PSI Structural Biology Knowledgebase

PSI | Structural Biology Knowledgebase
Header Icons
E-Collection

Related Articles
Families in Gene Neighborhoods
June 2015
Signaling: A Platform for Opposing Functions
May 2015
Nuclear Pore Complex: A Flexible Transporter
February 2015
Nuclear Pore Complex: Higher Resolution of Macromolecules
February 2015
Nuclear Pore Complex: Integrative Approach to Probe Nup133
February 2015
Piecing Together the Nuclear Pore Complex
February 2015
iTRAQing the Ubiquitinome
July 2014
CAAX Endoproteases
August 2013
The Immune System: A Strong Competitor
June 2013
The Immune System: Strand Swapping for T-Cell Inhibition
June 2013
PDZ Domains
April 2013
Protein Interaction Networks: Adding Structure to Protein Networks
April 2013
Protein Interaction Networks: Morph to Assemble
April 2013
Protein Interaction Networks: Reading Between the Lines
April 2013
Protein Interaction Networks: When the Sum Is Greater than the Parts
April 2013
Alpha-Catenin Connections
March 2013
Cytochrome Oxidase
November 2012
Bacterial Phosphotransferase System
October 2012
Solute Channels
September 2012
Budding ensemble
August 2012
The machines behind the spindle assembly checkpoint
June 2012
G Protein-Coupled Receptors
May 2012
Revealing the Nuclear Pore Complex
March 2012
Topping off the proteasome
March 2012
Anchoring's the way
February 2012
Reading out regioselectivity
December 2011
An effective and cooperative dimer
November 2011
PDZ domains: sometimes it takes two
November 2011
Raising a glass to GLIC
August 2011
A2A Adenosine Receptor
May 2011
A growing family
February 2011
FERM-ly bound
February 2011
CXCR4
January 2011
Guard cells pick up the SLAC
December 2010
Zinc Transporter ZntB
July 2010
Zinc Transporter ZntB
July 2010
Importance of extension for integrin
June 2010
Spot protein-protein interactions… fast
March 2010
Alg13 Subunit of N-Acetylglucosamine Transferase
February 2010
Urea transporter
February 2010
Two-component signaling
December 2009
ABA receptor...this time for real?
November 2009
Network coverage
November 2009
Get3 into the groove
October 2009
Guanine Nucleotide Exchange Factor Vav1 and Rho GTPase Rac1
October 2009
GPCR subunits: Separate but not equal
September 2009
Proofreading RNA
July 2009
Ribonuclease and Ribonuclease Inhibitor
April 2009
The elusive helicase
April 2009
Click for cancer-protein interactions
December 2008

Research Themes Protein-protein interactions

Proofreading RNA

PSI-SGKB [doi:10.1038/fa_psisgkb.2009.28]
Featured Article - July 2009
Short description: The structure of RNA polymerase II caught in the 'backtracked' state reveals how it ensures faithful transcription.Science 324, 1203-1206 (2009)

The three states of the RNA polymerase II elongation complex. The dashed oval represents the empty nucleotide addition site.

RNA polymerases catalyze rapid RNA chain growth. RNA polymerase II adds between 20 and 70 nucleotides per second to RNA and does this by moving forwards and backwards over at template at each step. Transcribing RNA polymerases swap between three states: pre-translocated, reverse-translocated and post-translocated. Structures of the pre- and post-translocated states are available, but that of the intermediate step – reverse-translocated or 'backtracked' – has only just been solved and reveals why it is so good at proofreading RNA during transcription.

During pre-translocation, the nucleotide that has just been added is still within the nucleotide addition site of RNA polymerase II. During post-translocation, RNA polymerase II has a free nucleotide addition site that is available to new nucleotides. In addition to these two states, there is the backtracked state, when RNA polymerase II moves backwards over the template, and the 3′ end of the new RNA is extruded. The advantage of backtracking is that it allows the RNA transcript to be checked and is the dominant state when the template is damaged.

Wang et al. 1 used two approaches to produce the backtracked complex. They used RNA–DNA hybrids with mismatched nucleotides at the 3′ ends of the RNA and bound it to polymerase II, thus directly creating the backtracked state. They also used DNA–RNA hybrids with damaged DNA, presuming that the enzyme encountering the damage would retreat to the backtracked state. They found that the structures of both complexes were very similar.

One of the structures solved by Wang et al. is of a hybrid containing one mismatched residue at the 3′ end of the RNA. The authors found that the last correctly matched residue was positioned within the nucleotide addition site and that the mismatched residue is located two residues downstream at a site the authors call 'P', for proofreading. The mismatched residue's interaction with RNA polymerase II distorts the RNA–DNA helix, perhaps producing a structure that is poised for intrinsic cleavage.

With two mismatched residues, the last matched residue and the first mismatched residues are in a location similar to that seen with one mismatched residue. But the second mismatched residues could not be seen in the structure, presumably because they are highly mobile.

Overall, their results lead to two conclusions. The first is that RNA polymerase II backtracked by one residue is stable. This supports the idea that there is an equilibrium between forward and backward motion during transcription. It also confirms that backtracking by one residue is favorable whereas going back over several residues is not.

The second conclusion is that the one-residue backtracked state is readily cleaved in the presence of the elongation factor IIS (TFIIS) and that a dinucleotide is released. This adds weight to the theory that cleavage occurs in the RNA polymerase II active site and that it is important for removal of misincorporated nucleotides.

RNA polymerase II moves forwards and backwards on the template until a mismatch of RNA–DNA causes the helix to distort and shift the polymerase into the backtracked state. If it remains in this state for a long time, cleavage ensues. This one-residue backtracked state is a key contributor to proofreading by RNA polymerase II.

Maria Hodges

References

  1. D. Wang et al. Structural basis for transcription: Backtracked RNA polymerase II at 3.4 Angstrom resolution.
    Science 324, 1203-1206 (2009).

Structural Biology Knowledgebase ISSN: 1758-1338
Funded by a grant from the National Institute of General Medical Sciences of the National Institutes of Health