Martin, Craig

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Professor & Department Head, Department of Chemistry
Last Name
Martin
First Name
Craig
Discipline
Chemistry
Expertise
Enzymology of transcription
Kinetics and thermodynamics
Nucleotide analogs, SELEX, fluorescence spectroscopy
Structure and function in enzyme-DNA interactions
Introduction
My group is interested in understanding specific chemical interactions which underlie complex enzyme systems. A major focus of the lab is to understand structure and function in enzyme-nucleic acid interactions. In transcription, the enzyme RNA polymerase must not only bind specifically to its promoter DNA sequence but must then initiate the processive catalysis of template-dependent RNA synthesis, leave the initial recognition site (promoter), and continue on to a stable, sequence-independent complex.
The T7 family of RNA polymerases present an ideal model system for the study of transcription. The single subunit enzyme is relatively simple and can be readily purified in large quantities from overproducing strains of E. coli. The chemical synthesis of oligonucleotides containing specifically modified base substituents, has allowed us to map out functional groups on the DNA which are critical to recognition and initiation. We can, for example, remove a single hydrogen bond donor or acceptor from a known position in the DNA helix and then ask quantitatively how recognition is perturbed. We can also introduce fluorescent base analogs that report on the melted state of the DNA, in order to map movement of the transcription bubble.
Recent crystal structures of initiation and elongation complexes (with DNA and RNA) provide a wealth of testable models for various aspects of this complex chemical process. Stopped flow and quench flow kinetic studies have allowed a dissection of the mechanistic steps in the initiation of transcription, while site-directed mutagenesis has allowed us to probe possible structural interactions. Our ultimate goal is to directly link changing structural interactions between the enzyme and DNA to individual chemical events in the transcription process.
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2018 - 201912020 - 20211
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    High-salt transcription of DNA cotethered with T7 RNA polymerase to beads generates increased yields of highly pure RNA
    (2021-01) Cavac, Elvan; Ramírez-Tapia, Luis E.; Martin, Craig T.
    High yields of RNA are routinely prepared following the two-step approach of high-yield in vitro transcription using T7 RNA polymerase followed by extensive purification using gel separation or chromatographic methods. We recently demonstrated that in high-yield transcription reactions, as RNA accumulates in solution, T7 RNA polymerase rebinds and extends the encoded RNA (using the RNA as a template), resulting in a product pool contaminated with longer-than-desired, (partially) double-stranded impurities. Current purification methods often fail to fully eliminate these impurities, which, if present in therapeutics, can stimulate the innate immune response with potentially fatal consequences. In this work, we introduce a novel in vitro transcription method that generates high yields of encoded RNA without double-stranded impurities, reducing the need for further purification. Transcription is carried out at high-salt conditions to eliminate RNA product rebinding, while promoter DNA and T7 RNA polymerase are cotethered in close proximity on magnetic beads to drive promoter binding and transcription initiation, resulting in an increase in overall yield and purity of only the encoded RNA. A more complete elimination of double-stranded RNA during synthesis will not only reduce overall production costs, but also should ultimately enable therapies and technologies that are currently being hampered by those impurities.
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    3' end additions by T7 RNA polymerase are RNA self-templated, distributive and diverse in character––RNA-Seq analyses
    (2018-01) Gholamalipour, Yasaman; Mudiyanselage, Aruni K.; Martin, Craig T.
    Synthetic RNA is widely used in basic science, nanotechnology and therapeutics research. The vast majority of this RNA is synthesized in vitro by T7 RNA polymerase or one of its close family members. However, the desired RNA is generally contaminated with products longer and shorter than the DNA-encoded product. To better understand these undesired byproducts and the processes that generate them, we analyze in vitro transcription reactions using RNA-Seq as a tool. The results unambiguously confirm that product RNA rebinds to the polymerase and self-primes (in cis) generation of a hairpin duplex, a process that favorably competes with promoter driven synthesis under high yield reaction conditions. While certain priming modes can be favored, the process is heterogeneous, both in initial priming and in the extent of priming, and already extended products can rebind for further extension, in a distributive process. Furthermore, addition of one or a few nucleotides, previously termed ‘nontemplated addition,’ also occurs via templated primer extension. At last, this work demonstrates the utility of RNA-Seq as a tool for in vitro mechanistic studies, providing information far beyond that provided by traditional gel electrophoresis.