Zachary Burton Research Interests
Multi-subunit RNA polymerases are among the most complex and dynamic enzymes found in living systems. Human RNA polymerase II is comprised of 12 peptide subunits, each the product of a separate gene. The large enzyme is highly flexible and dynamic, as indicated by x-ray crystal structures of the homologous yeast RNA polymerase II, which is found in multiple conformations. Regulation of RNA polymerase II is important because messenger RNA synthesis is subverted during cancer and viral infection. Normal development requires constant modulation and re-direction of gene regulation programs. Interest in multi-subunit RNA polymerases within the scientific community was recently underscored by the awarding of the Nobel Prize for Chemistry to Dr. Roger Kornberg of Stanford University. Dr. Kornberg was the first to report x-ray crystal structures of yeast RNA polymerase II. The characteristic "crab claw" structure of multi-subunit RNA polymerases is evolved to support: 1) high fidelity and efficiency through the NTP-driven translocation mechanism; 2) high processivity by tight binding to DNA and the RNA-DNA hybrid; and 3) translocation stepped in single base increments.
NTP-driven translocation
Human RNA polymerase II catalyzes RNA synthesis by an NTP-driven translocation mechanism. NTPs base-pair to the DNA template at downstream sites prior to being translocated stepwise into the active site for incorporation into the RNA chain as an NMP unit, releasing pyrophosphate. Because NTPs are matched to their DNA complements prior to transfer to the active site, mis-loading of NTPs is suppressed, reducing transcription errors. NTP-dNMP base pairs act as allosteric effectors for translocation, and NTP-driven translocation is coupled to pyrophosphate release. Coupling NTP-driven translocation to pyrophosphate release links the beginning of the next bond addition to the final stage of the previous bond addition. In this manner, accurate NTP loading is coupled to the previous bond completion. Accurate NTP-driven translocation coupled to pyrophosphate release is a fidelity checkpoint in each bond addition.
Fidelity
Transcription errors are sensed as translocation blocks. When translocation is blocked, downstream NTPs generate translocation pressure against the block. Translocation pressure against the block appears to result in reverse translocation, releasing NTP-Mg2+ from the active site or reversing bond formation through endogenous pyrophosphorolysis. At any time prior to pyrophosphate release, transcription errors remain reversible. Induced fit (NTP-Mg2+ tightening) in the active site is first used to determine fidelity. Accurate NTP-driven translocation mediated by NTP substrates loaded at downstream sites is used to enforce fidelity at the pyrophosphate release step.
Transient state kinetics
Transient state or pre-steady state kinetic analysis allows synchronized millisecond events during elongation to be monitored on a millisecond time scale. This type of analysis provides exquisite insight into the RNA synthesis mechanism. Human RNA polymerase II utilizes two rate-limiting steps: 1) NTP-Mg2+ tightening prior to phosphodiester bond synthesis (about 30 per second); and 2) NTP-driven translocation coupled to pyrophosphate release (about 30 per second). NTP loading to the active site is not rate-limiting (faster than 1000 per second). The processive transition between one bond and the next has distinct kinetics not observed during the escape from a long term (30 second) stall. The processive transition is highly NTP-dependent, demonstrating the NTP-driven translocation mechanism.
Transient state inhibitors
Alpha-amanitin is the deadly human poison derived from the death cap mushroom. Alpha-amanitin is a cyclic octapeptide with a covalent cross-bridge between the 4 and 8 positions. Death cap mushrooms were used as a tool for political advancement in ancient Rome. Empress Agripinna poisoned her husband in the year 54 C.E. (Current Era) by feeding him a death cap mushroom. As Pliny the Elder explained, "Among those foods that are eaten carelessly, I would place mushrooms. Although mushrooms taste wonderful, they have fallen into disrepute because of a shocking murder. They were the means by which the emperor Tiberius Claudius was poisoned by his wife Agrippina. Thus she gave the world a poison worse still--her own son Nero."
The Burton laboratory has used alpha-amanitin as a transient state translocation blocker. In this experimental design, alpha-amanitin is added to RNA polymerase II at the same time as NTP substrates, and the reaction is monitored in the millisecond phase. The reaction struggles to advance and to stop, and the results reveal the elongation mechanism. In one protocol, the reaction is observed to first advance and then retreat, as NTP-Mg2+ is expelled from the active site in response to the translocation block. Expulsion of the substrate NTP-Mg2+ in response to the translocation blocker is dependent on accurately templated NTPs at downstream positions. This experiment proves the major tenet of the NTP-driven translocation model: NTPs interact accurately at downstream positions and influence the fate of the NTP-Mg2+ occupying the active site. Alpha-amanitin is a superb tool to unravel the RNA polymerase II mechanism, and the transient state inhibitor reaction design is a highly informative approach.
Transcription factors
RNA synthesis by human RNA polymerase II is modified by many transcription factors. Transcription Factor IIF (TFIIF) stimulates the rate of elongation by supporting forward translocation. TFIIS supports dinucleotide cleavage. Hepatitis delta antigen is derived from hepatitis delta virus, a viroid satellite particle maintained during hepatitis B virus infection. Hepatitis delta antigen is another RNA polymerase II elongation factor. Alpha-amanitin is a potent inhibitor of RNA polymerase II. Using the human system, elongation factors and inhibitors are potent probes of the elongation mechanism.
NTP loading
NTPs load to the active site of human RNA polymerase II through the main enzyme channel. Others have supported a model in which NTPs load through the secondary pore to the RNA polymerase II active site, but this assertion is not correct. The secondary pore is a deep and narrow channel from the "funnel" to the deeply buried active site. The minimum diameter of the pore is about 7 angstroms. The minimum diameter of an NTP is about 6 angstroms. There is no space to exchange multiple NTPs through the pore. The pore is about 15 angstroms deep. Negative electrostatics of the pore is not conducive to NTP loading. An NTP substrate has no reference to the DNA template until it has fully penetrated the pore. There are 4 NTP substrates (ATP, GTP, CTP, and UTP), so mis-loading of NTPs is more frequent than accurate loading according to the secondary pore NTP loading hypothesis. NTPs cannot access downstream template sites through the secondary pore, but NTPs have been demonstrated to interact at downstream positions while the active site is occupied by an NTP. NTPs load through the main enzyme channel, not the secondary pore, and this conclusion is proven by Burton laboratory experiments, which clearly falsify the secondary pore NTP loading hypothesis. To support fidelity and the NTP-driven translocation mechanism, the secondary pore has evolved to exclude NTP entry. The pore is the route for pyrophosphate waste excretion after bond addition.