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RNA POL II
Description of the structure and function of the RNA Polymerase 2
| Question | Answer |
|---|---|
| Excluding plants, how many RNA polymerases do eukaryotes have? | 3 |
| What is transcribed by RNA Pol I? | rRNA exclusively |
| What is transcribed by RNA Pol II? | All protein coding mRNA which require a 5' cap and a 3' poly A tail |
| What is transcribed by RNA Pol III? | tRNA and other small rRNAs |
| What are the 3 main steps of transcription? | -Initiation -Elongation -Termination |
| What characterizes transcription initiation? | -DNA promoter recognition and binding -Opening and loading of the DNA into POL II |
| What characterizes transcription elongation? | Polymerization of RNA |
| What characterizes termination of transcription? | mRNA transcript is released |
| What elements are modeled in the structure? | -More than 3500 amino acids -22 nucleotides, of which 13 are DNA and 9 are RNA -8 Zn2+ ions -1 Mg2+ ion |
| How many subunits does POL II contain? How many are modeled in this structure? | -12 -10 -4 and 7 are not modeled |
| How are the structures of the individual domains stabilized? | By interacting with the other domains of the complex |
| What is found at the active site? | -The template DNA make an abrupt turn, exposing the template base -a single metal ion is positioned to participate in catalysis |
| What must the incoming RNTPs pass through to arrive at the active site? | The "funnel", which is the solvent-exposed space between the "wall" and the "bridge" domains. |
| What is the main difference between this solved structure and the structure of a free POL II? | The positioning of the clamp, which undergoes a 30 degree rotation upon binding |
| What are the "switch" regions? How many are there? | -Regions in the clamp that do not move with the main bulk of the clamp. -5 |
| What causes the ordering of the "switch" regions? | Binding to the DNA in the upstream DNA/RNA hybrid |
| Where are the switch regions located? | The base of the clamp |
| What is the bridge helix? | A long alpha helix spanning the cleft of the enzyme |
| What changes accompany the ordering of the switch regions? | Changes in the salt linkages with the bridge helix |
| Why are the conformational changes, triggered by DNA-binding, important to the function of the enzyme? | The conformational changes trap the DNA and nascent RNA in the enzyme, preventing disassociation and positioning the template for transcription |
| Are all nucleotides in the structure well ordered? | No. The ordering of the nucleotides begins at position +4, upstream of the active site |
| What interactions stabilize the DNA-RNA hybrid in the active site? | The DNA/RNA hybrid is sandwiched between the bridge helix and the wall domain of Rpb 2 |
| What form does the heteroduplex adopt? | An intermediate form between A- and B-DNA |
| The electron density is strongest around the active site. Why is this significant? | This suggests a low degree of mobility, which is likely required to achieve a high degree of transcriptional fidelity |
| Which regions of the heteroduplex make contact with the protein? | The DNA makes protein contact along its entire length while the RNA only contacts the enzyme close to the active site. |
| How are the conflicting requirements for tight binding for fidelity of transcription and mobility reconciled? | -Interactions between the enzyme and the substrate are through the phosphate backbone and lack sequence specificity -Several residues interact with 2 phosphates simultaneously, leading to processivity -The dramatic bend in the template DNA |
| What key characteristic of this complex is shared with DNA Polymerases? | The enzyme relies on 2 divalent cations to carry out catalysis. |
| What is different about the solved structure and that of the DNA Polymerase? | Only one divalent cation is present in the RNA Polymerase (analogous to metal A in DNA polymerases) |
| What happened to the presumptive metal B? | The authors propose that it left the active site bound to the pyrophosphate produced by the most recent round of RNA elongation |
| What role does metal A play? | Metal A coordinates the phosphodiester bond formation |
| What role does the presumptive metal B play? | Metal B coordinates the phosphates of the incoming nucleotide and does not participate in the phosphodiester formation. |
| How does the enzyme discriminate between ribonucleotides and deoxyribonucleotides at the active site? | Asn479 is speculated to be located in a region that allows it to hydrogen bond with the 2' hydroxyl of the incoming ribonucleotide |
| How does the enzyme discriminate between ribonucleotides and deoxyribonucleotides after incorporation? | Several residues are within H-bonding distance to 2' hydroxyls downstream of the active site |
| How does the shape of the enzyme contribute to selectivity of RNA over DNA? | The shape of the enzyme is highly complementary to the shape of the DNA/RNA hybrid |
| How does the enzyme ensure incorporation of the correct nucleotide? | The incorporation of an incorrect nucleotide would destabilize the growing RNA strand, leading to degradation. |
| What is the proposed role of the bridge helix? | Maintaining contact with the DNA during translocation of the enzyme |
| How does the bridge helix perform its function? | By bending as the enzyme translocates along the DNA |
| What is the proposed catalytic cycle of RNA Pol II? | The same as DNA Polymerase |
Created by:
mbell133
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