7
$\begingroup$

The recent question about forward vs. reverse strand got me thinking about directionality conventions in synthetic biology.

As noted in the answer to that question, if we consider only DNA in isolation, the strands are symmetric and there is no reason to prefer one over the other. A great deal of work in synthetic biology, however, is done on small plasmids, and there is definitely a directional context for these systems: the origin of replication.

The origin of replication is typically separated from the insertion point in an engineered vector, but the replication process is directional and goes around the whole plasmid. I am thus thinking that there may in fact be a difference in behavior caused by the interaction of transcriptional activity and the replication process, especially for a high-copy plasmid, where replication is much more highly active.

Does anybody know if this is, indeed the case, and to what degree is it worth being concerned about?

$\endgroup$
9
$\begingroup$

I like this question, and I had a similar thought when reading that "Forward or Reverse Strand" post. I've since found a 2005 publication by Mirkin and Mirkin1, which investigates the interaction between the replication fork and transcription machinery in context of an E. coli plasmid.

An excerpt from the abstract:

Studying Escherichia coli plasmids, which carry constitutive or inducible promoters in different orientations relative to the replication origin, we show that the mutual orientation of the two processes determines their mode of interaction. Replication elongation appears not to be affected by transcription proceeding in the codirectional orientation. Head-on transcription, by contrast, leads to severe inhibition of the replication fork progression.

To break this down, replication in E. coli proceeds at approximately 20 times the rate of transcription.2,3 From this, there are two kinds of collisions possible: (1) a "head-on" collision where replication and transcription complexes proceeding in different directions meet, or (2) a "co-directional" collision where the faster replication complex catches up to the slower transcription complex proceeding in the same direction. Mirkin and Mirkin find that co-directional collisions have little effect on the procession of the DNA polymerase III holoenzyme, whereas head-on collisions cause replication to stall.

One can clearly see that when transcription from the P7 promoter is codirectional with replication, replication was not affected (Fig.3A). Head-on transcription, in contrast, imposed severe constraints on the replication fork progression (Fig.3B).

Altogether, it is a gem of a paper, and I suggest reading the full discussion to best understand the limitations of their experiments and their caveats in interpreting the results. (e.g. the different rates of procession of DNA Pol I and III, and the frequency of polymerase switching.)


References

  1. Mirkin EV, Mirkin SM. Mechanisms of transcription-replication collisions in bacteria. Mol Cell Biol. 2005 Feb;25(3):888-95.
  2. Gotta SL, Miller OL Jr, French SL. rRNA transcription rate in Escherichia coli. J Bacteriol. 1991 Oct;173(20):6647-9.
  3. Hirose S, Hiraga S, Okazaki T. Initiation site of deoxyribonucleotide polymerization at the replication origin of the Escherichia coli chromosome. Mol Gen Genet. 1983;189(3):422-31.
$\endgroup$
2
  • $\begingroup$ That's a fantastic find! $\endgroup$ – jakebeal Apr 3 at 17:20
  • $\begingroup$ Nice paper! Strange though that it doesn't mention how such collisions affect plasmid copy number and gene expression levels (quantitaviely speaking). These would be relatively easy things to measure, especially compared to the assays they did do. $\endgroup$ – gaspanic Apr 3 at 20:40

Your Answer

By clicking “Post Your Answer”, you agree to our terms of service, privacy policy and cookie policy

Not the answer you're looking for? Browse other questions tagged or ask your own question.