Environmental sensing and response of conserved RNA-processing machines to specific external chemical cues

Molecular mechanisms involved in sensing and responding to changes in environmental conditions are of high interest in the field of molecular biology and biochemistry because of the immediate cascade of events and macromolecular interactions that occur in a relatively short time frame to provoke a physiological response. RNA-processing molecular machines are important players in the physiological response to environmental stress; however, relatively little is known about the specificity of their RNA substrate recognition and/or the ability for other chemical substrates to influence their activity. The research presented herein describes features of two highly relevant models to investigate substrate specificity and/or interactions with regulatory molecules: polynucleotide phosphorylase and the ribosome. Both of these machines are multi-subunit and homologs retain similar functions in all three domains of life. The ribosome is the translational machinery of the cell, whose primary function is to translate the genetic code from mRNA to protein; however, it also maintains the capacity to respond to small molecule inducers and regulatory elements to influence expression of nearby genes. Polynucleotide phosphorylase (PNPase) is a homotrimeric protein whose primary function is to degrade and recycle mRNA; yet it also plays an important role in RNA quality control by sequestering oxidatively damaged RNAs. While much is known about the primary functions of these molecular machines, the bases for their selectivity and recognition of environmental cues that induce these alternative functions have been less explored. In Chapter 1, I describe my work to elucidate the influence of cellular factors and metabolites on a coupled transcription-translation system, the tna operon. The operon is a well-characterized negative feedback loop regulating tryptophan breakdown in the presence of excess nutrients in the environment. While much effort has been made to characterize the system’s response to tryptophan, a systematic characterization of operon induction by the full panel of naturally-occurring amino acids has yet to be performed. Based on this approach, I discovered that L-cysteine can also induce the tna operon along with a suite of other amino acids that are influenced by factors involved in coupled transcription-translation. This work provides novel insight on bacterial gene regulation dynamics and provides a strategy to reduce cross-talk in engineered systems that utilize the tna operon. In Chapter 2, I explore how PNPase differentially detects and degrades modified RNA bases harbored on RNA oligonucleotides. Curiously, the chemical modifications influence the fate of RNA processing by this enzyme. I show that the enzyme demonstrates sequence specificity and that, while one RNA modification of guanine (8-oxoguanine) disables degradation, processing of another modification (1-methylguanine) enhances degradation relative to its unmodified counterpart. The biological implications of selective degradation by this enzyme raises new questions about its role in transcriptome maintenance and its influence on cellular metabolism. A more complete understanding of the full breadth of these multifunctional complexes may allow control of traditionally intractable systems or provide new targets for drug design and development by novel mechanisms.

Name thesis:degree_name

Doctor of Philosophy

Grantor

The University of Texas at Austin

Year dc:date.issued

2020

Author dc:creator

• Sherman, Mark Waring

Advisor dc:contributor.advisor

• Contreras, Lydia M.

Committee members dc:contributor.committeemember

• Tamamis, Phanourios
• Whitman, Christian P
• Maynard, Jennifer A

• Ribosome
• Tryptophan
• Oxidized RNA
• Tna operon
• Polynucleotide phosphorylase

Language dc:language.iso

en

Identifier URI

https://doi.org/10.26153/tsw/58142

OAI identifier oai:identifier

oai:repositories.lib.utexas.edu:2152/130794