T7 RNA Polymerase

From Proteopedia

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Revision as of 21:12, 22 January 2018

T7 RNA Polymerase Initiation Complex

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 "" 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.

1 Function
2 Disease
3 Relevance
4 Structural highlights

Function

Disease

Relevance

Structural highlights

This is a sample scene created with SAT to by Group, and another to make of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.

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"

Proteopedia Page Contributors and Editors (what is this?)

Craig T Martin, Karsten Theis, Jaime Prilusky, Michal Harel, Ann Taylor

Retrieved from "http://52.214.119.220/wiki/index.php/T7_RNA_Polymerase"

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 ==T7 RNA Polymerase Initiation Complex==
 <StructureSection load='1qln' size='450' side='right' caption='Promoter bound polymerase' scene=''>
-'''Promoter binding'''. T7 RNA polymerase <scene name='77/778917/Basic_cartoon_view/1'>binds and melts into dsDNA</scene>, 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) "<scene name='77/778917/Specificity_loop_first_view/1'>specificity loop</scene>." It is the combination of the N-terminal domain, with the positioned specificity loop, that forms the specific binding surface.
+'''Promoter binding'''. T7 RNA polymerase <scene name='77/778917/T7rp_promo_bound_basic/1'>binds and melts into dsDNA</scene>, 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) "<scene name='77/778917/T7rp_promo_bound_specif_loop_z/1'>specificity loop</scene>." 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 "<scene name='77/778917/Intercalating_loop/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 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.

T7 RNA Polymerase

From Proteopedia

Revision as of 21:12, 22 January 2018