Promoter binding. T7 RNA polymerase , by recognizing the upstream duplex region of the promoter (-17 to -5), and then melting a bubble (-4 to about +3), within the larger duplex. The duplex promoter domain binds primarily to the N-terminal domain of the enzyme, with the exception of the (C-terminal domain) "." It is the combination of the N-terminal domain, with the positioned specificity loop, that forms the specific binding surface.
Intercalating loop stabilizes the melted complex. Strong interactions with the duplex region of the promoter places the "intercalating loop" into the DNA between residues -4 and -5. The intercalating loop, also called the Valine Loop, has hydrophobic residues Val, Ile, etc that stack on and stabilize the exposed face of the base pair at position -5, stabilizing the locally melted structure.
Positioning of the +1 base in the active site. Melting of a bubble within the DNA allows the (single stranded) template strand to enter the active site, and allows template strand bases +1 and +2 to orient in the active site, poised for initiation.
Catalysis. The enzyme then binds the first two substrate NTP's, as directed by the template (typically two GTP's, encoded by CC in the template strand). A phosphoryl transfer reaction occurs to form the product dinucleotide (pppGpG).
Movement. At this point, the complex is in the pre-translocated state and to add the next base, the enzyme must translocate forward along the DNA (or equivalently, the RNA/DNA slides backwards), forming the post-translocated state. In the latter state (only) the active site now accommodates binding of the next NTP to the (+3) template base.
This cycle of NTP binding, catalysis (bond formation via phosporyl transfer), and forward translocation repeats over and over, throughout extension of the RNA.
The initial bubble grows. From 2mer, the system progresses ->3mer->4mer->5mer->6mer ... During this time, the newly formed RNA-DNA duplex (hybrid) grows, from 2 bases, to 3 bases, to 4 bases, etc., and during the time, the duplex is short and otherwise unstable. The enzyme active site presumably stabilizes these short hybrids, but evidence also suggests that the intercalating loop, upstream and the active site, downstream, stabilize the bubble and keep it from collapsing and competitively displacing the short, nascent RNA.
The growing hybrid induces protein domain movement. Also note that the initial active site accommodates only about a 3 base RNA-DNA duplex, as the N-terminal domain lies in the path of that hybrid (remember that forward translocation of the polymerase is really reverse translocation of the RNA-DNA hybrid). Beyond about 3 bases, the hybrid pushes on the N-terminal domain, inducing into to both translate backwards and rotate. This can be seen in structures of the complex with 7 and 8 bases of RNA synthesized.
Function
Disease
Relevance
Structural highlights
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