Date of Award
Master of Science (MS)
Luis R. Cruz-Vera
Peptides--Synthesis, Genetic translation, Escherichia coli
The tnaCAB operon in Escherichia coli is regulated via an L-tryptophan dependent ribosome arrest mechanism. TnaC, which codes for a 24-residue leader peptide, has a critical role in regulating the expression of this operon. When inducing concentrations of free L-Trp are reached within the cell, TnaC-ribosome complex is arrested at the tnaC stop codon due to L-Trp binding and interacting with the nascent peptide and translating ribosome. This interaction induces conformational changes in the ribosome active site affecting release factor 2 (RF2)-aided translation termination. Considering that the expression of the tnaCAB operon is among the highest within the cell and that arrested ribosomes are retaining RF2, we hypothesize that the free pool of RF2 is reduced, and translation termination of UGA-containing genes is disrupted. The goal of this project is to determine if TnaC acts as a trans-regulator of global protein expression through decreasing intracellular RF2 level. Using a RF2-Luciferase (LuxAB) reporter gene fusions, we found that the expression of this construct was not affected by increasing expression of the tnaCAB operon. This indicates that our fusion system was not able to detect changes in the intracellular RF2 concentration under inducing L-Trp concentrations. Next, we wanted to determine if the expression of the tnaCAB operon affects translation termination of UGA-containing genes. We used two fusion constructs: 1) trpB-trpA-luxA and 2) sapA-sapB-luxA. The expression of the downstream gene, in this case trpA and sapB, depends on efficient translation termination at the stop codon of the upstream gene, trpB and sapA respectively. We found that the expression of the sapB-luxA construct, unlike the trpA-luxA construct, is more affected under the expression of the tnaCAB operon. These results show that translation termination of nascent peptides with a C-terminal proline residue is affected during the expression of the tnaCAB operon. On the other hand, translation termination of nascent peptides, which lack a C-terminal proline residue, is not affected. In conclusion, our data suggests that RF2 retaining during expression of the tnaCAB operon alters expression of other genes.
Ghazal, Mohamed Yousef, "RF2 sequestering by stalled ribosome during TnaC synthesis affects the expression of C-terminal proline-containing nascent peptides" (2021). Theses. 357.