Zachary F. Burton
Professor
- B.S. 1975, University of California, Los Angeles
- Ph.D. 1980, University of California, Los Angeles
- Postdoctoral Associate, 1980-83, University of Wisconsin
- Postdoctoral Associate, Special Fellow, Leukemia Society of America 1984-86, 1983-87, University of Toronto
burton@msu.edu
107 Biochemistry Building
Michigan State University
East Lansing, MI 48824-1319
Office: 517-353-0859
Lab: 517-353-0859
Lab Home Page
Teaching Goals
Textbook
Publication search:
Zachary Burton Research Interests continued
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.
Full text of research interests