Login

Join for Free!
114655 members


Generation of DNA nucleotides

Discussion of all aspects of biological molecules, biochemical processes and laboratory procedures in the field.

Moderator: BioTeam

Re: Generation of DNA nucleotides

Postby gs99 » Tue Jan 12, 2010 5:01 pm

(Edit 1/15/10. There was a post that originally appeared before this one, from "rania".
It was later recognized as a bot and therefore deleted. See 1/13/10 post by mith)

Rania wrote:Why don't you just buy these?

I'm interested only in the interaction of DNA Polymerase with nucleotides.

All the diagrams show sequential processing, where DNA Pol:
(1) "reads" the nt on the template
(2) determines the complement
(3) "obtains" a copy of that complement nt molecule (as a neucleoside triphosphate)
(4) attaches the new molecule to the new DNA strand
(5) proceeds to the next nt on the template
In step 4, it appears that two phosphates are removed from each new nt molecule and hydrogen bonds are added.
http://en.wikipedia.org/wiki/File:DNA_polymerase.svg

Any of that incorrect?

Being a computer programmer, I can easily visualize "sequential processing".
But - not being a chemist - I'm looking for the operational mechanics.
How does DNA Pol "obtain" the appropriate nt molecule?

I'm aware of the four "natural forces". Are there additional forces at play here?

The answer seems to preclude any other proteins, since this part of DNA replication works in PCR where known objects are included.

ps. to Rania,
Thanks for your reply,
Rania wrote:Let us know when you figure it out.

but everything that followed the first occurrence of that sentence was completely over my head.
At least I couldn't see that it applied to my question.
Welcome to the forum!
Last edited by gs99 on Fri Jan 15, 2010 4:07 pm, edited 1 time in total.
gs99
Garter
Garter
 
Posts: 35
Joined: Thu Dec 31, 2009 2:48 pm

Postby kolean » Tue Jan 12, 2010 11:59 pm

You definitely need to know about chemistry, especially organic chemistry. There are covalent bonds, such as the bonds for the amino acids to form a protein. This protein then has amino acid side chains that will stick out of the surface of the protein (globular especially) and some that will be inside of clefts, and perhaps a metal ion will also be inside. Now when the protein is specialized for a function, like the DNA pol, it has specific placement of the amino acid side chains, so that there will be certain forces to attract specific charged/uncharged molecules to the function it is to do. The complementary binding of the nucleotides A to T is not covalent binding (sharing of an electron), but opposite attraction in hydrogen bonding.
http://upload.wikimedia.org/wikipedia/c ... ir.svg.png
this shows the hydrogen bonding: hydrogen has a positive force, while the oxygen has a negative force, and since they are opposites, they attract to each other. Though not enough to swap electrons and bond covalently.

And because there is no covalent bonding (though the phosphate backbone will covalent bond to the next nucleotide incorporated into the strand), it has to be guided to the right space of forces around it, and has to be especially complementary (correct hydrogen bonding to the correct complementary base) bound before that catalyzed reaction of nucleotide addition takes place.
kolean
Coral
Coral
 
Posts: 345
Joined: Thu Jun 25, 2009 3:15 am

Re: Generation of DNA nucleotides

Postby gs99 » Wed Jan 13, 2010 3:23 am

kolean wrote:You definitely need to know about chemistry, especially organic chemistry.

Agreed!

kolean wrote:Now when the protein is specialized for a function, like the DNA pol, it has specific placement of the amino acid side chains, so that there will be certain forces to attract specific charged/uncharged molecules to the function it is to do.


Awesome!

I assume each side chain operates by a specific force to do a specific activity, is that correct?
So a specific side chain doesn't do multitasking?
I didn't find any details for this. I'm remembering they are a collection of atoms...

I'm guessing these steps that need to be done by the side chains:

A moves the DNA pol to the beginning of the template
(or the DNA pol attracts the beginning of the template) (Either way, this is the first "attraction" to get things rolling)
B maintains proper closeness to the template
(These objects are floating in nucleoplasm; there are no handrails to hold as astronauts have on a space walk)
C reads the template nt, and gives message to
D that determines what is the complement, gives message to
E that turns on forces to attract a complement neucleoside triphosphate floating in the area
(Or possibly E has additional parts for each type: Ea, Et, Ec, and Eg)
F grabs the new molecule and positions it for an "operation"
G removes two phosphates
H guides the molecule to the right space of forces around it, so covalent bonds can be made at the backbone end
I attracts the proper hydrogen atoms to connect the two nt molecules
J grabs the template somehow and moves it to the next nt

The side chains are on different sides of the DNA pol
http://upload.wikimedia.org/wikipedia/e ... TaqPol.jpg
so I assume the forces are exerted from various sides.
This may sound simple, but doesn't that complicate handling of the molecules?

Some of these steps are cell-like (eg. sending messages), but how else do these simple side chains work with each other?

Kolean, is this what you meant?
Thanks again for your direction.

ps, My Voet "Biochemistry" book is in. I'll be taking some time to review it.
gs99
Garter
Garter
 
Posts: 35
Joined: Thu Dec 31, 2009 2:48 pm


Re: Generation of DNA nucleotides

Postby JackBean » Wed Jan 13, 2010 8:52 am

gs99 wrote:The side chains are on different sides of the DNA pol
http://upload.wikimedia.org/wikipedia/e ... TaqPol.jpg

Which side chains do you mean? Do you realize, that on the picture is only the C-alpha backbone and no amino acid's side chains?

Look to the Voet, read it carefully from the very first page. After will you have some insight into basic chemistry and biochemistry come back and ask ;)
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.
User avatar
JackBean
Inland Taipan
Inland Taipan
 
Posts: 5665
Joined: Mon Sep 14, 2009 7:12 pm

Re: Generation of DNA nucleotides

Postby gs99 » Wed Jan 13, 2010 6:42 pm

JackBean wrote:Which side chains do you mean? Do you realize, that on the picture is only the C-alpha backbone and no amino acid's side chains?


This page indicates that all amino acids have side chains.
http://en.wikipedia.org/wiki/File:Amino_acids.png

Kolean had said (DNA pol) "has specific placement of the amino acid side chains, so that there will be certain forces to attract specific charged/uncharged molecules to the function it is to do."

I got the impression that the "certain forces" relate to the side chains; perhaps I'm mistaken.

Are there different definitions of "side chains"?

At any rate, I was envisioning the various steps done by the DNA pol enzyme, not taking anything for granted. In slow motion, recognizing everything that it does.

Can anybody reading this (with advanced knowledge of biochemistry) explain in common language how DNA pol performs the steps I mentioned?

If you can explain it, should you be entitled to a Nobel prize?
gs99
Garter
Garter
 
Posts: 35
Joined: Thu Dec 31, 2009 2:48 pm

Postby JackBean » Wed Jan 13, 2010 9:45 pm

I know, that all AAs have side chains, but I'm asking, whether you know, what are these side chains, as you're talking about them and refering to picture, where they are NOT.
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.
User avatar
JackBean
Inland Taipan
Inland Taipan
 
Posts: 5665
Joined: Mon Sep 14, 2009 7:12 pm

Postby kolean » Wed Jan 13, 2010 11:25 pm

Check this out:
http://en.wikipedia.org/wiki/File:Proteinviews-1tim.png
This is the same enzyme/protein, but biochemists show it different ways. The last one is the one I was talking about. See how the blue is for the side chains that have a basic charge, and red is for acidic side chains, and polar is green, and nonpolar is white, and these are only on the surface (that is why it is called a surface view) and that is what can interact with other proteins. But not only do you have the different charges from the side chains, you also have the shape and the space the amino acids side chains occupy and don't occupy. I like to think of it as a collosal puzzle with many tiny pieces that all fit together somehow, and it is not static - but moving also!
oh yeah, check out the diagrams of amino acids and their side chains:
http://en.wikipedia.org/wiki/File:Amino_acids.png
(and that is a backbone diagram of amino acids. you'll have to know that there are hydrogens missing, and every intersection has a carbon atom there).
kolean
Coral
Coral
 
Posts: 345
Joined: Thu Jun 25, 2009 3:15 am

Postby mith » Wed Jan 13, 2010 11:39 pm

sorry, rania is a bot. post has been deleted.
Living one day at a time;
Enjoying one moment at a time;
Accepting hardships as the pathway to peace;
~Niebuhr
User avatar
mith
Inland Taipan
Inland Taipan
 
Posts: 5345
Joined: Thu Jan 20, 2005 8:14 pm
Location: Nashville, TN

Re: Generation of DNA nucleotides

Postby gs99 » Thu Jan 14, 2010 1:21 pm

mith wrote:sorry, rania is a bot. post has been deleted.

When I first read that post, I wondered about the content.
From the initial question - Why don't you just buy these? - I didn't know where it was coming from.
And then certain things that followed, the sentence that was repeated and other verbiage that seemed to be from other discussions.
The appearance was something not edited by a normal person.
I did make a comment. If I understand what a bot is, I feel better now. I had been concerned about hurt feelings.
Thanks for your efforts.
gs99
Garter
Garter
 
Posts: 35
Joined: Thu Dec 31, 2009 2:48 pm

Re: Generation of DNA nucleotides

Postby gs99 » Thu Jan 14, 2010 2:21 pm

kolean wrote:See how the blue is for the side chains that have a basic charge, and red is for acidic side chains, and polar is green, and nonpolar is white, and these are only on the surface (that is why it is called a surface view) and that is what can interact with other proteins.
JackBean wrote:I know, that all AAs have side chains, but I'm asking, whether you know, what are these side chains, as you're talking about them and refering to picture, where they are NOT.

From my observation, it seems there is a difference of opinion here.
I'm not going to focus on that, because I don't have the training to understand it today.

But I have come to a conclusion about my questions and would welcome your comments.

This morning, I came to this comparison of chemistry and biochemistry. This is not something I've seen in a book or on a web page.

CHEMISTRY
Heating the PCR tube to 95c to separate the DNA double helix.
I'm guessing that no matter what is in the PCR tube, hydrogen bonds will be dissolved, including those in DNA.

BIOCHEMISTRY
The enzyme Helicase (along with other enzymes etc.) is sent to a specific point in the DNA.
The cell knew it would be needing these proteins, so it generated them ahead of time.
At the correct time, these proteins are activated and move to where they are needed.
With no change of temperature, Helicase proceeds to separate the DNA in its own way.
That's just one small (but very important) role in DNA replication.

My conclusion is that proteins have "micro-intelligence" that activates whatever forces they have.
The Amino Acids are made of ordinary atoms.
A chemist could mix these elements in a zillion ways, changing temperatures etc. and they still couldn't do anything special.
But combine them in the special patterns of a protein, and things happen!
They can move. They can read. They can change other molecules. With "military precision".
Cells are viewed as the smallest living thing.
But the animations produced by HHMI and others portray proteins (and other components within the cell) acting as if they were alive.
Proteins made of molecules, made of atoms.

If this were a movie, the credits would take hours.
gs99
Garter
Garter
 
Posts: 35
Joined: Thu Dec 31, 2009 2:48 pm

Re: Generation of DNA nucleotides

Postby jonmoulton » Thu Jan 14, 2010 5:11 pm

gs99, invoking "micro-intelligence" isn't needed to explain the arrival of activated nucleotides during replication. The Polymerase doesn't "send out for" the right nucleotide triphosphate; instead, random nucleotide triphosphates bump into the template nucleotide. If the nucleotide triphosphate hydrogen bonds correctly with the template nucleotide, the new phosphodiester linkage forms. If the nucleotide triphosphate is the wrong one then usually it will not slip into the correct orientation to form the new bond, so it is quickly displaced and another random activated nucleotide slips into position at the polymerase active site and bumps against the template nucleotide. Eventually (in a tiny fraction of a second) a correct nucleotide is fitted and reacted and the polymerase advances. If a mistake is made (and this sometimes happens), it is almost always caught by the "proofreading" function of the polymerase and corrected. If not, a mutation has occurred (a rare event). The process is driven by diffusion and thermally-driven shaking and collision, not any form of intelligence on the part of a protein. If you put a thousand Lego blocks in a sack, roll it around and dump out the blocks, some of them will have fit together. It is really a similar process, with greater selectivity about which parts fit together and happening at the blazing speed of molecular interactions.
User avatar
jonmoulton
Viper
Viper
 
Posts: 423
Joined: Fri Feb 15, 2008 5:38 pm
Location: Philomath, Oregon, USA

Re: Generation of DNA nucleotides

Postby gs99 » Fri Jan 15, 2010 4:33 pm

jonmoulton wrote:another random activated nucleotide slips into position at the polymerase active site and bumps against the template nucleotide.

(1) Seems like a lot of "slipping into position" and "bumping" with incorrect molecules.
This results in a 75% failure rate. And worse if the nt triphosphates aren't consistently homogenized.
That doesn't sound good for an organism to survive.

(2) Why is randomness used here when it's not used elsewhere?
All that I've read indicates specific things happening, and happening in a specific sequence.
Do the proteins fold in random ways?
Do Single-strand binding proteins decide to appear randomly?
Does Primase operate randomly?

Various web sites describing the cell and proteins give a strong impression that randomness is not involved.
For example, this recent article had headlines:
"Scientists Uncover Role of Protein Critical for Activating DNA Replication",
http://www.sciencedaily.com/releases/20 ... ience+News)
and the article includes this comment:
"In multicellular organisms, the duplication of the DNA in chromosomes starts at multiple sites, called origins, within the genome.
For the genome to retain its integrity each time a cell divides, it's crucial that each origin "fires," or becomes active, just once and only during a timeframe in the cell cycle known as the S-phase.
A large number of proteins cooperate and interact with military precision to ensure this "once-only" condition."

jonmoulton wrote:The Polymerase doesn't "send out for" the right nucleotide triphosphate;

Many web sites like this one from University of Nebraska give the opposite impression.
In this case, it's explaining Transcription:
http://www-class.unl.edu/biochem/gp2/m_ ... ne_a2.html
"As RNA polymerase reads each nucleotide it brings in the complementary nucleotide and bonds them together forming the mRNA strand."
There are a lot of web sites apparently giving wrong information to the public!

jonmoulton wrote:The process is driven by diffusion and thermally-driven shaking and collision

http://highered.mcgraw-hill.com/sites/0 ... works.html
The end result of diffusion is a scattering of molecules.
How does that drive the appropriate nucleotide to where it's needed?
And what exactly is "thermally-driven shaking and collision"? (no results in Google)
Can you please explain these buzz words to the class?

jonmoulton wrote:invoking "micro-intelligence" isn't needed

See first definition: "to call for with earnest desire"
http://dictionary.reference.com/browse/invoke
I posted a question here about nucleotides in DNA replication, "calling for help", you might say.
I have an earnest desire to obtain answers; hoping some reader would volunteer information.
In preparing various posts, I never thought of the word "invoke", but I see that it could fit.
That's how a forum works. And I appreciate all the replies, especially those with references.

So what did you think of my comparison of biochemistry with chemistry?
You may know, I do not have training in either; just read books and web pages.

I thought myself clever in noticing that in PCR, DNA pol is the only protein.
Therefore, I was apparently incorrect in thinking that a separate protein "delivers" the nucleotide to DNA pol. In scientist fashion, I published the results.
(The reasoning came from Translation, where tRNA "brings" the Amino Acids to Ribosome, according to something I read somewhere. Has that been changed also? If it is done randomly with 20 some possibilities, what is the failure rate?)

Back to biochemistry being different than chemistry.
Atoms brought together in a special way. (synthesis of protein)
Working together. Signalling one another. Cooperating.
Can anybody argue that it doesn't happen?
How many times in this discussion has it been mentioned about how many proteins may be needed to accomplish DNA replication?
Which protein would you say is expendible?
Can a chemist with a masters degree prepare a mixture of these same elements to do the same work?

Perhaps we could call it "micro-works" - they do the work!

All living things - from bacteria to elephants - have "micro-works",
or whatever.
gs99
Garter
Garter
 
Posts: 35
Joined: Thu Dec 31, 2009 2:48 pm

PreviousNext

Return to Molecular Biology

Who is online

Users browsing this forum: No registered users and 4 guests

cron