Chemical studies on Escherichia coli DNA-dependent RNA polymerase

Wasylyk, Bohdan (1975) Chemical studies on Escherichia coli DNA-dependent RNA polymerase. PhD thesis, University of Glasgow.

Abstract

The interaction of E. coli DNA dependent RNA polymerase with nucleic acids was studied by chemical modification of both the enzyme and DNA. Chemical modification requires large quantities of a pure homogeneous preparation of a protein. Core enzyme was purified from up to 1 kg of E. coli, in yields of about 10 mg per 100 g cells, and to 95 % purity. Core-enzyme was used because it is the simplest active form, with most of the properties, of the complete enzyme. Work on the binding of synthetic peptides and proteins to nucleic acids suggests that tryptophan and tyrosine form stacking interactions with nucleic acid bases, and that lysine interacts ionically, N-bromosuccinimide was used to modify the tryptophan and tyrosine groups of RNA polymerase. A 30 fold molar excess of reagent rapidly and completely inactivated the enzyme. The modification resulted in oxidation of 1-2 tryptophans and 8 (+/-2) cysteines, loss of 9% of the protein fluorescence, and no loss of overall structure (as judged by the accessibility of thiols to reaction with 5, 5'-dithiobis(2-nitrobenzoic acid). Substrates did not protect against inactivation. The oxidation of 8 thiols, which accounted for up to 40 % of the inactivation, was limited to 4 thiols and 10 % inactivation by reversibly protecting the surface SH groups with 5,5'-dithiobis(2-nitrobenzoic acid). Inactivation of protected enzyme required a 30 fold molar excess of N-bromosuccinimide, and led to a loss of 11 % of the protein fluorescence, and no change (<2%) in the overall conformation (as judged by far ultraviolet circular dichroism). Amino acid analysis showed that between 3 and 6 tyrosines, and no methionines (+/-2) or histidines (+/-5) were oxidised. The error in determining tryptophan (+/-2) was too large to detect the oxidation previously observed by extinction changes. Stacking of aromatic amino acids with nucleic acid bases would contribute to the binding energy to DNA and nucleotides, and would help unwind the double helix, N-bromosuccinimide inactivation of thiol protected RNA polymerase resulted in no decrease in DNA binding activity (as judged by salt elution from DNA agarose), and no loss in ATP binding. No difference in inactivation was observed when assayed with native and denatured DNA, and inactivation was also independent of the initiation circumventing dinucleotide G-A, suggesting that the major cause of inactivation was not loss of unwinding or initiating activity. Further work is required to resolve the cause of inactivation. Methyl-[14C] acetimidate modification of 50 lysines resulted in complete inactivation of core enzyme. Both purine nucleoside triphosphates and denatured DNA protected against inactivation. ATP, GTP, CTP, and denatured DNA together resulted in maximal protection against inactivation, and protection of about 8 lysines against modification. Since amidination should not disrupt ionic interactions greatly, lysine may have another role in RNA polymerase. Protection against inactivation was used to obtain a dissociation constant of 80 +/- 20 ?M for denatured DNA from core enzyme. The kinetics of formaldehyde melting of DNA was studied as a probe of protein-DNA interactions. The kinetic analysis of Trifonov et al. (1968) did not adequately describe the unwinding of the small, double-helical, DNA from phage T7, despite taking precautions in the experimental techniques. Calf thymus DNA, and calf thymus DNA with nucleoside triphosphates and low concentrations of holoenzyme, gave interpretable kinetics. However the shortcomings of calf thymus DNA as a template, and the difficulties with explaining the kinetics with larger concentrations of enzyme, and with T7 DNA, detract from the method as a probe of RNA polymerase-DNA interactions.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: Adviser: A DB Malcolm
• Keywords: Biochemistry
• Date of Award: 1975
• Depositing User: Enlighten Team
• Unique ID: glathesis:1975-73110
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 14 Jun 2019 08:56
• Last Modified: 14 Jun 2019 08:56
• URI: https://theses.gla.ac.uk/id/eprint/73110