I'll start by saying I'm a total stranger to this field, so I'd love to be corrected if I misunderstood something along the way.

I'm writing a python code to identify all the reading frames in a sequence of nucleotides. To my understanding, a reading frame is defined by a start codon and a stop codon, which made total sense to me until I learned you can have nested/overlapping reading frames.

My question is:

Assuming the start codon "AUG", and the stop codon "UAA":


How do you identify the reading frames? Is each stop codon referring to a single start codon? If so, how can you tell to which one?

I tried looking this up and could only come up with articles explaining the effects of overlapping reading frames, rather than how are they interpreted. I would greatly appreciate even a nudge in the right direction.

Please let me know if I was not clear enough or if I got something wrong.


EDIT: After reading the answers here and researching further I learned that ORF identification on DNA sequences isn't as simple as finding a single start and stop codon, and is complicated beyond the scope of my question. I'll probably default to using online tools to do this from now on.

  • $\begingroup$ Hi @JayLeeman better to post the code. Simple answer is to translate from each start point and assess length of the exon against the number of stop codons, i.e. maximising the length of the exon. There are 6 possible reading frames, 3 forward, 3 reverse. If you have an idea of the structure of the protein, e.g. surface antigen you can assess GPI anchors and hydrophobic alpha helices as the end of the gene $\endgroup$
    – M__
    Sep 28 '20 at 11:07
  • $\begingroup$ There is no reason at all for an exon to have either a valid stop or start codon. If introns are in the sequence the whole enterprise is a waste of time. $\endgroup$
    – swbarnes2
    Sep 28 '20 at 23:11
  • $\begingroup$ @swbarnes2 This actually clears a lot out of me, thanks! Just clarifying: If I'm reviewing a DNA sequence, it still has introns. If there are introns, how can I locate the ORF's without being able to splice the introns "manually"? $\endgroup$
    – JayLeeman
    Sep 29 '20 at 5:57
  • $\begingroup$ You cannot programicly know where introns are. You can only make good guesses. Or, you have to look up what introns people have empirically determined in your sequence. $\endgroup$
    – swbarnes2
    Sep 29 '20 at 17:04
  • $\begingroup$ I'll look into that, sounds extremely interesting. Thank you! $\endgroup$
    – JayLeeman
    Sep 29 '20 at 19:05

There are 6 reading frames. Three forward, three in the reverse direction.

An Open Reading Frame, an ORF, is a sequence which begins with a start codon, and ends with a stop in the same frame. You find them by looking for sequences that fit that pattern.

Is each stop codon referring to a single start codon? If so, how can you tell to which one?

You could have two different transcripts for the same gene with different start codons but the same stop codon. But in general, the protein translation apparatus will not accidently read through a stop codon.

And yeah, I hope you are doing this for fun, because obviously people have already made publicly available tools to find ORFs.

  • $\begingroup$ Yes I am doing this fun, and looking at orf finders results at the same time to compare and figure out how it does what it does. I'm still having some trouble understanding: Given a single frame (out of the six) - I may encounter a frame with 100 start codons and 300 stop codons, and rest coding for amino acids. Can we actually locate the ORF's in a case like this? If so, these are the "rules" I'm looking for. Thanks for the reply! $\endgroup$
    – JayLeeman
    Sep 29 '20 at 6:04
  • $\begingroup$ If you want to know how ORF finders work, why don't you...look at how ORF finders work? $\endgroup$
    – swbarnes2
    Sep 29 '20 at 16:54

An RNA sequence has six possible reading frames depending on the offset (0, 1, 2) and the direction (forward, reverse complement). All frames can potentially be used for protein translation, but only one frame per protein (i.e. there is no shifting going on when translation is happening, excluding shifts introduced by splicing).

If you need to find all the possible protein sequences within an RNA sequence, there are plenty of programs already available (e.g. getorf and transseq from the EMBOSS tool kit).


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