...Oh my!

January 10, 2006 6:30 PM

Plasmids, prokaryotes, and markers...

I'm taking an engineering class and something came up in today's lecture that has bothered me since lab days.

Engineered plasmids often contain an MCS site, where there are 50-60 bp of restriction enzyme-compatible code for the insert. Yet the MCS often interrupts a coding sequence for a marker — in lab courses, this is usually, say, a LacZ marker.

In prokaryotes, mRNA transcripts are essentially whole. There isn't (correct me if I'm wrong) post-transcriptional processing of the variety we see in eukaryotes, including exon splicing.

In our prokaryotic vector, then, the MCS then seems to be part of the coding sequence for the LacZ marker code, since interruption signals an insertion event.

Is this MCS code translated into the final LacZ marker enzyme? It would otherwise seem to be "garbage" as far as functional protein code goes. Is it not nonsense code? Or is it excised in some way before or after the marker is transcribed or translated?

Disclaimer: This is not related in any way to any homework assignment. Sorry if this is an obvious question. Thanks in advance for informed answers.

posted by Rothko to Science & Nature (16 answers total)

Response by poster: (My first guess is that the MCS is clipped out of the protein post-translation.)
posted by Rothko at 6:34 PM on January 10, 2006

I *really* want to know what kind of engineering this is. I have better than rudimentary knowledge of mechanical (statics and dynamics and thermo), electrical and nuclear engineering, and yet I have NO idea what your questiono is.
posted by achmorrison at 6:43 PM on January 10, 2006

It's molecular biology, achmorrison, and the different ways DNA is turned into proteins in prokaryotes (simple, single-celled organisms like bacteria) and eukaryotes (mostly everything else, including us.) But my lab work is molecular ecology, so I can't be of much help, sorry Rothko.
posted by docgonzo at 6:48 PM on January 10, 2006

As I understand it, if the marker is in the Lac gene, the Lac gene becomes ineffective. The promoter region used to activate the Lac gene, however, now serves to activate the foreign gene you just spliced in.
posted by devilsbrigade at 6:51 PM on January 10, 2006

devilsbrigade has it right. The inserted sequence is expressed because of the Lac promoter preceding it. The Lac gene is not expressed so its absence becomes the marker. You can then tell transformed colonies because they will not turn xGal media blue (or whatever else it is you're using to show Lac-)
posted by TimeFactor at 7:14 PM on January 10, 2006

The "marker" is a translated protein enzyme product of the lacZ gene, namely beta-galactosidase, which breaks down a lactose analogue (X-gal) into galactose and a blue dye. If beta-gal is produced, then X-gal is converted into a blue dye, otherwise you're left with white X-gal "starch."

In the case of the lac operon, the "start" codon for the beta-gal protein occurs downstream of the MCS site so the MCS isn't transcribed. A rough diagram would be:

5' - Promoter -- MCS -- lacZ/beta-gal -- lacY -- lacA - 3'

Since the MCS itself is just "noise" nothing gets transcribed there. The action begins when the polymerase hits the "start" codon at the lacZ seq. Now if you insert a sequence into the MCS site with its own "start" and "stop" codons, then the polymerase transcribes your inserted seq and falls off before it reaches the lac sequences.

5' - Promoter -- M (inserted seq) CS -- lacZ -- lacY -- lacA - 3'

Remember that the promoter is more of a "hey over here" signal to the polymerase as opposed to a "start transcribing here" signal.

It's been a while so forgive me if I've got this all wrong.
posted by junesix at 7:23 PM on January 10, 2006

Short answer is the MCS site is part of the entire lac operon but the actual lac product transcription sites do not include the MCS. I'll try to find an image but everything is either too simplistic (lac operon, no MCS) or too detailed (full plasmid map, no detail on lac operon).
posted by junesix at 7:29 PM on January 10, 2006

I think Rothko is asking about the case when the gene is not successfully cloned into the MCS. In that case should be Promoter -> MCS -> LacZ. I think he wants to know why the extra base pairs of the MCS don't screw up the transcription and translation of the functional LacZ protein.

I've never learned the details of how promoters function, but my guess is that they can handle a certain amount of "slop," and that the RNA polymerase just skips over the extra base pairs until it runs into the start codon, at which point it starts transcribing.

I don't own it but I imagine Alberts covers this in detail.
posted by epugachev at 7:30 PM on January 10, 2006

Looks like I'm all wrong - the MCS is in the lacZ sequence. Ignore/delete my above two comments.
posted by junesix at 7:33 PM on January 10, 2006

Best answer: I haven't been able to find out a definitive answer yet, but let me throw in two possibilities. Let's first remember that MCS isn't just one restriction site, but a collection of sites, thus the name Multiple Cloning Sites. As you can see from the diagram, they're sort of all jammed together. This presents a couple possiblities:

These might still somehow code for the same or similar amino acids LacZ should have in that region. Seems a little unlikely, but a possiblity.

LacZ, when all folded up and doing its enzyme thing, might not care about 20 extra amino acids hanging off of it and would still function normally. As long as the MCS doesn't shift the reading frame or code for a stop, things could be alright. Some proteins are just that way.

It's a great question though, so I'm going to dig a little more into this.
posted by Mercaptan at 8:12 PM on January 10, 2006

Best answer: The MCS sequence is translated, not excised, according to this < href="http://oregonstate.edu/instruction/bb451/spring2004/bluescript.html">page and that's what I remember from doing this stuff eons ago (although with different vectors). The sequence is short, it's in the correct reading frame for what's downstream, and it occurs right at the beginning of the gene and I guess that's enough to keep from killing the B-galactosidase activity of the resulting protein. I can remember having lots of false positives (i.e plasmid but no insert) just from frame shift errors when the restriction enzyme cut ends would degrade and join bluntly.
posted by TimeFactor at 8:15 PM on January 10, 2006

Best answer: I swear there was a valid link in there when I previewed.
posted by TimeFactor at 8:16 PM on January 10, 2006

Aye, it is not transcribed into mRNA in this case for reasons listed below. Also remember that due to your noted lack of exon splicing in prokaryotes (something I studied way too much in eukaryotes), that the MCS sites being in the coded regions is irrelevant- the polymerase wouldn't care if the codons are nonsense or not. It is not their job to care- it is solely to figure out where to start and end & transcribe.
posted by jmd82 at 8:19 PM on January 10, 2006

I asked a similar question in a prokaryotic gene regulation class I took last year and the answer is, basically, what Mercaptan and TimeFactor said. The MCS is not excised; it is transcribed and translated along with the rest of the sequence. The presence of the MCS on its own is simply not enough to completely inactivate the beta-galactosidase protein, as the insertion of an entire gene would. This seems strange at first, but makes more sense if you think about the fact that the MCS is only ~20 bp long, whereas an average gene is a few thousand bp.

It could be that the insertion of a MCS into lacZ results in a protein that has somewhat lower than wild-type activity but is still able to split X-gal. That's just speculation on my part, however.
posted by purplemonkie at 8:28 PM on January 10, 2006

Apologies to Rothko for my incorrect answer earlier and I'm glad knowledgeable folks were able to get it.

Creeps into corner and makes self very small.
posted by junesix at 8:37 PM on January 10, 2006

Jmd82, the MCS is transcribed and translated, it just doesn't kill the beta-gal as TimeFactor describes. BG is a monster of a protein, I forget exactly how big, and pretty tolerant of insertions. If you're cloning something small, you'll often get light blue instead of white colonies because the BG is not completely dead.
posted by sennoma at 10:58 PM on January 13, 2006