Date of Award
2023
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Biotechnology Science and Engineering
Committee Chair
Luis R. Cruz-Vera
Committee Member
Roy Magnuson
Committee Member
Jerome Baudry
Committee Member
Bernhard Vogler
Committee Member
Matthew Sachs
Subject(s)
Peptides--Biotechnology, Genetic translation, Genetic transcription--Regulation
Abstract
Regulatory arrest peptides (RAPs) fine-tune gene expression based on physiological cues from internal and external environments. These peptides, generated from upstream open reading frames in bacteria and eukaryotes, function by conditionally arresting the ribosome to control the fate of associated messenger RNAs (mRNA). Commonly, RAPs contain two functional domains composed of conserved residues that are critical for ribosome arrest called the sensor and stalling domains. The sensor domain allows the peptide to monitor outside or within the ribosome for its inducer molecule by folding into secondary structures that produce a binding site. The stalling domain slows the rate of translation elongation or termination by clashing with ribosomal RNA (rRNA) nucleotides at the peptidyl-transferase center (PTC) during arrest. Despite this knowledge, the precise mechanism by which inducible arrest peptides recognize a specific ligand is not well understood. I reason that if conserved TnaC residues, as well as the structure of the L-tryptophan (L-Trp) inducer molecule, are involved in sensing L-Trp, then mutating binding site residues or finding small molecules that interact at the binding site like L-Trp will allow us to understand the molecular mechanisms that RAP sequences use to detect small molecules. Using microbiological and genetics experiments, I explore this general hypothesis by altering conserved and non-conserved TnaC residues to identify the functional segments involved in L-Trp binding. I studied how these changes affect the capacity for sensing L-Trp and other molecules. I observed that the addition of rare codons to the arresting domain of the tnaC gene can produce ribosome arrest that regulates gene expression. I observed that the substitution of proline residues at the sensing domain increased the RAP's capacity to detect L-Trp. Using bioinformatics and biochemical assays, I also determined that small molecules with the potential to interact at the L-Trp binding site can block the action of L-Trp to arrest the ribosome. In summary, my results indicate that altering the sensor domain can make it more sensitive for its ligand and that other structurally similar molecules can function as antagonists for binding. My results could be used to generate better molecular sensors and find drugs that inhibit gene expression in bacteria.
Recommended Citation
Gentle, Heather Nicole, "Understanding the sequence specificity of ribosome arresting peptides" (2023). Dissertations. 273.
https://louis.uah.edu/uah-dissertations/273