Re: what happens to the hydrogen during protein synthesis?

Question: what happens to the hydrogen during protein synthesis?
From: Brunetta
Grade: undergrad
City: No city entered., State/Prov.: No state entered. Country: UK
Area: Biochemistry Message ID Number: 1069780324.Bc

what happens to the hydrogen (which helps to form  bonds between
base pairs), when the bonds are broken for transcription (during
protein synthesis).

Hi Brunetta,

The process of transcription involves the copying of a DNA sequence into RNA by RNA polymerase, during which the double-stranded DNA must be separated so that the polymerase can identify and select the appropriate RNA building block to add in the growing chain. Translation is the process of protein synthesis where information encoded in mRNA is translated into an amino acid code by the ribosome, the chemistry of which involves transferring individual amino acids from tRNAs into a growing chain of amino acids called a polypeptide chain. I read your question a couple times and am not completely sure what you're studying at the moment, transcription or translation, and whether we're talking about the hydrogen bonds that hold individual base pairs together in DNA and RNA, or whether we're talking about the balance of hydrogen atoms in the chemical reaction that occurs in the ribosome, joining single amino acids together. So I answered both questions. :)

Hydrogen bonding and Transcription:

As I mentioned, the process of transcription involves the copying (transcribing) of genetic information encoded in double-stranded DNA into RNA. For a discussion of the differences between DNA and RNA, you should refer to a question posed by someone already to the madsci.org website:

How is DNA better suited than RNA to carry genetic info?

The actual process of copying the DNA sequence into RNA requires that the double-stranded DNA be first separated into the single DNA strands. One of the strands will be used as a template from which RNA will be synthesized, and the other DNA strand just hangs around until the job is done. Next, the new RNA building blocks, the individual ribonucleotides, form a single base-pairing interaction with the DNA strand before being attached to the growing RNA chain. In this way, each A, T(U), G, and C gets added in the proper order; the RNA that gets made is an exact mirror copy (like a negative image) of the DNA strand being used as a template, and is identical in sequence to the other strand of DNA.

Whether we're talking about simple organisms like bacteria, or complex species like mammals, the basics of transcription are the same. The transcription reaction is catalyzed by an RNA polymerase (so named because it makes a polymer of individual RNA nucleotides). Depending again on the complexity of the organism, this can be a single protein (as is the case in viruses and bacteria) or can be a mix of multiple different proteins each carrying out a separate part of the total transcription reaction. (For more details, feel free to refer to the textbooks at the bottom).

I casually mentioned that the first part of the process involves unwinding of the double-stranded DNA, the breaking of hydrogen-bonds that hold the individual base-pairs together. Hydrogen bonds are best described as a loose interaction between a hydrogen atom on one molecule and a lone pair of electrons on another molecule (generally we're talking about lone pair electrons on oxygen or nitrogen). Hydrogen-bonds are non-covalent, which means they are low energy and transient, similar to the ionic interactions that exist between common ionic salts. You'll recall from your basic chemistry books that a single positively charged cation (like Na+) will associate with a single positively charged anion (like Cl-), and that these interactions are easily broken (like when you add NaCl to water and the crystals rapidly dissolve. In much the same way, hydrogen bonds are readily formed and broken; this is most apparent in a simple solution of H2O. The structure of H2O has two sets of lone-pair electrons, each capable of acting as a "hydrogen-bond acceptor" with another H2O molecule. The tetrahedral geometry of the two lone pair electrons and two hydrogen atoms around the oxygen are also maintained when two H2O molecules are H-bonded (in other words, the two oxygen atoms, the hydrogen and the lone pair electrons are all in a linear orientation). In this rough schematic (you'll have to imagine what the proper tetrahedral geometry looks like), the molecule on the left is the "hydrogen-bond donor" and the one on the right is the H-bond acceptor:

        ..         ..
       :O - H - - :O - H 
        |          |
        H          H

The curious thing about H-bonds is that the hydrogen atom actually tends to wander towards the neighboring molecule, becoming partially shared between the two water molecules in this example. (In technical terms, the hydrogen is partially abstracted from one of the molecule). So the reality is closer to this equilibrium, with the hydrogen living somewhere in the middle:

        O - H - -  O - H    < == >   O(-) - -  H - O(+) - H    
        |          |                 |             |
        H          H                 H             H

I want to emphasize that, though I've left off the lone pair electrons to simplify things, they are still present on both molecules; the result of transferring one hydrogen means the winner takes on a positive charge and the loser here takes on a negative charge. In reality, the equilibrium is shifted towards the left (the hydrogen mostly sits on its original oxygen molecule) and has a partial tendency to shift to the neighboring molecule. This partial shared character gives the H-bond interaction its strength and a defined orientation and distance between the two molecules.

Put another way, the reaction in equilibrium is: 2(H2O) < == > OH^- + H3O⁺

This is basic acid-base chemistry of water; the consequence for biological reactions is that anytime a reaction mechanism needs either a H+ or a OH- added or subtracted, the fact that the reactions occur fully surrounded by water provides an almost infinite source, or a sink, for extra hydrogens and hydronium ions.

The relevance to DNA, RNA, and transcription is that the two strands of DNA are held together by hydrogen bonding interactions between opposing strands. Note that every hydrogen atom that is attached to an oxygen or a nitrogen is capable of forming a hydrogen bond, and generally does so very readily with water. When two DNA strands come apart each nucleotide base is more than happy to satisfy its hydrogen bonding interactions with bulk water. The extra energy that is gained in a double-helix structure (from base stacking interactions) favors the double-stranded form of the DNA over that in water. In terms of hydrogen bonding interactions, they are satisfied in both the single stranded form (by H-bonding with water) and in the double-stranded form (by H-bonding with the other DNA strand). The same is true of RNA, though the structure of RNA is a bit more complicated.

Translation and the mechanism of peptide-bond formation:

I also thought your question might be concerned with the process of ribosome-mediated translation of mRNA and the chemistry associated with attaching one amino acid to another. The answer to this question is relatively straight forward, but the effort required by researchers to arrive at the answer is truly staggering; that's why I've decided to give you a brief historical perspective on the study of peptide bond formation and ribosome function before giving you the punch line.

Understanding the mechanism by which peptide bonds are formed in living organisms is one of the broadest and most storied histories of modern biomedical research. The basic reaction of linking one amino acid to another is referred to as peptide bond formation:

       R1                     R2                  R1          R2
       |                      |                   |           |
 - N - C - C = 0 + H - N - C - C = O  =>  - N - C - C - N - C - C = O + H2O
   |       |             |       |            |       ||  |       |
   H       OH            H       OH           H       O   H       OH 

The net balance yields a single bond between the two amino acids plus one molecule of water, with the -OH coming from the carboxyl group of the first amino acid and the second hydrogen coming from the nitrogen of the other amino acid.

It has been known for almost 40 years that the chemical activity giving rise to peptide bond formation during messenger RNA (mRNA)-directed protein synthesis can be attributed to the large ribosomal subunit. It has been known for even longer that the ribosome is comprised of both protein and RNA components. (In bacteria, the large ribosomal subunit contains ~35 different proteins and two RNAs; mammalian ribosomes are bigger and even more complex). The amino acids are carried into the ribosome covalently attached to tRNA; each tRNA associates with the mRNA (using three RNA bases per amino acid), with each tRNA adding another single amino acid to the growing chain of new protein. So the equilibrium above is a simplification; the amino acids each are already covalently attached to their own tRNA molecule:

       R1                     R2                  R1          R2
       |                      |                   |           |
 - N - C - C = 0 + H - N - C - C = O  =>  - N - C - C - N - C - C = O +  OH
   |       |             |       |          |       ||  |       |        |
   H       O             H       OH         H       O   H       OH       tRNA1
           |                     |                              |
           tRNA1                 tRNA2                          tRNA2

Note that the balance of the ribosome mediated reaction is different from that of the previoius schematic; the hydrogen from the NH2 of the second amino acid gets transferred to the tRNA which leaves with the extra oxygen from the first reaction. (This is shown in detail in the figure below). The tRNA coming into the ribosome in the first cycle of the reaction becomes the carrier of the growing peptide chain in the next cycle of the reaction. Understanding which of the macromolecular components of the large ribosomal subunit contribute to its peptidyl transferase site (where the next amino acid is added in the process), where is that site located on the ribosome, and how the whole process works has taken a much longer time to develop.

By 1980, the mystery been reduced to about a half dozen proteins and the 23S rRNA as being the possible catalytic center for ribosome function. In the early 80's two groups (researchers in the lab of Dr. Thomas R. Cech at the University of Colorado and the lab of Dr. Norman R. Pace at the University of Indiana) made the remarkable discovery of catalytic active RNA in two different types of systems; catalytic RNA is the term given to RNA that has enzymatic activity, carrying out a bond cleavage reaction without the assistance of proteins in . At the time, this was a revolutionary discovery (for which Dr. Cech, but not Dr. Pace, unfortunately, received the Nobel Prize in Chemistry in 1988). These were RNA molecules that could break bonds, and it wasn't long before the idea was revived that 23S rRNA might be the functional component of the ribosome, where RNA would be the molecule

In 1984, Dr. Harry F. Noller and colleagues at U.C Santa Cruz, using an RNA-labeling experiment, showed that a highly conserved region (domain V) in the center of 23S rRNA was intimately involved in the peptidyl transferase activity. This was also supported by the observation that mutations in that RNA loop in living E. coli cells renders them resistant to many inhibitors of peptidyl transferase activity. The evidence implicating the 23S rRNA as being the catalytic center of the ribosome continued to mount.

In 2000, the crystal structure was solved of the complete ribosome from an archaebacterial organism by the group of Dr. Thomas A. Steitz at Yale, offering the first atomic level view of a functional ribosome. The two seminal publications for which Dr. Steitz will likely receive the Nobel Prize some day were published in the journal "Science":

1)  Nenad Ban, Poul Nissen, Jeffrey Hansen, Peter B. Moore, 
     Thomas A. Steitz, Science.(2000) v.289(5481):905-20.
     "The Complete Atomic Structure of the Large Ribosomal 
     Subunit at 2.4 Å Resolution "

2)  Poul Nissen, Jeffrey Hansen, Nenad Ban, Peter B. Moore,
     Thomas A. Steitz, Science.(2000) v.289(5481):920-30.
     "The structural basis of ribosome activity in peptide
     bond synthesis."

In the second of these two papers, the detailed catalytic mechanism of RNA-mediated peptidyl bond formation is finally elucidated. (Incidentally, the process of determining atomic level structures by crystallography is EXTREMELY difficult; the Herculean effort required to yield the first real structural detail of a functional ribosome can be literally counted in hundreds of man-years within Dr. Steitz's group alone, in addition to the efforts of scores of other scientists in addition to the ones I've already mentioned). The reaction is indeed catalyzed by the RNA component of the ribosome:

So how does the ribosome actualy perform the chemical reaction? In the mechansim proposed by Dr. Steitz's group, one of the nucleotides on the big rRNA, A2486, is thought to have an equilibrium between an unprotonated N3 and a neutral protonated N3. If you take a look at the figure I've uploaded along with this response, shown in panel (A) of the figure taken from the Nissen article [Science.(2000) v.289(5481):920-30], "the N3 of A2486 abstracts a proton from the -NH2 group as the latter attacks the carbonyl carbon of the peptidyl-tRNA". In panel (B), "the protonated N3 stabilizes the tetrahedral carbon intermediate through hydrogen bonding to the oxyanion". Finally, in panel (C) "the proton is transferred from the N3 to the peptidyl tRNA 3' OH as the newly formed peptide deacylates." The balance is no net change in the number of hydrogens, just a rearrangement of bonds. The driving force for the reaction comes from another source (hydrolysis of ATP), which is a topic for another day. Again, we see an important role of hydrogen bonding in carrying out these rather elegant chemical reactions.

For additional reading on either transcription or translation, check out the following textbooks, or refer to any introductory level biochemistry or molecular biology textbook:

     "Biochemistry" 4th ed., Lubert Stryer, New York: W.H. Freeman,
            QP514.2.S66 1995

     "Genes VII", Benjamin Lewin, New York: Oxford University Press,
            QH430.L487 1999