Beta-Prime Subunit of Bacterial RNA Polymerase: Difference between revisions

'''[[RNA polymerase]]''' (RNAP) is a molecular machine that copies DNA into RNA comprised and is found in every living organism. The bacterial RNAP complex consists of six subunits (ββ’α2ωσ) and three channels. RNAP initially binds to DNA at the promoter, forming the closed complex<ref name='genetics'>Snyder, L. & Champness, W. (2007). Molecular genetics of bacteria (3rd ed.). Washington, D.C.: ASM Press.</ref>. The DNA surrounding the promoter sequence unwinds and forms the open complex (http://www.pingrysmartteam.com/RPo/RPo.htm - please note that different nomenclature is used)<ref>2006 Pingry SMART Team: RNA Polymerase Holoenzyme Open Promoter Complex (Rpo) Jmol Tutorial</ref>. RNAP releases from the promoter and transitions into the elongation complex (EC). The EC moves along the template strand, adding ribonucleotides to the 3’ hydroxyl of the RNA transcript.  

This tutorial uses the β’ subunit of the RNAP elongation complex of ''Thermus thermophilus''. The β’ subunit contains structures crucial for transcription, including the sites for ribonucleotide addition and catalysis. Double-stranded DNA (dwDNA) enters RNAP through the active site channel, while ribonucleotides (NTPs) enter through the secondary channel. As the dwDNA enters, it separates into the template and non-template strands. The template strand forms an approximately 90 degree kink in the active site channel. At the kink, one DNA base pair becomes available for NTP pairing and translocates to the +1 site. An NTP enters the active site and induces conformational change of the trigger loop into the trigger helix. The trigger helix forms a three-helical bundle with the bridge helix. This bundle changes dimensions of the active site and facilitates positioning of the NTP for addition to the growing RNA strand. Upon addition of the nucleotide, the dwDNA and RNA/DNA hybrid translocate through RNAP with stabilization from the rudder. The growing RNA strand is separated by the lid and exits RNAP through the exit channel. The DNA template strand rejoins the non-template strand as it exits the active site channel.  

<swf width="422" height="317">http://myweb.msoe.edu/~hoelzer/CrestUWMilwaukee2011-Video1.swf</swf>

 

<swf width="422" height="317">http://myweb.msoe.edu/~hoelzer/CrestUWMilwaukee2011-Video2.swf</swf>

<StructureSection load='2o5j' size='400' side='right' caption='' scene='β’ Subunit of Bacterial RNAP' scene='User:Catherine_L_Dornfeld/Sandbox_1/2o5j_overview_1/4'>

<scene name='User:Catherine_L_Dornfeld/Sandbox_1/2o5j_dna_1/2'>DNA</scene> in the active site channel provides the genetic information for RNA transcription. The active site channel is 27 Å wide and accommodates both <scene name='User:Catherine_L_Dornfeld/Sandbox_1/2o5j_dwdna_1/2'>downstream DNA</scene> (dwDNA) and an <scene name='User:Catherine_L_Dornfeld/Sandbox_1/2o5j_hybrid_1/2'>RNA/DNA hybrid</scene><ref>PMID: 10499798</ref>. The <scene name='User:Catherine_L_Dornfeld/Sandbox_1/2o5j_template_1/1'>DNA template strand</scene> (blue) provides the complementary sequence for the RNA transcript and threads through the active site channel adjacent to the active site. The <scene name='User:Catherine_L_Dornfeld/Sandbox_1/2o5j_nontemplate_1/1'>non-template strand</scene> (dark blue), or coding strand, is held away from the active site by the clamp helices and rudder.  

The template strand is kinked at the junction between the dwDNA and RNA/DNA hybrid<ref name='elongation'>PMID: 17581590</ref>. The lone unpaired acceptor template base at the <scene name='User:Catherine_L_Dornfeld/Sandbox_1/2o5j_kink_1/1'>+1 site</scene> is located at the kink<ref name='elongation' />. The base pair at the <scene name='User:Catherine_L_Dornfeld/Sandbox_1/2o5j_plus2site_1/1'>+2 site</scene> is distorted<ref name='elongation' />. Upstream of the kink is the <scene name='User:Catherine_L_Dornfeld/Sandbox_1/2o5j_hybrid_1/2'>RNA/DNA hybrid</scene>. This hybrid structure is comprised of the template strand and the complementary RNA transcript connected by hydrogen bonds. The most recently formed hybrid bond is located at the <scene name='User:Catherine_L_Dornfeld/Sandbox_1/2o5j_minus1site_1/1'>-1 site</scene><ref name='elongation' />.   

The <scene name='User:Catherine_L_Dornfeld/Sandbox_1/2o5j_rudder_1/1'>rudder</scene> (coral) stabilizes the dwDNA and upstream RNA/DNA hybrid with numerous sidechain interactions. Two <scene name='User:Catherine_L_Dornfeld/Sandbox_1/2o5j_rudderarg_1/1'>arginine sidechains</scene> are shown contacting the dwDNA and RNA/DNA hybrid<ref name='elongation' />. The rudder meets the <scene name='User:Catherine_L_Dornfeld/Sandbox_1/2o5j_clamp_1/1'>clamp helices</scene> (dark grey) which interact with the σ subunit of RNAP.  

The upstream internal chamber can accommodate a 9-bp RNA/DNA hybrid<ref name='elongation' />. The RNA/DNA hybrid encounters the <scene name='User:Catherine_L_Dornfeld/Sandbox_1/2o5j_lid_1/1'>lid</scene> (light green) that sterically blocks continued elongation<ref name='elongation' />. The lid facilitates <scene name='User:Catherine_L_Dornfeld/Sandbox_1/2o5j_lidcleavage_1/1'> cleavage of the hydrogen bond</scene>, releasing the growing RNA transcript into the exit channel. As the bond is cleaved, the template strand moves one position upstream through the active site channel. This process is called translocation. This allows the lone <scene name='User:Catherine_L_Dornfeld/Sandbox_1/2o5j_acceptor_1/1'>unpaired template nucleotide</scene> to move into the +1 site adjacent to the active site where nucleotide addition occurs<ref name='elongation' />.

The <scene name='User:Catherine_L_Dornfeld/Sandbox_1/2o5j_secondary_1/1'>secondary channel</scene>, which is bordered by the <scene name='User:Catherine_L_Dornfeld/Sandbox_1/2o5j_rim_1/1'>rim helices</scene> (very dark blue), forms the entrance for both ribonucleotides (NTPs) and deoxyribonucleotides (dNTPs) into RNAP. The secondary channel's dimensions are 15 x 20 Å, preventing the entrance of dsDNA<ref name='loading'>PMID: 17581591</ref>. Nucleotides are coupled to a magnesium ion (MgII) as they enter. <scene name='User:Catherine_L_Dornfeld/Sandbox_1/2o5j_asn737_1/1'>Asn737</scene> (indigo) evaluates ribose hydroxyl groups in order to discriminate between NTPs and dNTPs<ref name='loading' />. The hydrogen bonds on the incoming NTP must complement those of the acceptor template base. As the NTPs enter, it is proposed that <scene name='User:Catherine_L_Dornfeld/Sandbox_1/2o5j_metthr_1/1'>Met1238 and Thr1088</scene> (cyan and magenta) aid in selection and orientation of the cognate NTP<ref name='loading' />. Once the cognate NTP is selected, its alpha and gamma phosphates form temporary phosphate contacts with RNAP near the active site<ref name='loading' />. The nucleotide adopts a relaxed conformation and resists loading into the catalytic position<ref  name='loading' />. This conformation is influenced by the "basic rim gate" consisting of four residues that surround the NTP phosphates<ref  name='loading' />. Two basic rim gate residues, <scene name='User:Catherine_L_Dornfeld/Sandbox_1/2o5j_basicgate_1/1'>Arg783 and Arg1029</scene>, are shown (light purple).  

The <scene name='User:Catherine_L_Dornfeld/Sandbox_1/2o5j_bridge_1/1'>bridge helix</scene> (magenta) separates the active and secondary channels while interacting with the <scene name='User:Catherine_L_Dornfeld/Sandbox_1/2o5j_trigger_1/1'>trigger helix</scene> (cyan). In the pre-insertion state, the trigger loop has an unstructured conformation<ref  name='loading' />. Loading of the NTP near the active site induces a conformational change in the trigger loop, and it becomes the two-helical trigger helix<ref  name='loading' />. The <scene name='User:Catherine_L_Dornfeld/Sandbox_1/2o5j_trigger2_1/1'>trigger helix</scene> "swings" into the secondary channel and changes the dimensions of the channel to 11 x 11 Å<ref  name='loading' />. This reduction in size prevents diffusion of NTP away from the active site while simultaneously preventing interference from other nucleotides<ref  name='loading' />. The presence of the trigger helix causes the NTP phosphate contacts to change<ref  name='loading' />. All three phosphates contact RNAP and adopt a more rigid conformation<ref  name='loading' />. The <scene name='User:Catherine_L_Dornfeld/Sandbox_1/2o5j_ntp_1/1'>NTP</scene> is now ready for catalysis.  

The <scene name='User:Catherine_L_Dornfeld/Sandbox_1/2o5j_activesite_1/1'>active site</scene> consists of three highly conserved aspartate sidechains (Asp739, Asp741, Asp743) chelated to a Mg2+ ion (MgI) required for catalysis<ref name='loading' />. <scene name='User:Catherine_L_Dornfeld/Sandbox_1/2o5j_mgions_1/1'>MgI and MgII</scene> (lime green) chelated to the NTP coordinate positioning of the NTP for catalysis<ref name='lehninger'>Nelson, D. L. & Cox, M. M. (2008). Lehninger principles of biochemistry (5th ed.). New York: W. H. Freeman and Company.

</ref>. The <scene name='User:Catherine_L_Dornfeld/Sandbox_1/2o5j_threeprime_1/1'>3' hydroxyl</scene> of the RNA transcript has nucleophilic activity and attacks the alpha-phosphate of the NTP<ref name='lehninger' />. A phosphodiester bond forms between the RNA transcript and the alpha-phosphate, and the beta- and gamma-phosphates leave as a pyrophosphate group<ref name='lehninger' />.

 

After catalysis the RNA/DNA hybrid moves in the <scene name='User:Catherine_L_Dornfeld/Sandbox_1/2o5j_minus1site_1/1'>-1 site</scene>, and the ribonucleotide in this bond provides the 3’ hydroxyl for the next incoming NTP<ref name='elongation' />.

This section will feature an animation demonstrating the process of nucleotide addition.  

*What is the function of the magnesium ion in the active site of RNA polymerase? How does it relate to the magnesium ion coupled to the incoming NTP?  

Steven Forst, Ph.D., University of Wisconsin-Milwaukee

 

Rick Gourse, Ph.D., University of Wisconsin-Madison

 

MSOE Center for BioMolecular Modeling: Mark Hoelzer, Margaret Franzen, Ph.D. and Tim Herman, Ph.D.  

 

NSF CREST Program