Why are proteins always synthesized from the N-terminus to the C-terminus? Can there be any “reverse” peptide-bond formation to synthesize proteins in the C-terminal to the N-terminal direction?

  • $\begingroup$ Are you talking about natural processes only, or also about chemical ones? Because chemistry really doesn't care (in either case actually) and can do both directions. Nature just stuck with the direction that was easier 'to implement'. $\endgroup$
    – Nicolai
    Jun 2, 2017 at 7:06
  • $\begingroup$ @nicolai I think the OP wants to know about natural processes (lest this question would be better for chemistry.SE). I tried to make the concept as clear as possible in my answer, yet it basically comes down to what you say ;) $\endgroup$ Jun 2, 2017 at 7:52
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    $\begingroup$ Actually 3′→5′ synthesis is possible … synthetically: news-medical.net/news/20120830/… $\endgroup$ Jun 2, 2017 at 12:25
  • $\begingroup$ I have edited your question to remove the irrelevant and incorrect preliminary statements. $\endgroup$
    – David
    Jun 7, 2017 at 21:18

2 Answers 2


Nice question! As you already know, DNA is always shown in 5'$\rightarrow$3' direction because it is always synthesized in this direction (amino acids are joined by CO-NH peptide bond). So, a polypeptide looks like this (source):


In fact, if you just look at the polypeptide in the reverse direction, you could view it in C terminus to N terminus direction. But we don't do so because that is not the conventional direction of the polypeptide's biosynthesis.

Tracing the Roots: To know why there is no "reverse" peptide bond (NH-CO), we first need to know how peptide bonds are formed in polypeptides. Polypeptides are formed in ribosome, and the process of formation of peptide bond occurs in the peptidyl transferase complex of ribosome. Since ribosome is a ribozyme, this reaction is also catalyzed by the catalytic sites of RNA (i.e. 2'-OH) instead of proteins. See the image below for the mechanism (from Marina V. Rodina):

peptidyl transferase mechanism

As visible from the diagram, nitrogen (in -NH2) from acceptor tRNA (A site) attacks the ester linkage at the peptidyl tRNA (P site). Carboxylic carbon cannot attack the nitrogen (for "reverse" bond formation) because it is already in ester linkage. One might ask then "if the nitrogen was joined to tRNA at P-site, the carboxylic carbon could have attacked on amino acid at A-site. Why is amino acid not joined to tRNA by the amine nitrogen?" To know why it is so, lets go a step further and see how tRNAs are charged i.e. how aminoacyl tRNA synthetase works. Aminoacyl tRNA synthetase charges tRNA in a two-step reaction. For displaying mechanism, I will take the example of histidyl-tRNA synthetase (diagrams from Proteopedia):

  1. amino acid + ATP → aminoacyl-AMP + PPi

    step 1step 2

  2. aminoacyl-AMP + tRNA → aminoacyl-tRNA + AMP

    charging tRNA

As is clearly visible now, whether amine nitrogen or carboxylic carbon will attach to phosphate of ATP is decided in the first step. See the image again:

step 1

You might ask that since nitrogen (in -NH2) also has a lone pair of electrons, it could also make a nucleophilic attack on the phosphate group, similar to the oxygen (in -COOH). Why doesn't this happen then? This cannot be accounted for by saying that the amine nitrogen is far away from the reaction site (look at the diagram again, and pay attention to how close the carboxylic carbon and amine nitrogen are. If we could move the amino acid even a little bit, this reaction could easily occur). Well, here's the twist: the optimum pH of this reaction lies near pH 6 i.e. in the acidic pH range (Lui et al, 1978). Now, as we know, under acidic pH, -NH2 exists in the protonated form i.e. -NH3+. In this condition, nitrogen lacks lone pair and thus cannot attack phosphate on ATP. Thus, it is not possible to attach amino acid to AMP via -NH2 group and, thus, form a "reverse" peptide bond. I hope this depth of the answer would be enough to satisfy your curiosity :)

  • $\begingroup$ Two comments. 1. You are explaining the directionality in terms of the way the activation of the amino acid occurs today. As long as you make that clear, that is fine. However the question of why it evolved that way is not answered. 2. Your arguments about ionization of amino groups at low pH do not hold water. The pH optimum of the charging reaction is irrelevant. What is relevant is the pH under which the reaction occurs, which is the cellular pH of, say 7.4–7.6. And if the amino group lacks a lone pair there, how does it manage to make a peptide bond, as per the first diagram? $\endgroup$
    – David
    Jun 8, 2017 at 20:24
  • $\begingroup$ @david 1. It evolved this way because evolving in the other direction was not possible (again, due to pH, I don't know if you understood what I wanted to convey through the pH factor) 2. I don't think you read Lui et al, the reaction does occur at a rather acidic pH (to make it faster). I don't know about peptidyl transfer reaction, most probably it occurs at cellular pH (about pH 7.5 or because the reaction occurs in almost isolated condition. Also, these are all suggested mechanisms, nobody has seen how the reaction actually occurs. $\endgroup$ Jun 9, 2017 at 5:26
  • $\begingroup$ All the stages of protein synthesis occur at the same pH in a cell. If a particular reaction requires an ionization state of a group in the substrate different from that in aqueous solution (e.g. an unionized amino group in the peptidyl synthetase reaction) that is provided by the enzyme environment. In evolutionary terms, when one assumes there was a non-enzyme catalysed reaction first, you still have a problem. And one unexplained assumption of your answer is that activation of amino acids for attachement to tRNA has to be via ATP (not acetyCoA or something else). $\endgroup$
    – David
    Jun 9, 2017 at 16:25
  • $\begingroup$ @david the truth is that we don't yet know the true reaction mechanism, I used histidyl-tRNA synthetase as an example. The main point is that the $\alpha$-amino nitrogen is in the protonated form; if the cell can't provide this proton, the enzyme must. I did not explain acetyl-coA or other residues since the major (and I think only) substrate for aminoacyl-tRNA synthetase is ATP (I don't know if you have studied about aminoacyl-tRNA synthetase, or maybe there are some other enzymes about which I don't know). $\endgroup$ Jun 10, 2017 at 4:09

This is just in addition to the other answers; some somewhat relevant thoughts I had.

Interestingly, even non-ribosomal peptide synthesis seems to proceed N to C terminal. Given the modular nature of such enzymes, synthesis in the reverse direction doesn't seem to be outside the realm of possibility, though I haven't found any known examples.

This might be a stretch as a counterexample, but if you consider the biosynthesis of the tripeptide glutathione, a γ-peptide bond is first formed between cysteine and glutamate, after which glycine is attached to the carboxyl group (ie C-terminus) of cysteine with an α-peptide bond.


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