T7 RNA Polymerase
From Proteopedia
(→T7 RNA Polymerase Initiation Complex) |
(→T7 RNA Polymerase Initiation Complex) |
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'''Movement'''. At this point, the complex is in the <scene name='77/778917/T7rp_init_pretranslocated/1'>pre-translocated state</scene> 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, to then <scene name='77/778917/T7rp_init_ggg/4'>form a 3 base transcript</scene>. | '''Movement'''. At this point, the complex is in the <scene name='77/778917/T7rp_init_pretranslocated/1'>pre-translocated state</scene> 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, to then <scene name='77/778917/T7rp_init_ggg/4'>form a 3 base transcript</scene>. | ||
| - | This cycle of NTP binding, catalysis (bond formation via phosporyl transfer), and forward translocation repeats over and over, throughout extension of the RNA. | + | This cycle of NTP binding, catalysis (bond formation via phosporyl transfer), and forward translocation repeats over and over, throughout extension of the RNA. |
| + | 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 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 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 <scene name='77/778917/T7rp_3mer_rna_backedout/1'>3 bases</scene>, to 4 bases, to 5 bases, to 6 bases, to<scene name='77/778917/T7rp_7mer_rna_backedout/1'> 7 bases</scene>, to <scene name='77/778917/T7rp_8mer_rna_backedout/1'>8 bases</scene>, etc., and during the time, the duplex is short and otherwise unstable. |
'''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. | '''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. | ||
Revision as of 22:41, 22 January 2018
T7 RNA Polymerase Initiation Complex
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References
1,"1aro","Free enz plus lysozyme","T7RP with the bound inhibitor T7 lysozyme, no DNA - Jeruzalmi, D. & Steitz, T. A. (1998) EMBO J 17, 4101-4113"
2,"1cez","Enz with DNA bound (ED complex)","Early structure of T7RP with promo bound - Cheetham, G. M., Jeruzalmi, D. & Steitz, T. A. (1999) Nature 399, 80-83"
3,"2pi5","Enz with DNA bound (ED complex)","T7RP with promoter and first two NTPs bound - Kennedy, W.P.,††Momand, J.R.,††Yin, Y.W. (2007) Mechanism for de novo RNA synthesis and initiating nucleotide specificity by t7 RNA polymerase. J.Mol.Biol. 370: 256-268"
4,"2pi4","ED complex with GTP + GTP","T7RP with promoter and first two NTPs bound - Kennedy, W.P.,††Momand, J.R.,††Yin, Y.W. (2007) Mechanism for de novo RNA synthesis and initiating nucleotide specificity by t7 RNA polymerase. J.Mol.Biol. 370: 256-268"
5,"1qln","ED with 3mer RNA","T7RP with promoter DNA and GTP, allowing formation of a 3 base transcript - Cheetham, G. M. & Steitz, T. A. (1999) Science 286, 2305-2309",true
7,"3e2e","Initial complex at +7","The structure of a transcribing T7 RNA polymerase in transition from initiation to elongation - Durniak, K.J., Bailey, S., Steitz, T.A. (2008) Science 322, 553-7"
6,"3e3j","Initial complex at +8","The structure of a transcribing T7 RNA polymerase in transition from initiation to elongation - Durniak, K.J., Bailey, S., Steitz, T.A. (2008) Science 322, 553-7"
8,"1msw","Elongation complex (Steitz)","Elongation complex model formed with mismatch bubble DNA - Yin, Y. W. & Steitz, T. A. (2002). Structural basis for the transition from initiation to elongation transcription in T7 RNA polymerase. Science 298, 1387-1395."
9,"1h38","Elongation w scaffold","Elongation complex model formed by multi-piece scaffold - Tahirov, T. H., Temiakov, D., Anikin, M., Patlan, V., McAllister, W. T., Vassylyev, D. G. & Yokoyama, S. (2002) Nature 420, 43-50"
10,"1s0v","Elongation w ab-me-ATP","Scaffold elongation complex with non-hydrolyzable substrate NTP - Temiakov, D., Patlan, V., Anikin, M., McAllister, W. T., Yokoyama, S. & Vassylyev, D. G. (2004) Cell 116, 381-391"
11,"1s76","Elongation w ab-me-ATP","Mismatched bubble elongation complex with non-hydrolyzable substrate NTP - Yin, Y. W. & Steitz, T. A. (2004) Cell 116, 393-404"
12,"1s77","Elongation w PPi","Yin, Y. W. & Steitz, T. A. (2004) Cell 116, 393-404"
13,"4rnp","Low res free enz"
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