DNA polymerase reads the template strand from 5' to 3' direction, BUT adds the nucleotides to a open 3'OH group so it attaches from 3' to 5'. It makes the rna molecule starting at 3' towards 5'. the same is true for rna polymerase (I think)
Your explanation as to why it couldn't bind in the 3 to 5 direction seems to be lacking. I appreciate that you attempted to answer. My professor didn't know and got mad that I asked. The way I see it, the only reason it works in the 5 to 3 direction is because it uses dNTP. Which is kind of like sayings, "it happens that way because that's the only way it happens." Well, I would say to that, why isn't their a different enzyme and cofactor to break the Oxygen bind on the other end? Thanks.
Hey, thank you for your video. But i have some questions. Normaly there is a triphosphate at the 5´-end, why the cell theoretically should be able to synthesize in this direction. After a proofreading, only one phosphate remains which is not "energy-rich" enough for a nucleophil attack on a 3´-OH, ok. But why cannot the cell use a ligase in this case to synthesize in 3´-5´ direction? By adding an additional phosphate it should work (would be the same like in the repair of single strand nicks). I do not see any biochemical problem. Another strategy for the cell could be the usage of phosphocreatine. So, in my opinion it is not impossible, but just not discovered/developed by the cell yet. I am looking forward to your reply/thoughts about this.
+Gen. Szadek Yes, it could be done. But it would require ATP hydrolysis to drive it if you were doing it via ligase, so it wouldn't be particularly efficient - especially compared to the rather elegant 5'-3' synthesis process that uses the same nucleoside triphosphate as a substrate and a source of energy for the reaction. I didn't intend to argue here that 3'-5' extension was impossible, just not easy.
Question, when we say DNA is replicated from the 5' end to 3' end, can't you also say it is 3' to 5'? Because DNA is made of 2 strands (1 pair) and they are anti-parallel. When the 2 strands separate so free nucleotides join, the nucleotides attach to both strands: on the leading strand, the nucleotides start at the free end (3') working inwards (to the 5' end), while the lagging strand starts at the fork end (3') and works outward (to the 5' End). So it works both ways? Also the actual replication is 3' to 5' so why do we say it is 5' to 3'? Thank you!
First, the 5' to 3' direction of replication refers to each strand, not to the molecule as a whole. Second, nucleotides are added to the 3' end of the strand, so it grows in the 5' to 3' direction.
The DNA polymerase walks down the single stranded template in 3'-5' direction, but the new strand that is being synthesized grows only 5'-3' direction.
Had the exact same question. DNA is read from the original strand from 3' -> 5' during replication so I couldn't wrap my head around why we were calling it 5' -> 3'. Thanks for clarifying Borislav!
The part that I don't understand is why 3' to 5' doesn't work. Specifically if DNA polymerase attempts to add a dNTP to the 5' end, why is the pyrophosphate leaving group already gone, since it wouldn't have been involved in the previous addition of a nucleotide to the 5' end of the chain.
I have a question? In the second stage when we try from 3' to 5', the 2 phosphate groups are absent in the A and in the first stage they are present. How it is? because the behavior must be same for the A in both cases. Meaning that in the second case why he dont attach the two other phosphate groups, how are they absent as they are not involved in any reaction untill that stage.
This question has already been answered in reply to "QuickStart Language" : Because triphosphates are unstable. The existing end wouldn't have them unless it had just been put there. Furthermore, in many situations, synthesis starts at a nick in existing DNA, and there wouldn't be three phosphates there in the first place.
then the "spontaneous hydrolysis" should be the key point of explaining why it goes from 5' to 3', because the rest is based on this hypothesis. Could you elaborate this part?
Wait, but when going from 3' to 5' instead, why did the terminal nucleotide not have three inorganic phosphates? If the preceding reaction was with the phosphate group of the preceding nucleotide and the hydroxyl group of the dATP (in the photo, dAMP), shouldn't the dAMP still be a dATP?
I am wondering that too. As far as i am concern, DNA Pol can´t work 3´ to 5´ because proofreading would terminate polimerization. So, evolution would have to choose between proofreading or polimerization 3´ to 5´. But without proofreading, there would be too many mutations...and (probably) no life.
I dont understand why there are no additional phosphates and magnesium co factor on the 5 prime carbon of the dntp in the reversendirection (3-5)... Yes I understand that without these the reaction can only proceed in the 5-3 direction... My question is therefore, why are the phosphates and mg co-factors for the reverse direction missing?
the dinucleotide triphosphates, and Mg cofactors are supplied by the DNA polymerase (not shown) so they won't be on the parent strand initially, just the incoming nucleotides.
The extra 2 phosphates are missing in 3' to 5' synthesis because the trisphosphate group spontaneously hydrolyses in aqueous conditions, so at some point you'd only end up with a single phosphate left that can't be attacked by OH, so 3' to 5' synthesis can't continue any further. Spontaneous hydrolysis also happens to the free nucleotides, but when you're synthesising from 5' to 3' all you need to do is find another nucleotide that hasn't been hydrolysed yet.
One thing is really throwing me off. Your explanation is pristine but a chemical workaround to moving from 5'--3' seems fairly straightfoward, and I can't figure out why nature doesn't just apply it. It seems to me that a simple solution would be to attach a pyrophosphate group to the terminal 5' nucleotide of the primer in order to make the reaction more thermodynamically favorable. Is this just the way nature has decided to carry out this process? If so, if I were to (in a lab setting of course), attach a pyrophosphate to last nucleotide of the 5' end would I effectively "trick" DNA polymerase to proceed in a 3'--5' manner?
The second and fourth answers here should help. biology.stackexchange.com/questions/477/why-is-dna-replication-performed-in-the-5-to-3-direction One explanation is that nucleoside triphosphates will readily hydrolyze in aqueous solutions, and placing the phosphate groups on the growing chain would be a waste were this to happen. But I question the validity of this; while ATP (as an example) does have a short half-life, DNA replication is much faster, and should outpace the hydrolysis of the hypothetical triphosphate end of the growing chain. The other explanation, which I prefer, is that 3'-to-5' directionality prohibits proofreading. If the triphosphate from the chain were to be hydrolyzed, only for the new base to be determined incorrect, excision of this base would leave the chain a dead end, since there would not be another triphosphate group at the 5' end. The picture in the link illustrates this more clearly. Although there are DNA repair mechanisms that may potentially address this (you can imagine that excision of a damaged dNTP in one strand would leave the 5' end of the downstream nucleotide without a triphosphate anyway, making it hard to insert a new undamaged dNTP), they require other enzymes that are more specialized for their tasks. As far as polymerase proofreading goes, 5'-to-3' is the only direction that can work.
excellent video! you saved me from exam! BTW, in the end of the lecture, you said that the phosphates are too far from OH, so it is very difficult from reacting. Is it that far? I mean, those molecules are very tiny, and I think it should be easy for phospherates to go to the next OH attached on P. That way, bond should be made.
Are dNTPs used in RNA synthesis too? I know RNAs use ribose sugar where dNTPs are obviously made up of deoxy Ribose sugar. So in mRNA sequencing, is the substrate still dNTPs or just NTPs?
It might be a combination of the facts that 1 the primer doesn't have a triphosphate group on its 5' end (and is thus energetically unfavorable compared to the 3' end) and 2 you would need an alternate DNA polymerase protein structure, which just hasn't evolved. Wouldn't you agree? So it's both evolutionary predetermined and energetically unfavorable and most likely evolutionary predetermined because it's energetically unfavorable.
oh and offcourse DNA polymerase wouldn't be able to continue after removing a mispaired nucleotide during proofreading since then there would be no energy left to continue.
@beccalishass 5' end of the INCOMING dNTP has a good leaving group (PP). If you're going to join 5' to 3', that means the incoming dNTP has to add on to the 3' end of the existing strand.
why is there only one phosphate not phospahtes on the top of strand when dna is synthesized from 3' to 5'? Is there other reaction that eliminates two phosphates?
Had the replication been 3 prime to 5 prime,there would have been no need for the last 5 prime NTP that exists in the chain to not have a triphosphate group. It could have been 5' pppApTpGpCOH 3' and then the incoming OH could very well have a pppA to attack.
Annu Aparajita Yes, I've considered that, but the ppp linkage is inherently unstable; if you had to leave one on the DNA all the time, it would rapidly degrade. While the nucleoside triphosphates, being just raw material for making DNA rather than the information storage molecule itself, can be synthesized as needed and used rapidly.
Yes that makes sense.This is also the explanation provided by Eric Lander.Another can be found here. sandwalk.blogspot.ca/2009/04/head-growth-and-tail-growth.html
whereas my alevel mark schemes just says: DNA polymerase is specific and only complementary with the 5' end, and shapes of the 5' and 3' ends are different...
Thank you very much professor! I'm a final year med student preparing for the USMLE Step 1 and I was confused as to the "why" of 5' to 3' replication. This was an excellent explanation.
To flag the point of origin. Without the RNA primer, DNApolymerase would not be able to attach itself to the DNA strand for replication. It's basically a 'flag' that specifies where to begin replication of DNA.
Thank you for your answer. You answer completes the explanation of the video. I am sad how I spent 9 minutes watching the video and asked the same question cd4500 asked.
This video was filmed with a nucleic acid
Alexei Christodoulides bAhahahah
LMAO
I started watching this, and at first I was thinking "yeah, yeah, whatever; this isn't really helping." But then I kept watching, and it became the clearest explanation of this concept that I have ever seen. Thank you very much.
same here. I think this is the best explanation I have ever seen. Thank you very much. May God bless you more....
Same here! , The expanation is really plane but it has what it should. 1 The exothermal character of the polimerization is mentioned and linked to making that oxygen on the first phosphate of the nucleotide's 5' position a good leaving group. 2 The nucleophilic character of the oxygen on the primer's 3' position is shown to be the cause for 3' in the primer to bind to the oxygen of the phosphate in 5' position of the nucleotide. 3.It is shown how the reaction will not procede trying to bind 3' in the nucleotide with 5' in the primer because the primer does not carry the triphosphate moiety ...hope I got it right ☺
4 minutes in and im thinking the same thing, "Let me peek at the comments and see if I should finish this video or he is just yapping" Lol alright I'll finish thanks man
Mad praise for this guy. He actually clearly explained using the displayed skeletal formula of each of the molecules what all my Year 1 lecturers so far have just dictated to me. Books aren't as easy to learn from I feel, so this video is greatly appreciated.
Will share with anyone else who is looking to understand this :D
professors always leave out the detail you put in. thank you for presenting this clearly.
This is the best explanation I've found on the biochemical reasons for the 5' reaction direction. Super helpful to my own students. Thanks for posting.
Can you clarify when did the nucleotide added to the growing strand lost the pyrophosphate?
As a chemist transitioning more towards biochemistry there was a need for a quick refresher on this subject. This presentation was great. Thanks.
You're right, if the additional phosphates were retained on the 5' end, you could add a nucleotide there. However, they're lost by spontaneous hydrolysis, so there typically is only a single phosphate on the 5' end of any nucleotide strand. dNTPs are constantly being synthesized. I suppose you could have an enzyme that would go around adding extra phosphates on 5' ends of strands where they'd been lost, but it would be rather a waste of energy, as lots of strands aren't going to be extended.
Hi There! 😁 Thanks a ton for your explanation. Am curious - What’s stopping the 5’ end from binding to an oxygen from water surrounding? Seems if water binds before 3’ oxygen - no space for reaction to continue. Thoughts?
I've always wondered WHY DNA has a leading and lagging strand but none of my professors ever get specific when the 5'-->3' synthesis question comes up. Thanks so much. This is so helpful!
Best,
TL
Great video explanation! My first med school exam is tomorrow and this was one of the questions the professor asked us to know for the exam.
Such a great video! Even with the kinda fuzzy graphics, your explanation made this concept super clear. Thank you!
This was very very helpful doctor ...thank you so much. Can you please make another video as to why RNA primee is really required for starting dna replication.
this video was made when i was four years old and now im using it 13 years later to help me get into medical school, really hope the man that made this is well, he has explained it better than any teacher ive had :)))))))
Happily retired and doing a lot of birdwatching, thanks!
The way Prof. Eric Lander explained in his Intro Bio class is that, indeed, 5' end on the growing DNA strand can have three phosphates attached PPP (as cd4600 pointed out) and the reaction can go from 3' to 5'. But here is the problem. This PPP end is highly reactive. So DNA went to all this trouble replicating only to have its 5' PPP-end react with something and halting replication.
While if PPP on a free nucleotide reacts - not a big deal - just one nucleotide was lost.
Thank you so much! This explanation cleared everything up for me! I never understood why DNA polymerase only made bonds at the 3' end and just had to go with it. Now I get it! Thank you so much!
I have seen a few very ridiculous videos but this one is really amazing and although it is beyond the depth of basic biology I needed this to understand something that is important to my studies, the comments are helpful as well. thank you from Houston.
The second half of this video is by far the best explanation I've seen for both why 5' to 3' is energetically favorable and why 3' to 5' is not.
also, if you would take a look at my other conclusion regarding the ribosomes, it would be great too. here goes,
Ribosomes read the mRNA from 5' to 3'. They do this by attaching to AUG on the 5' end of the mRNA molecule. They then read all the way till a stop codon is found. The Anticodon (on the tRNA) must be complementary to this, so it must be read from 3' to 5'. That is the anticodon for 5' AUG 3' is 3' UAC 5'.
@Platypusti - In principle, it could work. The main problem is the inherent instability of the triphosphate. That's why it's reactive, right? If you're going to put that triphosphate on the 5' end of the primer, you're going to have to use that primer very soon, before the extra phosphates come off spontaneously. As far as I know, there's no example in nature of an organism doing it that way, but I'm not saying there couldn't be.
Am just speech less.... I didn't find even a single vedio explaining clearly...I just want to thank you from bottom of my heart ❤️❤️❤️...👍🏻
@agathman
oh thats true, I think I just got very excited and wanted to type it all out fast!
So yea, the template strand is read from the 3' to the 5' direction. the dna polymerase (or rna polymerase) adds makes 5' to 3'. It does this by adding to the free 3' OH group.
Super helpful explanation to understand what's at the core of the phrase "DNA is built 5' ----> 3' not 3' ----->5' many thanks!!!!!
@basantologist Actually, this could be either one; the process of replication works the same on both strands. I have some illustrations that show the whole process of replication in E. coli, with both lagging and leading strands, which might be helpful to @AM loki. I'll put a link in the video description.
@AM loki You see, originally, the DNA has two strands which gets unzipped so the DNA polymerase can come and add the new nucleotides. In this video, you were shown only one of the template strands.. The other template strand, because it is anti parallel and the DNA polymerase can only go 5' to 3', it has to do it in segments which are called the Okazaki fragments. This occurs on the lagging strand. The strand shown here as the template was the leading strand.
This is really great, i found a lot of explanations regurgitating the same ideas that everyone teaches but wasnt getting at the key problem i had with why you couldnt flip the strand upside down and label it 5' at the top by mistake
Excellent explanation, straight to the point with clarity of information. Very nice.
At 7:28 min, why doesn't the nucleotide at the 5' end have three phosphates?
+QuickStart Language Because triphosphates are unstable. The existing end wouldn't have them unless it had just been put there. Furthermore, in many situations, synthesis starts at a nick in existing DNA, and there wouldn't be three phosphates there in the first place.
So it's not hard to prove that with a : simple white board and a black marker you can give the clearest explanations of the concepts without leaving slightest of doubts in the minds of the audience/viewers/seekers.
This professor for sure knows what is meant by "to understand" and that is precisely what he has tried make us do.
Compare it with all those flashy animations with loads of blabber going no where...
Thank you professor for this...
@GJEViLakaTHEKING -- I think you're misstating the conclusion. DNA polymerase (or RNA polymerase) moves along the template strand in the 3' to 5' direction (as seen from the template strand). The newly synthesized nucleic acid strand is antiparallel to that template, and is made by adding nucleoside triphophates to the 3' end of that new strand. Thus the new strand grows in the 5' to 3' direction. The 5' end of, for instance, an RNA molecule is made first; the 3' end is made last.
@GJEViLakaTHEKING Yes, absolutely. Anticodons are the one exception to the rule that nucleic acid sequences are given 5' to 3' -- generally we state anticodon sequences 3' to 5', so we'd say the methionine anticodon (pairs with AUG) is UAC. Normally you'd say CAU, since that's 5' to 3', but it would be confusing to say it that way.
Actually cd4600, that's THE more plausible explanation, because there is no reason to think that the receiving nucleotide is in the monophospate form, it should be in the triphosphate form, but then if something promotes the hydrolisis of this "last" nucleotide it would be imposible to continue adding more nucleotides to this chain in the 3' to 5' direction.
This was awesome review for my MCAT studying, thank you so much for going into detail about the concepts behind how AND why dna is replicated in such a manner!
Summary of lecture" when 5>3 occurs 5 prime has to 2 extra phosphate attached to it which which pulls off one oxygen from 5' phosphate hence it can now bond with 3' OH. Other way 3>5, 3' can bring same OH group but there at 5' it's stable it has no extra 2 phosphate which can pulls off oxygen from 5' phosphate and leave space for 3' OH. I hope you got it
When he explains why there can't be a nucleophilic attack from 3' to 5' - why is the assumption that the template nucleotide only has one phosphate? If it had 3 phosphates that would make it viable for nucleophilic attack from the dNTP being added. In one his replies to an earlier comment, he states that the reason this can't happen is because the triphosphate is too reactive to be on the 5' end because the pp gets lost by spontaneous hydrolysis. However, why would this not be the case for the 3' end?
Also i realised if you flipped the chain upside down you would have a carbon 2' as an extender when it should be 3' or 5' where extension occurs
You Explain things like an amazing Boss. It would be great if my university hired you as a prof!
I always have a hard time understand with the directions, so THANKS!
in video's conclusion,
DNA polymerase reads the template strand from 5' to 3' direction, BUT adds the nucleotides to a open 3'OH group so it attaches from 3' to 5'. It makes the rna molecule starting at 3' towards 5'.
the same is true for rna polymerase (I think)
Your explanation as to why it couldn't bind in the 3 to 5 direction seems to be lacking. I appreciate that you attempted to answer. My professor didn't know and got mad that I asked.
The way I see it, the only reason it works in the 5 to 3 direction is because it uses dNTP. Which is kind of like sayings, "it happens that way because that's the only way it happens." Well, I would say to that, why isn't their a different enzyme and cofactor to break the Oxygen bind on the other end? Thanks.
Hey, thank you for your video. But i have some questions. Normaly there is a triphosphate at the 5´-end, why the cell theoretically should be able to synthesize in this direction. After a proofreading, only one phosphate remains which is not "energy-rich" enough for a nucleophil attack on a 3´-OH, ok. But why cannot the cell use a ligase in this case to synthesize in 3´-5´ direction? By adding an additional phosphate it should work (would be the same like in the repair of single strand nicks). I do not see any biochemical problem. Another strategy for the cell could be the usage of phosphocreatine. So, in my opinion it is not impossible, but just not discovered/developed by the cell yet. I am looking forward to your reply/thoughts about this.
+Gen. Szadek Yes, it could be done. But it would require ATP hydrolysis to drive it if you were doing it via ligase, so it wouldn't be particularly efficient - especially compared to the rather elegant 5'-3' synthesis process that uses the same nucleoside triphosphate as a substrate and a source of energy for the reaction. I didn't intend to argue here that 3'-5' extension was impossible, just not easy.
Ooooouf ouuf ouf chill there mate, your over exaggerating it now 😂
7:35 my mind voice
This video has been waiting for me for 9 years
you must be the happeiest person in the world at that moment
This is exctly the explanation i was looking for , thanks a lot!
This is a must-see! the best explanation of the topic from 14years ago?
I love you sir!
Really the best explanation I have seen so far!!!!
That was the missing part of the explanation above. Thank you both.
video quality: 240p
explanation quality: 2160p
cannot thank you enough
Thank you so much! My professor neglected to draw out the structures for why 3`-->5` wouldn't work so it was very confusing!
Excellent video! No lecturer has done this explanation as well! Thanks alot!
Question, when we say DNA is replicated from the 5' end to 3' end, can't you also say it is 3' to 5'? Because DNA is made of 2 strands (1 pair) and they are anti-parallel. When the 2 strands separate so free nucleotides join, the nucleotides attach to both strands: on the leading strand, the nucleotides start at the free end (3') working inwards (to the 5' end), while the lagging strand starts at the fork end (3') and works outward (to the 5' End). So it works both ways? Also the actual replication is 3' to 5' so why do we say it is 5' to 3'?
Thank you!
First, the 5' to 3' direction of replication refers to each strand, not to the molecule as a whole. Second, nucleotides are added to the 3' end of the strand, so it grows in the 5' to 3' direction.
The DNA polymerase walks down the single stranded template in 3'-5' direction, but the new strand that is being synthesized grows only 5'-3' direction.
Had the exact same question. DNA is read from the original strand from 3' -> 5' during replication so I couldn't wrap my head around why we were calling it 5' -> 3'. Thanks for clarifying Borislav!
Thanks so much for explaining this. Clear and straight to the point
Very helpful,greetings from Sicily.
The part that I don't understand is why 3' to 5' doesn't work. Specifically if DNA polymerase attempts to add a dNTP to the 5' end, why is the pyrophosphate leaving group already gone, since it wouldn't have been involved in the previous addition of a nucleotide to the 5' end of the chain.
I have a question? In the second stage when we try from 3' to 5', the 2 phosphate groups are absent in the A and in the first stage they are present. How it is? because the behavior must be same for the A in both cases.
Meaning that in the second case why he dont attach the two other phosphate groups, how are they absent as they are not involved in any reaction untill that stage.
This question has already been answered in reply to "QuickStart Language" :
Because triphosphates are unstable. The existing end wouldn't have them unless it had just been put there. Furthermore, in many situations, synthesis starts at a nick in existing DNA, and there wouldn't be three phosphates there in the first place.
You way of teaching is amaizing sir
I think this explanation is better than the one who relates this to proofreading.
Reasonable and nice illustration!
Thank you so much, I have wondered why this is the case for so long!
then the "spontaneous hydrolysis" should be the key point of explaining why it goes from 5' to 3', because the rest is based on this hypothesis. Could you elaborate this part?
cd4600 i had the exact same question. glad you mention it
and ill believe in irakli roraldzes explanation thanks so much
Good job describing why 3'-5' won't work. Thanks!
Where does the hydrogen from the hydroxyl go when it attaches with phosphorus?
then how do the first primer nucleotide comes in place , done by Dna primase?
He is very clever the way he explains it.
Wait, but when going from 3' to 5' instead, why did the terminal nucleotide not have three inorganic phosphates? If the preceding reaction was with the phosphate group of the preceding nucleotide and the hydroxyl group of the dATP (in the photo, dAMP), shouldn't the dAMP still be a dATP?
I am wondering that too. As far as i am concern, DNA Pol can´t work 3´ to 5´ because proofreading would terminate polimerization. So, evolution would have to choose between proofreading or polimerization 3´ to 5´. But without proofreading, there would be too many mutations...and (probably) no life.
VERY VERY HELPFUL AND WELL EXPLAINED, I FINALLY UNDERSTAND IT!!!!
I dont understand why there are no additional phosphates and magnesium co factor on the 5 prime carbon of the dntp in the reversendirection (3-5)...
Yes I understand that without these the reaction can only proceed in the 5-3 direction...
My question is therefore, why are the phosphates and mg co-factors for the reverse direction missing?
the dinucleotide triphosphates, and Mg cofactors are supplied by the DNA polymerase (not shown) so they won't be on the parent strand initially, just the incoming nucleotides.
The extra 2 phosphates are missing in 3' to 5' synthesis because the trisphosphate group spontaneously hydrolyses in aqueous conditions, so at some point you'd only end up with a single phosphate left that can't be attacked by OH, so 3' to 5' synthesis can't continue any further.
Spontaneous hydrolysis also happens to the free nucleotides, but when you're synthesising from 5' to 3' all you need to do is find another nucleotide that hasn't been hydrolysed yet.
One thing is really throwing me off. Your explanation is pristine but a chemical workaround to moving from 5'--3' seems fairly straightfoward, and I can't figure out why nature doesn't just apply it. It seems to me that a simple solution would be to attach a pyrophosphate group to the terminal 5' nucleotide of the primer in order to make the reaction more thermodynamically favorable. Is this just the way nature has decided to carry out this process? If so, if I were to (in a lab setting of course), attach a pyrophosphate to last nucleotide of the 5' end would I effectively "trick" DNA polymerase to proceed in a 3'--5' manner?
The second and fourth answers here should help. biology.stackexchange.com/questions/477/why-is-dna-replication-performed-in-the-5-to-3-direction
One explanation is that nucleoside triphosphates will readily hydrolyze in aqueous solutions, and placing the phosphate groups on the growing chain would be a waste were this to happen. But I question the validity of this; while ATP (as an example) does have a short half-life, DNA replication is much faster, and should outpace the hydrolysis of the hypothetical triphosphate end of the growing chain.
The other explanation, which I prefer, is that 3'-to-5' directionality prohibits proofreading. If the triphosphate from the chain were to be hydrolyzed, only for the new base to be determined incorrect, excision of this base would leave the chain a dead end, since there would not be another triphosphate group at the 5' end. The picture in the link illustrates this more clearly.
Although there are DNA repair mechanisms that may potentially address this (you can imagine that excision of a damaged dNTP in one strand would leave the 5' end of the downstream nucleotide without a triphosphate anyway, making it hard to insert a new undamaged dNTP), they require other enzymes that are more specialized for their tasks. As far as polymerase proofreading goes, 5'-to-3' is the only direction that can work.
excellent video! you saved me from exam!
BTW, in the end of the lecture, you said that the phosphates are too far from OH, so it is very difficult from reacting. Is it that far? I mean, those molecules are very tiny, and I think it should be easy for phospherates to go to the next OH attached on P. That way, bond should be made.
Are dNTPs used in RNA synthesis too? I know RNAs use ribose sugar where dNTPs are obviously made up of deoxy Ribose sugar. So in mRNA sequencing, is the substrate still dNTPs or just NTPs?
So in RNA synthesis, the nucleoside triphosphates have ribose in them, so they're called rNTPs rather than dNTPs.
Great thank you for the reply!
Since primers are made of RNA, would the 3' end of the primer have a OH group at Carbon 2???
Yes, and also at the 3' carbon. DNA polymerase will only add to the 3' carbon.
I FINALLY get this concept. thank you!!
excellent presentation and use of resources
It might be a combination of the facts that 1 the primer doesn't have a triphosphate group on its 5' end (and is thus energetically unfavorable compared to the 3' end) and 2 you would need an alternate DNA polymerase protein structure, which just hasn't evolved. Wouldn't you agree? So it's both evolutionary predetermined and energetically unfavorable and most likely evolutionary predetermined because it's energetically unfavorable.
oh and offcourse DNA polymerase wouldn't be able to continue after removing a mispaired nucleotide during proofreading since then there would be no energy left to continue.
@beccalishass 5' end of the INCOMING dNTP has a good leaving group (PP). If you're going to join 5' to 3', that means the incoming dNTP has to add on to the 3' end of the existing strand.
why is there only one phosphate not phospahtes on the top of strand when dna is synthesized from 3' to 5'? Is there other reaction that eliminates two phosphates?
저도 그 부분이 잘...^^;;
Thank you for the awesome video! You make it look easy!
Explanation clear as day !
but which side does okazaki fragments occured? the pyrimidines or the purines?
#please notice this
Had the replication been 3 prime to 5 prime,there would have been no need for the last 5 prime NTP that exists in the chain to not have a triphosphate group.
It could have been
5' pppApTpGpCOH 3' and then the incoming OH could very well have a pppA to attack.
Annu Aparajita Yes, I've considered that, but the ppp linkage is inherently unstable; if you had to leave one on the DNA all the time, it would rapidly degrade. While the nucleoside triphosphates, being just raw material for making DNA rather than the information storage molecule itself, can be synthesized as needed and used rapidly.
Yes that makes sense.This is also the explanation provided by Eric Lander.Another can be found here.
sandwalk.blogspot.ca/2009/04/head-growth-and-tail-growth.html
Just the first time it won't favor , but if somehow first dntp is attached then it will go
whereas my alevel mark schemes just says: DNA polymerase is specific and only complementary with the 5' end, and shapes of the 5' and 3' ends are different...
All true, but I think there's a deeper chemical reason, as I've suggested in the video.
thank you for the video for a better explanation!
Thank you very much professor! I'm a final year med student preparing for the USMLE Step 1 and I was confused as to the "why" of 5' to 3' replication. This was an excellent explanation.
Low-key Loving JK, I did not expect to find BTS here! Good to know we Army's take our curricular activities just as seriously, lol!
Deniane John Wow hello fellow army lol
Hi Allen, very good explanation. thank you. Please produce more like this, ideally with a better quality video. Looking forward! Thx
can u plz tell me wat is the exact role of rna primer in the process of replication?
To flag the point of origin. Without the RNA primer, DNApolymerase would not be able to attach itself to the DNA strand for replication. It's basically a 'flag' that specifies where to begin replication of DNA.
Excellent video! Intuition abounds!
Thank you for your answer. You answer completes the explanation of the video. I am sad how I spent 9 minutes watching the video and asked the same question cd4500 asked.
really helpful and interesting thank you
Thank to you for your teaching it's very brilliant and great i can understand this i said onemore time thank you very much Mr.Agathman
so in a nutshell, 5 prime end has a favourable leaving group (O) and therefore.........still a tad lost
Why can't every teacher break concepts down like this
Yeah, I like that explanation.
Fantastic explanation. Thank You!
Excellent
Can anyone simplify this to AS know how?
Pleas can your write 📝 the sol
So it's an SN2 mechanism right?
Yes
Fantastic video. Thank you!
@AM loki If anyone feels I am wrong, do feel free to correct me for I am also a student., but I'll attempt to explain.
great vid, nicely explained.
Impressive explanation .. !
Wow thank you so much, liked and subscribed
beautifully explained.
You the man!
Brilliantly explained :)