Biology subjects

Jack, B. R.

Publications and source records attributed to Jack, B. R..

4 recordsLinked to original sources

bioRxiv2019

Transcript degradation and codon usage regulate gene expression in a lytic phage

Many viral genomes are small, containing only single- or double-digit numbers of genes and relatively few regulatory elements. Yet viruses successfully execute complex regulatory programs as they take over their host cells. Here, we propose that some viruses regulate gene expression via a carefully balanced interplay between transcription, translation, and transcript degradation. As our model system, we employ bacteriophage T7, whose genome of approximately 60 genes is well annotated and for which there is a long history of computational models of gene regulation. We expand upon prior modeling work by implementing a stochastic gene expression simulator that tracks individual transcripts, polymerases, ribosomes, and RNases participating in the transcription, translation, and transcript-degradation processes occurring during a T7 infection. By combining this detailed mechanistic modeling of a phage infection with high throughput gene expression measurements of several strains of bacteriophage T7, evolved and engineered, we can show that both the dynamic interplay between transcription and transcript degradation, and between these two processes and translation, appear to be critical components of T7 gene regulation. Our results point to a generic gene regulation strategy that may have evolved in many other viruses. Further, our results suggest that detailed mechanistic modeling may uncover the biological mechanisms at work in both evolved and engineered virus variants.

systems biology↗

bioRxiv2018

Pinetree: a step-wise gene expression simulator with codon-specific translation rates

MotivationStochastic gene expression simulations often assume steady-state transcript levels, or they model transcription in more detail than translation. Moreover, they lack accessible programming interfaces, which limits their utility.\n\nResultsWe present Pinetree, a step-wise gene expression simulator with codon-specific translation rates. Pinetree models both transcription and translation in a stochastic framework with individual polymerase and ribosome-level detail. Written in C++ with a Python front-end, any user familiar with Python can specify a genome and simulate gene expression. Pinetree was designed to be efficient and scale to simulate large plasmids or viral genomes.\n\nAvailabilityPinetree is available on GitHub (https://github.com/benjaminjack/pinetree) and the Python Package Index (https://pypi.org/project/pinetree/).

systems biology↗

bioRxiv2018

Combinatorial Approaches to Viral Attenuation

Attenuated viruses have numerous applications, in particular in the context of live viral vaccines. However, purposefully designing attenuated viruses remains challenging, in particular if the attenuation is meant to be resistant to rapid evolutionary recovery. Here we develop and analyze a new attenuation method, promoter ablation, using an established viral model, bacteriophage T7. Ablating promoters of the two most highly expressed T7 proteins (scaffold and capsid) led to major reductions in transcript abundance of the affected genes, with the effect of the double knockout approximately additive of the effects of single knockouts. Fitness reduction was moderate and also approximately additive; fitness recovery on extended adaptation was partial and did not restore the promoters. The fitness effect of promoter knockouts combined with a previously tested codon deoptimization of the capsid gene was less than additive, as anticipated from their competing mechanisms of action. In one design, the engineering created an unintended consequence that led to further attenuation, the effect of which was studied and understood in hindsight. Overall, the mechanisms and effects of genome engineering on attenuation behaved in a predictable manner. Therefore, this work suggests that the rational design of viral attenuation methods is becoming feasible.\n\nImportanceLive viral vaccines rely on attenuated viruses that can successfully infect their host but have reduced fitness or virulence. Such attenuated viruses were originally developed through trial- and-error, typically by adaptation of the wild-type virus to novel conditions. That method was haphazard, with no way of controlling the degree of attenuation, the number of attenuating mutations, or preventing evolutionary reversion. Synthetic biology now enables rational design and engineering of viral attenuation, but rational design must be informed by biological principles to achieve stable, quantitative attenuation. This work shows that in a model system for viral attenuation, bacteriophage T7, attenuation can be obtained from rational design principles, and multiple different attenuation approaches can be combined for enhanced overall effect.

microbiology↗

bioRxiv2017

Reduced protein expression in a virus attenuated by codon deoptimization

The engineering of hundreds of synonymous codon changes into a viral genome appears to provide a general means of achieving attenuation. The mechanistic underpinnings of this approach remain enignmatic, however. Using quantitative proteomics and RNA sequencing, we explore the molecular basis of attenuation in a strain of bacteriophage T7 whose major capsid gene was engineered to carry 182 suboptimal codons. As expected, there was no evident effect of the recoding on transcription. Proteomic observations revealed that translation is halved for the recoded major capsid gene, and a smaller reduction applies to a few genes downstream, potentially caused by translational coupling. Viral burst size is also approximately halved, and the fitness drop accompanying attenuation is compatible with the reduced burst size. Overall, the fitness effect and molecular basis of attenuation by codon deoptimization are compatible with a relatively simple model of reduced translation of a few genes and a consequent diminished virion assembly. This mechanism is simpler than that operating in eukaryotic viruses.

systems biology↗