Publications by authors named "Mark W Sherman"

4 Publications

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The Tryptophan-Induced Ribosome Stalling Sequence Exposes High Amino Acid Cross-Talk That Can Be Mitigated by Removal of NusB for Higher Orthogonality.

ACS Synth Biol 2021 Apr 9. Epub 2021 Apr 9.

McKetta Department of Chemical Engineering, University of Texas at Austin, Austin, Texas 78714, United States.

A growing number of engineered synthetic circuits have employed biological parts coupling transcription and translation in bacterial systems to control downstream gene expression. One such example, the leader sequence of the tryptophanase () operon, is a transcription-translation system commonly employed as an l-tryptophan inducible circuit controlled by ribosome stalling. While induction of the operon has been well-characterized in response to l-tryptophan, cross-talk of this modular component with other metabolites in the cell, such as other naturally occurring amino acids, has been less explored. In this study, we investigated the impact of natural metabolites and host factors on induction of the leader sequence. To do so, we constructed and biochemically validated an experimental assay using the operon leader sequence to assess differential regulation of transcription elongation and translation in response to l-tryptophan. Operon induction was then assessed following addition of each of the 20 naturally occurring amino acids to discover that several additional amino acids (, l-alanine, l-cysteine, l-glycine, l-methionine, and l-threonine) also induce expression of the leader sequence. Following characterization of dose-dependent induction by l-cysteine relative to l-tryptophan, the effect on induction by single gene knockouts of protein factors associated with transcription and/or translation were interrogated. Our results implicate the endogenous cellular protein, NusB, as an important factor associated with induction of the operon by the alternative amino acids. As such, removal of the gene from strains intended for tryptophan-sensing utilizing the leader region reduces amino acid cross-talk, resulting in enhanced orthogonal control of this commonly used synthetic system.
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http://dx.doi.org/10.1021/acssynbio.0c00547DOI Listing
April 2021

RNA oxidation in chromatin modification and DNA-damage response following exposure to formaldehyde.

Sci Rep 2020 10 6;10(1):16545. Epub 2020 Oct 6.

McKetta Department of Chemical Engineering, University of Texas at Austin, Austin, TX, 78714, USA.

Formaldehyde is an environmental and occupational chemical carcinogen implicated in the damage of proteins and nucleic acids. However, whether formaldehyde provokes modifications of RNAs such as 8-oxo-7,8-dihydroguanine (8-oxoG) and the role that these modifications play on conferring long-term adverse health effects remains unexplored. Here, we profile 8-oxoG modifications using RNA-immunoprecipitation and RNA sequencing (8-oxoG RIP-seq) to identify 343 RNA transcripts heavily enriched in oxidations in human bronchial epithelial BEAS-2B cell cultures exposed to 1 ppm formaldehyde for 2 h. RNA oxidation altered expression of many transcripts involved in chromatin modification and p53-mediated DNA-damage responses, two pathways that play key roles in sustaining genome integrity and typically deregulated in tumorigenesis. Given that these observations were identified in normal cells exhibiting minimal cell stress and death phenotypes (for example, lack of nuclear shrinkage, F-actin alterations or increased LDH activity); we hypothesize that oxidative modification of specific RNA transcripts following formaldehyde exposure denotes an early process occurring in carcinogenesis analogous to the oxidative events surfacing at early stages of neurodegenerative diseases. As such, we provide initial investigations of RNA oxidation as a potentially novel mechanism underlying formaldehyde-induced tumorigenesis.
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http://dx.doi.org/10.1038/s41598-020-73376-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7538935PMC
October 2020

Computational evolution of an RNA-binding protein towards enhanced oxidized-RNA binding.

Comput Struct Biotechnol J 2020 27;18:137-152. Epub 2019 Dec 27.

Artie McFerrin Department of Chemical Engineering, Texas A&M University, 3122 TAMU Room 200, College Station, TX 77843, United States.

The oxidation of RNA has been implicated in the development of many diseases. Among the four ribonucleotides, guanosine is the most susceptible to oxidation, resulting in the formation of 8-oxo-7,8-dihydroguanosine (8-oxoG). Despite the limited knowledge about how cells regulate the detrimental effects of oxidized RNA, cellular factors involved in its regulation have begun to be identified. One of these factors is polynucleotide phosphorylase (PNPase), a multifunctional enzyme implicated in RNA turnover. In the present study, we have examined the interaction of PNPase with 8-oxoG in atomic detail to provide insights into the mechanism of 8-oxoG discrimination. We hypothesized that PNPase subunits cooperate to form a binding site using the dynamic SFF loop within the central channel of the PNPase homotrimer. We evolved this site using a novel approach that initially screened mutants from a library of beneficial mutations and assessed their interactions using multi-nanosecond Molecular Dynamics simulations. We found that evolving this single site resulted in a fold change increase in 8-oxoG affinity between 1.2 and 1.5 and/or selectivity between 1.5 and 1.9. In addition to the improvement in 8-oxoG binding, complementation of K12 Δ with plasmids expressing mutant PNPases caused increased cell tolerance to HO. This observation provides a clear link between molecular discrimination of RNA oxidation and cell survival. Moreover, this study provides a framework for the manipulation of modified-RNA protein readers, which has potential application in synthetic biology and epitranscriptomics.
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http://dx.doi.org/10.1016/j.csbj.2019.12.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6965710PMC
December 2019

Discovery and Characterization of Native Promoters for Tunable Gene Expression.

Appl Environ Microbiol 2019 11 16;85(21). Epub 2019 Oct 16.

McKetta Department of Chemical Engineering, University of Texas at Austin, Austin, Texas, USA

The potential utilization of extremophiles as a robust chassis for metabolic engineering applications has prompted interest in the use of for bioremediation efforts, but current applications are limited by the lack of availability of genetic tools, such as promoters. In this study, we used a combined computational and experimental approach to identify and screen 30 predicted promoters for expression in using a fluorescent reporter assay. The top eight candidates were further characterized, compared to currently available promoters, and optimized for engineering through minimization for use in Of these top eight, two promoter regions, and , were stronger and more consistent than the most widely used promoter sequence in , Furthermore, half of the top eight promoters could be minimized by at least 20% (to obtain final sequences that are approximately 24 to 177 bp), and several of the putative promoters either showed activity in or were specific, broadening the use of the promoters for various applications. Overall, this work introduces a suite of novel, well-characterized promoters for protein production and metabolic engineering in The tolerance of the extremophile, , to numerous oxidative stresses makes it ideal for bioremediation applications, but many of the tools necessary for metabolic engineering are lacking in this organism compared to model bacteria. Although native and engineered promoters have been used to drive gene expression for protein production in , very few have been well characterized. Informed by bioinformatics, this study expands the repertoire of well-characterized promoters for via thorough characterization of eight putative promoters with various strengths. These results will help facilitate tunable gene expression, since these promoters demonstrate strong and consistent performance compared to the current standard, This study also provides a methodology for high-throughput promoter identification and characterization using fluorescence in The promoters identified in this study will facilitate metabolic engineering of and enable its use in biotechnological applications ranging from bioremediation to synthesis of commodity chemicals.
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http://dx.doi.org/10.1128/AEM.01356-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6803307PMC
November 2019