I've a question about ORF and translation into protein. Say I have an RNA transcript that contains two ORFs, one in phase 1 ; one in phase2 ; as:

ORF number 1 in reading frame 1 on the direct strand extends from base 529 to base 759.

ORF number 2 in reading frame 3 on the direct strand extends from base 849 to base 1223.

So ORF number 1 comes earlier in the transcript, but ORF number 3 is longer ..

Is it always the first ORF that is translated ?

Edit >

  • It's in an eukaryoytic organism and same gene (it's the same transcript)

  • I use ORF finder (http://www.bioinformatics.org/sms2/orf_find.html) on my transcript sequence and it gives me two potential ORFs (ORF 1 and ORF 2 within two different frames)


  • 3
    $\begingroup$ This is impossible to answer. Are you talking about bacteria or eukaryotic cells? Are these different genes? $\endgroup$ – Chris Jun 30 '15 at 14:20
  • $\begingroup$ There are several parameters such as presence of proximal Ribosome Binding Site in prokaryotes or Kozak sequence in eukaryotes, secondary structures etc. $\endgroup$ – WYSIWYG Jun 30 '15 at 14:21
  • $\begingroup$ Please edit your question and clarify. The answer could be ORF1, ORF2 or both or neither depending on the details. Is this in a species that has introns? Is the RNA spliced? Mature? Are these the same gene? What exactly do you mean by ORF? Are they both terminated by a STOP codon? $\endgroup$ – terdon Jun 30 '15 at 15:17
  • $\begingroup$ Making this even worse, is this a viral RNA that separates ORFs by IRES? $\endgroup$ – user137 Jun 30 '15 at 22:11
  • $\begingroup$ An open reading frame is one that extends for some distance without containing one of the three translational termination codons. This has nothing to do with translation initiation. Translation in both prokaryotes and eukaryotes requires an AUG initiation codon. You don't mention if there are any in-frame AUG codons in your hypothetical transcript. Without this information nobody would be able to provide a meaningful answer. $\endgroup$ – mdperry Jun 30 '15 at 22:27

If both of the ORFs that the web tool predicted start with an AUG initiation codon, then the textbook answer is that the 5' cap on eukaryotic mRNAs is the first feature that is recognized by the translational apparatus, and the ribosome scans along until it finds the first AUG (it is a simplified description). However, if there are several AUGs near each other then the one that matches the best to the Kozak PWM (position weight matrix) will be preferred. The Kozak sequence can be approximated by: CCATGG which is also recognized by the restriction enzyme NcoI. Highly expressed genes tend to match most closely to the Kozak consensus.


The most common case like this is a prokaryotic mRNA, where polycistronic genes show up regularly. In that case the ribosomes are free to attach to the RNA and both genes are usually co-translated.

enter image description here

The proportion of translation between the two genes does vary depending on the affinity of the ribosome to the mRNA, which is partially determined by the ribosome binding site sequence.

If there is a lot of transcription activity on the mRNA, many ribosomes at once can translate any single ORF, as you can see in the micrograph above.


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