Date of Award
Doctor of Philosophy (PhD)
This thesis examines three factors that affect the prokaryotic transcription cycle; Microcin J25, a peptidyl inhibitor of RNAP; Q antiterminator protein, a bacteriophage protein that modifies RNAP to readthrough termination signals in phage late genes; and Gp33, a T4 phage protein that is a co-activator of T4 phage late gene transcription. We determined that the Microcin J25 peptide (MccJ25) has a lassoed-tail structure using mass spectrometry and NMR. The 21 amino acid MccJ25 peptide is produced from a 58 amino acid pro-peptide, McjA, that is truncated and post-translationally modified by two maturase enzymes. We synthesized McjA and constructed a protein-A tagged biosynthesis cassette to be used in the analysis of MccJ25 maturation. Escherichia coli (Ec) RNAP has not, thus far, been amenable to crystallization despite considerable effort. One potential hindrance to crystallization may be the carboxy terminal domain of the alpha subunit (α-CTD, residues 249-329) that is connected to an amino-terminal domain (α-NTD, residues 8-235) by a 14-residue linker. We successfully expressed, purified and crystallized a version of Ec RNAP without the α-CTD. The Q antiterminator proteins from lambdoid phage, modify Ec RNAP to read through termination sites within the phage late gene. A structure of Q protein could reveal how Q antitermination occurs and may provide insight into the mechanism of termination. We developed an improved expression and purification protocol for λQ, and performed biophysical characterization on both λQ and the related 82Q. The T4 phage protein Gp33 is a co-activator that interacts with both Ec RNAP and T4 DNA replication machinery to upregulate transcription from T4 late promoters. Gp33 is thought to have an analogous function to E. coli σ704 in that it binds the beta flap of RNAP and helps recruit RNAP to promoters. Ec RNAP bound to Gp33 is recruited to the promoter by a unique mechanism through a protein-protein interaction between Gp33 and the DNA-bound sliding clamp of the T4 DNA replisome, Gp45. We obtained crystals of Gp33 bound to the Ec Beta flap that diffracted to 3.0 Ångstroms. The preliminary structure shows Gp33 as a mainly helical protein that wraps around the Beta flap.
Twist, Kelly-Anne Fisi, "Structural Studies of Three Factors That Affect the Prokaryotic Transcription Cycle; Microcin J25, LAMBDA Q and T4 GP33" (2009). Student Theses and Dissertations. 255.