I‘ve always been a helix-turn-helix kinda guy. That’s the way I roll.
My thesis research involved a couple of repressors – homeodomain proteins—that bound to DNA via helix-turn-helix DNA-binding domains. They controlled cell-type fates. Combinatorially.
(I also worked on HDAC-mediated transcriptional silencing, although at the time I was totally unaware that that was what I was working on. Awwwkward. That’s a story for another time.) For my post-doc , I was surrounded by people studying the co-operative binding and sequence-specificity of various helix-turn-helix proteins, and how they achieve transcriptional activation. There was some pretty seminal work done, to which I contributed precisely nothing. Not a productive post-doc, you could say.
Anyhoo, I never really paid much attention to Zinc fingers. Which was rather remiss of me, since the sort of modular DNA-binding activity that they have is pretty much ideal for building networks that regulate gene expression. But hey, grad students can be rather parochial.
Then our dearest Sal posted an OP here on Zinc fingers, playfully entitled “Giving Evolutionary Biologists the Finger!”
In it, Sal wrote some truly weird shit about the need to precisely cut and paste DNA sequences to make tandem duplications. That’s a topic that we have covered previously, ad nauseam.
However, in a novel flight of fancy, he wrote some moderately ignorant stuff about DNA binding: Sal reckoned that duplicating a Zn finger would “in general reduce its binding affinity”
Well, just from first principles, I could point out that he was wrong:
Well, I pointed out that if the protein is already functional as a transcription factor, the tandem repeat would in general reduce it’s binding affinity for the original Transcription Factor binding site, and worse the transcription factor starts binding to some other site in the genome! OOPS!
No Sal. The tandem finger would bind tighter and more selectively.
This did motivate me to do a quick google search for “Jonathan Miller” as I believed he actually discovered Zinc fingers and I remembered the guy, albeit vaguely. Imagine my shock when up pops a picture of my ex-girlfriend, credited with being the person who first demonstrated a Zn-finger protein (TFIIIA) binding to DNA (via DNAseI footprinting). I had no bloody idea at all. So I admit that I am coming at Zinc fingers from a position of ignorance. Pathetic, unforgivable ignorance, given my personal history. What a twit.
All this biography is to make the simple point that when I told Sal that adding an extra finger would generally increase binding, I was merely speaking from first principles. I had no clue about the actual data.
I even used a cartoon “grade school” example to illustrate how the binding affinities might be expected to change when a finger gets duplicated. I was just making stuff up, really:
Sal’s primordial uni-finger recognizes a cognate and nine off-target sites. The energies are, in order,
5, 4, 4, 3, 3, 2, 2, 2, 1, & 1.
The DNA that codes for the finger duplicates in frame, but imprecisely.
In general there will be no steric hindrance, so ALL BINDING ENERGIES WILL INCREASE.
Now they are :
7, 5, 5, 8, 6, 3, 3, 3, 2, & 2.
The selectivity has gone up (i.e. total range is higher). The new duo-finger binds to its target quite a bit better. Excitingly, it also binds to an off-target site much better — this second site is now the best binder!
This new, avid binding could be good, bad or indifferent for the cell’s fitness.
If it is bad, it gets selected out. Either the second site evolves, the duo-finger evolves, or the duo-finger goes extinct.
If it is indifferent, drift at the second site.
If it is good, then novel effing function just arose!
In all cases except the ‘goes extinct’ scenario, you have a new and improved Zinc finger protein.
But let’s step back for a moment and think about first principles, and the evolution of DNA-binding specificity.
With a C2H2-Zinc finger, you have this 28 amino acid motif that recognizes a 3 base pair partially overlapping sequence.
There’s the possibility of growing the specificity via tandem duplication and divergence.
Now the extent to which this is possible depends on a couple of things…
I want to emphasize at this juncture that all of these things are variable over a continuum. They are not dichotomous, but for simplicity of exposition we may sometimes talk as if they were. (It’s a bit like using teleological language…)
There’s the question of how evolvable the sequence specificity of a given C2H2 finger might be.
The unknown variable: how much of the binding energy comes from sequence-specific contacts (e.g. hydrogen bonds to the bases, ACGT), and how much of the binding energy comes from sequence-independent contacts (hydrogen bonds to the phosphate backbone, for example).
To the extent that sequence-specific contacts supply the energy, then the ligand-DNA interaction will be something of a frozen accident, and co-evolution may be challenging: the landscape is rather craggy and difficult to traverse, with steep-sided, more isolated peaks. To the extent that sequence-independent contacts supply the energy, then the ligand-DNA interaction will have some spare affinity that it can “waste” on sub-optimal sequence-specific interactions, and co-evolution can occur across a rolling foothills-type landscape.
There’s also the question of how useful, in general, adding another C2H2-Zn repeat might be.
What happens if we add another Zn finger (via unequal cross-over)?
To the extent that sequence-specific contacts supply the energy, then the effect of the extra finger will vary by site, and the additional binding affinity will be pretty much advantageous or not, IMMEDIATELY. The sequence space is still not easily navigable; it is something of a crap-shoot. To the extent that sequence-independent contacts supply the energy, then all of the original binding sites will see increased affinity, and the sequence-specific aspects can co-evolve over subsequent time: co-evolution can occur across a rolling foothills-type landscape.
This leads to an interesting idea.
If sequence-specific contacts are the dominant supplier of binding energy, then we would expect C2H2 Zinc fingers to have evolved to recognize a limited sub-set of all possible 64 triplets (the frozen accidents), and we would expect the variety of successful combinations of C2H2 ZnF repeats to be rather limited. (We might also expect organismal “complexity” to be limited too, but that’s getting into postdiction territory. That’s naughty )
If, on the other hand, sequence-independent contacts supply much of the binding energy, then we might expect Zinc fingers to have evolved to recognize the majority of the possible 64 triplets, and we would expect a much larger variety of successful combinations of ZnF repeats to be found, particularly in regulatory networks that arose via duplication.
We might expect the explosion of “complexity” to coincide with the wholesale duplication of ZnF proteins, but again, heh, post-diction territory. Let’s behave ourselves and not go there.
To answer colewd’s question,
Does anyone have experimental evidence that these sequences have flexibility? Are they highly preserved?
A five minute PubMed search led me to this paper. Najafabadi et al 2017
For a layman’s introduction, there is an accompanying “Highlight” article (like a News and Views article in Nature).
The take-home: in metazoans, but not in plants and fungi, Zinc finger proteins get a lot of their binding energy from sequence-independent contacts. In metazoans, but not in plants and fungi, fingers exist that bind to virtually all possible triplets. Special shout-outs go to opossums, lancelets and iguanas.
Thus, my cartoon example above probably represents a situation midway between plants and metazoans. In plants, there would be a bit more variation in the post-duplication binding energies (and it would be slightly less easy to evolve away from a “disadvantageous” binding interaction). In metazoans, however, the post-duplication binding energies would all be higher (utterly refuting Sal’s original point), there would be rather modest differences in the by-site increments, and it would be very easy to evolve away from a “disadvantageous” binding interaction. Given the Creationist tendency to focus on a particular small subset of chordates, I don’t expect they’ll find much solace in the green plant data. Of course, this paper might be wrong. What evidence can we bring to bear on this question? (Cutting and pasting paragraphs that you don’t understand from random papers does not constitute evidence, folks.)
These titles. What’s next, “cucking creationism”?
I said RELATIVE, not absolute. Your whole OP, as far as me, is a misrepresentation.
What’s the alternative, point mutations? And 13 sequences of 84 nucleotides at that. You didn’t cover the topic, you didn’t provide probability estimates of how 13 sequences in CODING regions would appear like this. Mini satelites are mostly in non-coding regions.
For Jock’s claim that what I said was wierd, the sequences in question were highlighted here:
http://theskepticalzone.com/wp/wp-content/uploads/2019/04/znf136_zfC2H21.png
stcordova,
Just a wee edit.
Wow, and Salvador doubles down on things I, and others, already explained a billion times. It’s has to be whatever he said because, well, he doesn’t understand the answers at all.
Salvador, you’re astoundingly obtuse. Sorry, but you being this unreachable is beyond comprehension. You have authentic mental issues. I sincerely tell you that you might need counselling.
No you idiot. The alternative is ectopic recombination, which ensures, merely by virtue of DNA complementarity, that each copy will be the same 84 nucleotides long.
Of course not you idiot. They did not appear by point mutations! Both DNA_Jock and me provided plenty of explanations as to how tandem duplications can happen by ectopic homologous recombination, with drawings and all, to try and get you to understand something that high-school students understand after just one attempt, when I explain it to them.
You don’t get it because you have no fucking idea of any of it. You don’t have the minimal understanding. I think that you don’t know that DNA is a double helix, or what double helix means, or that there’s complementarity between the two DNA strands, or what a strand even might be, or what complementarity as a word even means. I thinks that you don’t know that these molecules are dynamic and imagine them to be stones. I think you don’t know the difference between DNA and protein, and that you lack the slightest understanding of biochemical interactions. I think you use words that you don’t understand, hoping that nobody will notice.
So what? Ectopic DNA recombination doesn’t require a region to be non-coding, doesn’t require a region to be coding, doesn’t require a region to be a gene, doesn’t require a region not to be a gene. All it requires is DNA complementarity between the misaligned, recombining, regions.
Entropy,
You really believe what are writing here?
Yes, you idiot.
Of course you idiot.
You never had a fucking idea.
Uh-uh.
No
You are obtuse.
You have authentic mental issues. I sincerely tell you that you might need counselling.
Yes. Absolutely. The requirements to understand the many examples I and DNA_Jock used to try and help Salvador out of his ignorance do not require much basic knowledge of DNA. So, if Salvador didn’t lack such basic knowledge, then he would have to be dishonest to the point of not caring risking ridicule, as long as his intended audience, creationists as ignorant as himself, could not understand the examples either. I’m not kidding at all.
Looks like the other way around hoodoo, since you’d have to understand what’s going on before offering your “rebuttals.” If you understood, you’d know that you’re doing nothing but show off your ignorance, and that you’re stupid enough not to notice. You’re ridiculing yourself while pretending to make an example of moderation issues out of this.
I think Sal is wrong. At first glance, duplicating the finger should have little to no effect on binding. Perhaps it might have a slight tendency to concentrate the TF at the promoter.
If there was a second binding site that would drastically increase affinity, so one has to calculate the probability that that site would be present by luck in the right place 1/64? But non-cannonical sites can have some affinity and that and increased affinity could be selected for. (my very poor intuition tells me the seq spec vs non-spec binding doesn’t matter)
So duplicating a finger could have a range of possible effects, from no effect at all to increased affinity
Entropy,
From where I sit you don’t appear to be reading for comprehension and you are talking over Salvador. DNA Jock is trying at this point to converse. The ad hominem strategy you guys use it not very effective as most people here see through it.
RodW,
So maybe some experiments would help drive some consensus?
There have been many hundreds of experiments that I think would all support that.
I dont have the time to slog through the posts and all the comments but Sals only hope is to claim that the spacing between the fingers has to be extremely precise, but he’s fighting a loosing battle
Since most of what DNA_Jock wrote in this discssion that pertains to me is NOT the meaning of what I said, but rather arguments he incorrectly attributes to me (for whatever reason), I’m pretty much boycotting this discussion.
I mentioned in the passing that DNA_Jock got caught for the second time misrepresenting what I said. He had at least the courtesy to apologize the first time I called him out about degenerate zinc fingers.
I give him the space to assert he thinks he describes my arguments more accurately than I do (and I’m the one putting them on the table!).
But it’s pointless for me to get him to change his misrepresentation of what I said regarding RELATIVE affinity, not absolute affinity.
In any case, lest the readers think I didn’t successfully counter what he and Entropy have said, here is a sample addressing his main misrepresentation:
So, thanks any way to DNA_jock for the comments. It gives me input in what sort of misrepresentations and distortions of my claims will be made and thus I have a head start in dealing with the sort of garbage that will be possibly coming down the pike when I have to defend what I’ve been saying.
Bill Cole, if you have questions directed at me about this stuff, you know which thread to find me on.
I hope you’ll enjoy my mention of Vitamin D Receptor and Alternative Splicing in connection with the discussion of Zinc Fingers.
RodW,
How do you know?
Then, either you don’t understand the answers either, or you haven’t seen them. Have you seen them? If so, did you understand them?
So did I for quite a while, until I’ve got tired of Salvador’s inability to understand combined with his arrogant dismissals. Since he doesn’t understand he should not use that fucking triumphant tone. Yet, there he goes, arrogantly claiming that there’s no answers, yet showing that he did not understand them.
Once something is explained, he should not go back to his original bullshit. Yet, he goes on as if nothing was explained at all. Not only that, he adds bullshit that shows that he did not understand any of it.
This is not fair. I tried very hard to get Salvador to understand how ectopic/homologous recombination easily results in as many copies easily, all with “perfect” lengths. I tried several times before, and I tried several times in his latest OP. So, which ad hominem strategy are you talking about? After trying so hard, Salvador, instead of explaining where he gets lost, claims to understand, only to come back with even more claims that show, clearly, that he did not get it at all.
Again, it’s not ad hominem if Salvador is unable to understand one iota and demonstrates so. I insist: did you read the examples? If so, did you understand them? If you did, you’d be horrified that Salvador repeats things we already solved and explained to him too many times.
Why did you ask if I really thought that Salvador was ignorant of all of that if you were going to ignore my answer? I am truly convinced that Salvador doesn’t understand the most basic biology. Again, the only other option is that he doesn’t care as long as people like yourself are unable to notice it.
Neither is a good sign about Salvador. Neither would be a good thing about you, if you prefer not to check what we explained, and if you think that my answer to you wasn’t sincere either. It’s very offensive that you asked, I answered, and now you think that I lied to you. Sorry. I really think that Salvador doesn’t get the most basic biology. I’m not lying.
How you don’t know should be the proper question. Why don’t you know Bill? What have you missed?
Is this the new ID strategy? Play dumb until you frustrate the hell out of everyone, and then when they use some harsh language to point out your ignorance just focus on the insults so you don’t have to address the science.
That’s not new. That’s been the MO for evangelical religionists since the dawn of time. It’s part of their whole apologetic, to pretend to be morally superior in some way (often through faked courtesy, “hate the sin not the sinner” is the quintessential example), and then when people lose patience, spend eons emphasizing that one lapse of calm as somehow the intrinsic consequences of the God-hating sinful ways. It’s how their religion was forced to evolve when it became socially unacceptable to burn people for disbelief.
T_aquaticus,
This is the first post of Jock and one of the first 6 posts. You can judge if this was an ID strategy. From what I can tell is that Sal wanted editorial feedback. The ad hominem arguments were front and center before Sal got started answering questions.
colewd,
You are making Rumraket’s point.
That first comment of mine on the “Giving Evolutionary Biologists the Finger” thread contained four links to a conversation back in December 2017 wherein Entropy and I painstakingly explained to Sal exactly how he was wrong about the “precision” needed to generate tandem repeats.
Please review that conversation. If you have any difficulty understanding how Sal is utterly wrong, just ask.
Sal returns a year later with the same discredited argument, biologists point this out, and all you can do is tone troll? You are really making Rumraket’s point.
Absolutely and also to: “… find out what sort of falsehoods, misrepresentations, and other crap my critics might throw at it so I can better compose my essays.”
Glad to hear our feedback is appreciated!
Performance art should be done on Noyau, phoodoo. It is allowed on Moderation Issues too, I guess.
DNA_Jock,
Discredited how? By a counter argument? By an experiment?
Are you the arbitrator of truth in biology? You started here saying that your knowledge of zinc fingers was nominal at best and then you tried to say you salvaged your argument by understanding of first principles of protein interactions.
This is complete nonsense. You have no idea how a cell will react to changes in transcriptional binding unless you have done several experiments or can cite several experiments that support your claim. How many have you cited so far that specifically show how a change in binding affect the cell.
Sal is doing experimental work at the NIH now on this specific subject.
Is it just possible that he may be a little more up to date than you and Entropy?
Is it just possible that random mutation and natural selection may have a hard time explaining zinc fingers origin.
No, phoodoo, substantive content.
You are confusing two different errors by Sal. The argument that was discredited in 2017 relates to all tandem repeats, not just Zinc fingers. Which you would be aware of, had you bothered to look, because that error is an absolute howler, that you should be able to understand. Do you understand what “cross-over” is in DNA? Do you understand how DNA recombines?
Aha. I may have been too subtle. The point of this OP is that even a neophyte such as myself can spot that Sal’s “the tandem repeat would in general reduce it’s binding affinity for the original Transcription Factor binding site,” is obviously, demonstrably wrong from first principles
Now, there’s always the possibility that I was wrong with my “first principles” argument. Because I am shamefully unfamiliar with this area, I was genuinely worried that I might be wrong, and I would have to come back here and eat crow. BUT what the paper that I cite in this OP demonstrates is that I was correct about binding energies. Far more correct than I imagined, and (completely unexpectedly and fascinatingly) it is entirely possible that the reason metazoans are more complicated than fungi and plants (in particular their nervous systems) is BECAUSE of just how earth-shatteringly wrong Sal was about Zinc finger binding energies.
Is he? For real? I would love to see independent confirmation of this. Is he named on an approved NIH grant? He has a tendency to inflate his credentials and exaggerate the degree of support that he garners at the NIH.
For instance, you may have been mislead by his saying ” I was called upon to investigate research developments such as ENCODE and other projects at the National Institutes of Health.”
If he really were working at the NIH, that would make his incompetence all the more embarrassing.
Apparently not. He did not find Najafabadi et al 2017, but instead has been relying on older, off-topic citations.
Yes, it is possible. I encourage you to make the case, because Sal most certainly has not.
DNA_Jock,
Yes, and I also understand it well enough that there is a lot of uncertainty of exactly how recombination works. There is very little certainty in biology as we understand very little of it at this point.
Jock you are always adding spin to conversations by using labeling and other techniques. Why do you do this? Whats your end game?
I am attempting to understand his argument. I would encourage you to do the same. Your alternative is to continue to degrade the site you spend your time moderating.
You may be confusing two separate concepts here. Crossing over happens in vivo during meiosis. Recombinant DNA is produced in the lab.
colewd:
Alan:
Recombination happens through crossing over. In vivo.
Google recombinant DNA. This is different from genetic recombination.
keiths:
Alan:
Colewd was talking about recombination. It’s right there in his comment, Alan:
The confusion was yours, not his.
By explaining how recombination works and can result in tandem duplications when there’s duplicate sequences close to each other. This knowledge comes form years of experimental and observational work on molecular biology.
This was not about being an arbitrator of “truth,” but about understanding how some phenomena happen, the “how” including understanding of a very basic concept: DNA complementarity.
DNA_Jock didn’t say that he “salvaged” his argument, he said that he explained something based on first principles, then it happened to be confirmed in the zinc finger universe. The first principles are solid, they are generic, they do not need knowledge of zinc-fingers in particular, they just need understanding of some basic physical/chemical phenomena, more specifically those we re-learn in biochemistry.
Of course your misunderstanding sounds like non-sense, this is where, given that DNA_Jock has worked on molecular biology, you should have assumed that you misunderstood and asked DNA_Jock if you’ve got it right, rather than assume that your misunderstanding was right.
Given that DNA_Jock understand those first principles, and has worked on helix-turn-helix proteins (a motif frequently found in guess what? Transcription factors!), I’d think that DNA_Jock knows what happens to a cell under those circumstances.
Is he? Doesn’t look that way. He doesn’t even understand how homologous recombination happens. He insists on point mutations, as if ectopic recombination was some foreign word. Written in some ancient and obscure language. WHile I doubt that he’s working at NIH, if he is, maybe he’s doing experiments by following recipes but has no idea what’s going on. I’ve seen that. I’ve sent students to study first principles after noticing that they were doing their experiments with “kits,” but without understanding what was going on.
No, it simply ins’t possible at all. His lack of understanding of the most basic first principles makes the contrary point. What good would it be for Salvador to be up-to-date to the most current kits for some molecular biology, if he cannot understand what’s going on in physical/chemical terms? If he doesn’t understand why homologous recombination is called homologous? Without an understanding of first principles, no amount of modern kits and equipment will help anybody understand anything.
Random mutations and natural selection only explain some parts of the evolutionary history of zinc fingers. Seems like you, like Salvador, have a very hard time understanding that point mutations are not the one and only source of variants available for natural selection.
keiths,
DNA_Jock asked both about “cross over” and how DNA recombines. If he was asking the same question twice rather than asking about two separate concepts then I misunderstood.
ETA In any event “Yes, and I also understand it well enough that there is a lot of uncertainty of exactly how recombination works. There is very little certainty in biology as we understand very little of it at this point” is not accurate.
Alan,
Jock and colewd were talking about recombination. You were confused by that and brought in recombinant DNA, thinking it was the same thing. It’s not, as you have since discovered.
keiths,
Maybe.
Sorry, but this is just not true. We understand recombination enough to explain how tandem duplications can continue to grow (and shorten).
Lack of explanations at the quantum mechanical level is no excuse for Salvador’s inability to understand how ectopic recombination results in tandem duplications.
Quote-mining wikipedia/articles, like Salvador does, doesn’t compensate for the lack of understanding, it further demonstrates that the understanding’s just not there.
Both DNA_Jock and me understand Salvador’s “argument.” Salvador’s “argument” is a load of arguments from incredulity “supported” by poorly-if-at-all-understood molecular biology jargon. Seems like the jargon is there purely for rhetorical effect.
Alan,
Not “maybe”. You made a mistake, and I corrected you. It’s not a crisis (or at least it shouldn’t be). Accept it and move on.
keiths,
There are two concepts. If DNA_Jock was indeed only referring to genetic recombination during meiosis rather than the recombinant properties of DNA in general then I misunderstood. I’m sure Jock will confirm at some point.
Alan,
This is comical. (And by the way, your refusal to admit and correct mistakes is one of the things that makes you such a terrible moderator.)
Yes, and you confused the two. Colewd was talking about recombination, which is why he used the word “recombination”:
You confused recombination with recombinant DNA:
The confusion was yours, not his.
It’s not the end of the world, Alan.
keiths,
No, it’s no big deal.
So far, I seem to be seeing Sal say separately that tandem repeat will both diminish binding affinity for the target and increase it.
Allan Miller,
It’s that clear to you! 😱
Keiths is correct — I was talking about one concept, towit what happens in vivo: Recombination via cross-over.
Let’s see if colewd or Sal can complete the sentence
“The difference between gene conversion and cross-over depends ….”
using ten words or less.
Hint: you must use the word “junction”.
Ironically, recombinant DNA technology involves the sort of cutting and pasting operations that Sal thinks are required to grow tandem repeats, but which are in fact rather rare in vivo and ABSOLUTELY NOT how tandem repeats grow and shrink.
Although a few sad looneys have used homologous recombination to build novel constructs in vitro, or to screen genomic libraries.
Turns out yeast is really good at homologous recombination.
DNA_Jock,
Thanks for the correction. 😉
Chock full of content! Lots of words! Amazing points!
Oh, look at all the SUBSTANTIVE content! Blockbuster post, Jock must be so impressed!
Bravo!
Alan:
Heh. Alan can’t bear to acknowledge the person who actually corrected him.
Here’s an article that showcases the degree of polymorphism in the DNA-binding of human transcription factors (focus on HTH’s, but also looked at Zinc fingers)
Barrera et al, 2016: “Survey of variation in human transcription factors reveals prevalent DNA binding changes”
Hat-tip to Sal
DNA_Jock,
Nice article.