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I feel that I might have a complete misunderstanding here. If DNA has two strands, how does the machinery of RNA transcription determine which one to transcribe?

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I'll keep this short and simple. The direction of transcription (which determines which strand is used as the template) is controlled by the promoter, which is a region of specific DNA motifs at the 5' end of a gene. RNA polymerase binds to the promoter, which orients it on the correct strand and in the correct direction, after which it can proceed to transcribe the gene.

enter image description here

That great little animation is from this website.

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To add to canadianer's answer, in fact genes can be found on both strands of the DNA in most eukaryotic cells, in the sense that the sense and anti-sense strands are not always the same strand. The direction is therefore completely determined by the promoter. Furthermore, there are bidirectional promoters.

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  • $\begingroup$ This is interesting +1!! Can you add a reference to the sense/anti-sense being not confined to a particular strand? $\endgroup$ – AliceD Sep 25 '15 at 13:37
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    $\begingroup$ @AliceD I'm pretty sure that he/she is just referring to the fact that, along an entire chromosome (which has two strands), some genes will be found on one strand and some on the other. The terms sense and antisense are relative to a specific gene, not an entire chromosome. Sometimes, uncommonly, two genes will overlap on opposite strands at the same loci. $\endgroup$ – canadianer Sep 25 '15 at 14:30
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    $\begingroup$ @AliceD that would be pretty much any genome paper or undergraduate text book, really. For a visual overview, have a look at this. You'll see genes in both directions, so on both strands (the direction is given by the arrows in the lines representing the genes). $\endgroup$ – terdon Sep 25 '15 at 14:31
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    $\begingroup$ @AliceD good grief, when did not knowing something not directly related to your field become a reason to feel dumb? Did I miss a meeting? My PhD was on gene prediction and genome annotation, so this is my bread and butter. Just don't ask me to describe the Krebs cycle :) $\endgroup$ – terdon Sep 26 '15 at 12:36
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    $\begingroup$ @terdon I have a BSc in Biochemistry. I left the field a decade ago and went into neuroscience. Honestly, I have struggled with this since the first time I started learning about DNA. It all didn't make sense. I honestly thought there was one coding strand, and the other was just a backbone for chemical stability..:-/ This post has enlightened me for sure. It all fell in place. Just here. $\endgroup$ – AliceD Sep 26 '15 at 12:51

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