Good evening, all! It’s been quite a while, hasn’t it? Still, it seems like just yesterday that I was inhaling metabolic pathways while trying to learn Chinese through osmosis (uh, I think they call that “passive acquisition”). A lot of things have changed in a semester, but, still, they’re essentially the same—I’m still a biochemistry student, and I still seem to forget about my tests until two days before.
With that in mind, please forgive me for the sloppiness of this post. It’s late, and I’m having to resist drawing in my sketch journal and starting another runthrough of Undertale to get this down on paper (er…). Such is the life I lead.
All right, let’s go!
So, DNA is freakin’ important. This comes as a surprise to exactly no one. No longer do we live in the age of, “But, proteins have so much more variability! DNA doesn’t matter!” We all know the significance of DNA as a genetic code, and we all know that is has a very specific sequence that tells us a lot of stuff about ourselves. (Those out there with savvier parents who know them well [cough] might even be getting their genomes analyzed as we speak.)
Still, knowing all of this, a pretty glaring question remains: how does our DNA determine who we are? Yeah, yeah, it codes for proteins (and a lot of other junk), but how? Aren’t we all dying to know? You know, since we’re fatally inquisitive and lacking in access to Wikipedia…
Well, inquisitive and Wikipedia-deprived reader, the answer depends on a fun little pair of processes called transcription and translation!
Transcription, the first step in the process, is where we take the bits of DNA that actually code for proteins and make readable codes out of them. If you’ve had biology (or if you’re reading that Wikipedia article I linked to), you already know where I’m going with this: we transcribe DNA into an mRNA (messenger RNA) template.
Pretty simple, right? That makes a lot of sense. Completely straightforward, totally understandable…
Okay, hold on, ’cause here’s where the actual biochemistry starts.
In prokaryotes, all of RNA synthesis is carried out using a single enzyme, RNA polymerase. You want mRNA? RNA polymerase is on it. tRNA? It’s got you. rRNA? … you get the idea.
This polymerase does its magic in essentially the way that you’d expect—it synthesizes a new mRNA strand in the 5′ to 3′ direction, stringing each new NTP (nucleoside triphosphate) onto the 3′-OH of the last using the energy released from releasing pyrophosphate from the NTP (you know, ATP —> AMP + 2Pi).
So far, no surprises. We’ve got an enzyme that can read a strand of DNA and copy down a complementary code. Seems pretty simple, right?
Yeah, but how do you tell it where to start?
Well, turns out, this is something rather simply solved in prokaryotic systems. A little factor called σ factor binds to the enzyme, creating RNA polymerase holoenzyme. The sigma factor then recognizes nifty sequences called “promotor sequences” in the DNA, which it binds to, forming a closed promoter complex. The enzyme then unwinds the DNA (~14 bp), forming the open promoter complex.
Now, before we go any further, let’s talk a bit about the promoter. The promoter is a region of DNA that contain consensus sequences, in this case the Pribnow Box (TATAAT) and the -35 Region (TTGACA). Now, it’s true that a stronger interaction between sigma factor and the -35 region means more efficient transcription, but there’s a more directly helpful thing here: A-T interactions are easier to break than others, which makes the Pribnow box a prime place for the enzyme to start unwinding the DNA double helix.
Now that we’ve got our enzyme bound and ready to go, what do we do? Well, first, an NTP (usually a purine) binds at the initiation site of the polymerase, pairing with the initiation site (first nucleotide) in the DNA sequence. Then the next NTP binds at the elongation site, and the enzyme joins them together by bonding the 5′-alpha-phosphate of the second nucleotide to the 3′-OH of the first and spitting out 2Pi. The enzyme translocates (moves) along the strand and keeps adding nucleotides until it’s added somewhere between 9 and 12. Then sigma factor falls off, and the rest of the enzyme (the core polymerase) does its thing until it’s finished the whole transcript.
Elongation is relatively uneventful; the enzyme proceeds down the DNA strand, leaving a growing RNA strand in its wake. The movement of the enzyme introduces tension in the DNA helix. Because of this, a gyrase enzyme introduces negative supercoiling ahead of the enzyme, and topoisomerases remove the negative supercoiling behind it. Crisis averted.
Finally, our lil’ prokaryotes need to terminate transcribing their mRNAs. They can do this one of two ways: either using a rho factor (rho-dependent) or using the sequence of the RNA itself (intrinsic).
Intrinsic termination makes use of secondary structures in the transcript to terminate transcription. G-C “hairpins” form in the mRNA, making the polymerase stall. Intervening strings of As then disassociate from the Us of the transcript, opting to form slightly stronger bonds with their complementary Ts in the other DNA strand.
Rho-dependent is slightly more complicated: rho-factor, an ATP-dependent helicase, binds to C-rich regions of the transcript and chases the polymerase down, trying to gain ground until it reaches the transcription bubble. After the polymerase stalls on tricky G-C regions, rho factor unwinds the DNA-RNA hybrid, releasing the new transcript from the DNA template.
All right, so we’ve gone through all of prokaryotic transcription relatively painlessly. What now? Am I going to quickly gloss over eukaryotic transcription, too? It can’t be too hard, can it?
That’s the kind of enthusiasm the world needs, dearest reader. Unfortunately, life’s a little more complicated than that.
Eukaryotic stuff is generally a lot more complicated than prokaryotic stuff, and if you’re expecting biochemistry to be any different, when you get to my next post, you’re gonna have a bad time.
Questions? Comments? Digital versions of coffee and sparkling water? Put ’em down there!