Biology: Biotechnology: Reverse Transcriptase

Reverse Transcriptase

05/06/2012

~ Debbie26

Brilliant!

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I have to tell you guys a story about a friend of mine - she's a professor at Cornell University, and often is sitting in on my classes, and when I teach this central dogma, and I always say DNA makes RNA and RNA makes protein Laurel just goes nuts. And Laurel is a virologist. And she always interrupts me and says that's heresy because to a degree she's right.
Now, don't get upset with me, you know, you've been telling us all this time DNA makes RNA and RNA makes protein - this is the secret to life - did you lie? No, it's true. All of that is true; but sometimes, and somewhere this is not true. And it brings us to the topic of retroviruses.
Retroviruses are a whole group of viruses that don't follow the central dogma. They kind of work it backwards. And in a retrovirus what they do is - is they do the opposite. They start out with RNA, and from RNA, they make DNA. Now the good part of this answer is RNA still makes proteins for them so I'm not a complete heretic. And retroviruses are a tool we use in molecular biology.
I want to tell you a little bit about how retroviruses work, and then later on we'll talk about how we use this concept of the retrovirus to help us out in biotechnology.
Many of you know of retroviruses, some of them are very common diseases - like the cold is a retrovirus. The one that gets the most publicity though is HIV. The HIV virus is a deadly retrovirus, and let's talk a little bit how HIV infects the cell. Now here's the thing - retroviruses depend for their function on an enzyme called "reverse transcriptase." Now don't forget, if you don't know anything about transposons you know that reverse transcriptase actually occurs in some eukaryotic cells too. In fact, some theories, some hypotheses suggest that RNA viruses - these retroviruses are actually kind of escaped, reversed transcriptase. Genes from a eukaryotic cell. Okay so reverse transcriptase is what I want to talk to you about, and how it works.
Here's HIV, and here's HIV infecting a cell. Now you all know from your whole idea of bacteriophages that viruses, in general, land on a cell, and DNA viruses at least take over the DNA of that cell by injecting and inserting their own DNA into the geno. That's a premise here I want to work with. Because in this case, It's not going to be quite the same - there's a little bit before that. But the bottom line is when phages and viruses infect, like little aliens; they do inject their genome into you. And that genome does become part of your DNA, and your DNA does replicate their DNA. Retroviruses are cooler still. What they do is inject into your system when they inject into one of your cells. They inject RNA - they don't inject DNA. So, they are going to put RNA into your cell. Now attached to that injection is what I'm showing here that is the reverse transcriptase. That enzyme is going to take RNA and it's going to the RNA it injected, and here's the thing, It's going to take that RNA, and it's going to make a copy of its own RNA using your viscells nucleotides. But it's not going to make RNA from that it's going to use deoxyribonucleotides. So let's see what's going to happen.
In comes the RNA. The RNA is going to assemble because of reverse transcriptase it's going to read along this RNA and pull in nucleotides, and so it will pull in a nucleotide, and make this hybrid, and this is an RNA - DNA hybrid. And from this strand, which can now dissociate you can now make a double stranded DNA strand. So, in essence, what this thing has done it has taken its RNA made a DNA from that, and now, typical viral things can happen. So from this DNA what can occur? Well, from this DNA we can insert it, and your cell or any cell that it's in considers it it's own DNA. And if it inserts that thing, which it does into a coding sequence or into a place where RNA prolimerates can come along and read it - think of this. If RNA prolimerates comes along and reads this, it's going to make RNA. So, at a later time, it will make RNA.
Now what can that RNA do? Think about it. That RNA that it made can make proteins. I've got a question for you. What good are proteins going to do a virus? What do you remember about viruses? What's a virus' outside coat made out of? Protein. And so we're going to make the proteins that can be assembled into the new, and actually to be quite honest with you, what it does is it makes this kind of a capsule - this is the final product. It's actually going to make this kind of capsule - it's going to make a protein capsule. But the other thing is this - what it next does is what did I say it made? I said it made from the DNA that it inserted it makes RNA. And what's going to go inside of that capsule? What's on the inside of all retroviruses - RNA. So once, it makes this RNA it can be encapsulated, and then butt of as a new virus. So these retroviruses work backwards but in working backwards, they propagate themselves by making now an intermediate called DNA rather than the opposite like you and I do.
Well that's a sad story and it's a story that affects millions of lives - retro viruses are very common but, you know, the thing about it is reverse transcriptase can be used as a tool. Wait until you see how we can do that.
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