T7 RNA Polymerase: Difference between revisions
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'''Intercalating loop stabilizes the melted complex'''. Strong interactions with the duplex region of the promoter places the "<scene name='77/778917/T7rp_promo_bound_val_loop_z/1'>intercalating loop</scene>" 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. | '''Intercalating loop stabilizes the melted complex'''. Strong interactions with the duplex region of the promoter places the "<scene name='77/778917/T7rp_promo_bound_val_loop_z/1'>intercalating loop</scene>" 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 | '''Positioning of the +1 and +2 bases 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. Note that GTP at position +2 is in the normal substrate position and is <scene name='77/778917/T7rp_mg_stabilizes_elong_ntp/1'>stabilized by coordination by the Mg(II)</scene> that will be used in catalysis. GTP at position +1, by contrast, sits where the 3' base of the elongating RNA normally sits. In an elongation complex, that base is held in place by the upstream duplex. During initiation, the <scene name='77/778917/T7rp_init_mg_coords_g1/1'>+1 GTP is coordinated by a second Mg(II)</scene>, but that Mg(II) is not coordinated by the protein, so there is little binding stabilization. For this reason, Km for the +1 base is much higher (binding is weaker) than for all other (elongating) bases. | ||
'''Catalysis'''. The enzyme then <scene name='77/778917/T7rp_gg_bound/1'>binds the first two substrate NTP's</scene>, as directed by the template (typically two GTP's, encoded by CC in the template strand). The <scene name='77/778917/T7rp_gg_phosphoryl_xfer/2'>3' hydroxyl of the +1 NTP attacks the alpha phosphate of the +2 NTP</scene> to initiate a phosphoryl transfer reaction. Release of pyrophosphate (PPi) leaves the product dinucleotide (pppGpG) in the active site. Note that one of the <scene name='77/778917/T7rp_mg_stabilizes_rxn/1'>Mg(II) ions is poised to stabilize</scene> the trigonal bipyramidal reaction intermediate (not shown) in this SN2 phosphoryl transfer reaction. | '''Catalysis'''. The enzyme then <scene name='77/778917/T7rp_gg_bound/1'>binds the first two substrate NTP's</scene>, as directed by the template (typically two GTP's, encoded by CC in the template strand). The <scene name='77/778917/T7rp_gg_phosphoryl_xfer/2'>3' hydroxyl of the +1 NTP attacks the alpha phosphate of the +2 NTP</scene> to initiate a phosphoryl transfer reaction. Release of pyrophosphate (PPi) leaves the product dinucleotide (pppGpG) in the active site. Note that one of the <scene name='77/778917/T7rp_mg_stabilizes_rxn/1'>Mg(II) ions is poised to stabilize</scene> the trigonal bipyramidal reaction intermediate (not shown) in this SN2 phosphoryl transfer reaction. | ||
