Transcription

The Martin Lab has a long history of studying fundamental mechanisms in transcription. Using powerful tools of biophysical chemistry and enzymology, we have focused on the (relatively) simple, single subunit RNA polymerase from bacteriophage T7. Our work is all with purified enzyme and largely with synthetic DNA templates, affording us exquisite control over the system.

Some words about transcription

Unlike a textbook enzyme where substrate binds, catalysis occurs, and product is released, a polymerase must move along the DNA, with repeated cycles of catalysis/movement (the primary job of a DNA polymerase). An RNA polymerase maintains a short (about 8 base pair) RNA-DNA hybrid, within a melted bubble in the DNA, but then releases RNA beyond (upstream, away from the active site) that point into solution. The DNA to which the RNA, and the substrate NTPs, bind is called the template strand – it provides the encoding information. The DNA displaced in the bubble is the nontemplate strand and is actually not needed in vitro (except at the promoter).

Initiation. Unlike a DNA polymerase, an RNA polymerase must initiate transcription de novo – that is, without a pre-formed primer. And it must do so at specific sequences (promoters) in the DNA. Thus, T7 RNA polymerase binds a promoter sequence that extends upstream about 17 bases from the transcription start site. It recognizes features in the duplex region from positions -17 to -5, and melts a “bubble” extending from position -4 to about +3 (transcription starts at position +1, and there is no position 0!). Some of the binding energy from the duplex DNA contacts is used to melt open and maintain this initial bubble. The primary initiation event involves the first two substrate NTPs, sitting down at positions +1 and +2, followed by a phosphoryl transfer reaction to create the first phosphodiester bond, releasing pyrophosphate from the +2 NTP. The triphosphate from the +1 NTP is retained passively – we call this the 5′ end of the transcript. At this point, the polymerase must move forward (translocate) along the DNA (or the DNA move backward within the polymerase), to allow positioning of the +3 NTP in the active site, encoded by the +3 base in the template strand. Another phosphoryl transfer reaction occurs to generate a 3 base transcript (a 3mer RNA).

Initial transcription. To translocate again, on duplex DNA, the downstream end of the bubble must melt to expose the base at +4 in the template DNA. Since RNA polymerase retains its duplex promoter interactions, the upstream end of the DNA bubble remains fixed and so the bubble is now expanding (forward). Repeated rounds of translocation/phosphoryl transfer expands the bubble (if this region artificially contains no nontemplate strand, everything still occurs normally, in the absence of a bubble). Interestingly, another thing is happening as the RNA grows during initial transcription: the RNA-DNA hybrid duplex grows from 2, to 3, to 4, etc base pairs. In T7 RNA polymerase, the growth of that hybrid, as it translocates backwards within the polymerase active site, very quickly causes it to “bump up” against a domain in the polymerase, pushing on it and causing it to move (translocate) and rotate. Addition of RNA bases at the active site lengthens the hybrid, which serves as a growing piston to drive this motion. This is critical, as energy from phosphoryl transfer is being converted into mechanical “strain” in the form of this piston motion.

Transition to elongation. Why did nature evolve this piston/strain process? To consider this, we need to back up briefly. During the process just described, we have a 2, 3, 4, … base duplexes (as the RNA-DNA hybrid is growing). Without an enzyme present, the bubble wouldn’t form in the first place, but even if it did, it would immediately collapse back down, displacing the newly synthesize 2, 3, 4 base RNA. Remember that the energy of promoter binding was used to melt the bubble and continues to be used to maintain the upstream edge of the bubble. Therefore, during initial transcription, when the RNA-DNA hybrid is short, nature must maintain those strong promoter contacts. Eventually the hybrid becomes long enough (see below) to resist collapse of the bubble, and at this point, the promoter contacts can be lost. Indeed, at this point, it is important to releaser the duplex promoter contacts, so that the enzyme can move 10, 100, 1000 bases downstream. But the promoter contacts are strong – we need an input of energy to drive release of those contacts, but the only favorable energy input in the system derives from the phosphoryl transfer reaction. The growing RNA-DNA hybrid piston is the mechanical/energetic coupling nature needs.