Biology subjects

Dykeman, E. C.

Publications and source records attributed to Dykeman, E. C..

3 recordsLinked to original sources

bioRxiv2024

An empirical model of aminoacylation kinetics for E. coli class I and II aminoacyl tRNA synthetases

Efficient functioning of the prokaryotic translational system depends on a steady supply of aminoacylated tRNAs to be delivered to translating ribosomes via ternary complex. As such, tRNA synthetases play a crucial role in maintaining efficient and accurate translation in the cell, as they are responsible for aminoacylating the correct amino acid to its corresponding tRNA. Moreover, the kinetic rate at which they perform this reaction will dictate the overall rate of supply of aminoacylated tRNAs to the ribosome and will have consequences for the average translational speed of ribosomes in the cell. In this work, I develop an empirical kinetic model for the 20 aminoacyl tRNA synthetase enzymes in E. coli enabling the study of the effects of tRNA charging dynamics on translational efficiency. The model is parametrised based on in vitro experimental measurements of substrate Km and kcat values for both pyrophosphate exchange and aminoacylation. The model also reproduces the burst kinetics observed in class I enzymes and the transfer rates measured in single turnover experiments. Stochastic simulation of in vivo translation shows the kinetic model is able to support the tRNA charging demand resulting from translation in exponentially growing E. coli cells at a variety of different doubling times. This work provides a basis for the theoretical study of the amino acid starvation and the stringent response, as well as the complex behaviour of tRNA charging and translational dynamics in response to cellular stresses. Author summaryElucidating the complex interplay between tRNA charging by aminoacyl tRNA synthetases and the overall ribosomal demand for tRNAs will have important consequences for understanding the effects of amino acid starvation and the stringent response. Here I introduce an empirical kinetic model of the 20 E. coli tRNA synthetases and examine tRNA charging dynamics during exponential growth. The results show that the model is in good agreement with a variety of experimental observations, such as tRNA charging fractions, average translational speed of the ribosome, and measured total cellular tRNA abundances.

systems biology↗

bioRxiv2024

Design of a self-regulating mRNA gene circuit

Protein expression from mRNA in vivo is predominately controlled via regulatory feedback mechanisms that adjust the level of mRNA transcription. However, for positive sense single-stranded RNA viruses, protein expression is often controlled via secondary structural elements, such as internal ribosomal entry sites, that are encoded within the mRNA. The self-regulation of mRNA translation observed in this class of viruses suggests that it may be possible to design mRNAs that self-regulate their protein expression, enabling the creation of mRNAs for vaccines and other synthetic biology applications where protein levels in the cell can be tightly controlled without feedback to a transcriptional mechanism. As a proof of concept, I design a polycistronic mRNA based on bacteriophage MS2, where the upstream gene is capable of repressing synthesis of the downstream gene. Using a computational tool that simulates ribosome kinetics and the co-translational folding of the mRNA in response, I show that mutations to the mRNA can be identified which enhance the efficiency of the translation and the repression of the downstream gene. The results of this study open up the possibility of designing bespoke mRNA gene circuits in which the amount of protein synthesised in cells are self-regulated for therapeutic or antigenic purposes.

synthetic biology↗

bioRxiv2021

RNA X-ray footprinting reveals the consequences of an in vivo acquired determinant of viral infectivity

The secondary structures of the bacteriophage MS2 ssRNA genome, frozen in defined states, were determined with minimal perturbation using constraints from X-ray synchrotron footprinting (XRF). The footprints of the gRNA in the virion and as transcript are consistent with single, dominant but distinct conformations, and reveal the presence of multiple Packaging Signals potentially involved in assembly regulation that have not been detected by other techniques. XRF also reveals the dramatic effect of the unique Maturation Protein (MP) on both the capsid lattice, and the gRNA conformation inside the phage compared with a virus-like-particle composed only of coat protein subunits. Aspects of genome organisation in the phage, their impacts on the capsid shell, and the distortion of lattice geometry by MP, are hallmarks of molecular frustration. Phage assembly therefore appears to prepare the particle for the next step of the infectious cycle.

molecular biology↗